Study On Synthesis Of Phenyllactic Acid By Lactic Acid Bacteria And Optimization Of Its Conditions And Detection Method Of Phenyllactic Acid

Phenyllactic acid?PLA?is a metabolite of lactic acid bacteria.Due to its broad-spectrum antibacterial properties,stable nature and high safety,it has been increasingly recognized as a food preservative in recent years.Lactic acid bacteria?LAB?are commonly used probiotics in the food industry,and generally recognized as safe.In recent years,it has been widely studied as a synthetic strain of PLA.A RP-HPLC method was established for PLA detection in LAB broth.The effects of three stationary phases,two organic phases,seven water phases and their proportions on the determination of PLA by HPLC were investigated.The double capped C18column?4.6×250 mm,5?m?with 1 m L/min flow velocity at 30?was selected as the stationary phase,isoelution with 0.05%TFA acetonitrile?75?25,v/v?.PLA samples were analyzed under 210 nm detection wavelength.The results showed that the linear range of the calibration curve for PLA was 0.2?5.0 mg/L with a correlation coefficient of 0.9998.The recovery rate was 99.88%?101.08%,LOD and LQD were 0.14 mg/L and 0.46 mg/L,respectively,relative standard deviation?RSD?of precision and repeatability were 0.30%and 0.37%at 1_std,precision and repeatability were 0.59%RSD and 0.79%RSD at 30_thd,stability of PLA standard stock solution was 2.45%RSD.Under the 95%confidence interval?coverage factor k=2?,when the amount of PLA in the fermentation broth was 0.14 mg/L,the expanded uncertainty was 0.0046 mg/L.The yield of PLA synthesizd by Lactobacillus brevis P3 and Lactobacillus plantarum ATCC8014 were 62.60 mg/L and 77.58 mg/L after 120 h culture,respectively.The results demonstrate that PLA can be detected stably,with high efficiency,low cost,simple operation,accurate and reliable results under the proposed method.By comparing 2D-HPLC with 1d-HPLC,it was found that the target peak of PLA detection in fermented food was easily interfered by impurity peak,and the sensitivity of the method can not meet the detection of PLA in fermented food.In this paper,a2D-HPLC method for the PLA detection in fermented foods was established,and the content of PLA in five common fermented foods was determined.The presence of PLA in the sample was verified by UPLC-MS/MS.Both 1D and 2D columns were C18columns.1D column used 0.1%formic acid and methanol to enrich the impurities in the sample.The six-way valve was switched.at 4.70 min.PLA entered the 2D-HPLC system and was eluted by 0.05%TFA and acetonitrile for analysis and detection.The method had a good fitting linearity in the test range?0.4?10.0 mg/L?,r=0.9999,the LOD and LQD were 0.16 mg/L and 0.52 mg/L respectively,and the recovery of 5different systems of food samples were 91.79%?107.37%.The method has good accuracy and validity verified by UPLC-MS/MS.It has been found that the determination of PLA in food by 2D-HPLC possesses the advantages of high resolution,simple pretreatment,accurate results and high detection efficiency.The change of colonies number,the yield of PLA and p H changes of L.plantarum ATCC8014 and L.brevis P3in 120 h were measured under different temperature,p H and salt concentration.The synthesis rate of PLA first increased,then decreased and then gradually tended to 0.Based on the Logistic equation and Luedeking equation,it was found that the growth rate of L.plantarum ATCC8014 was lower than that of L.brevis P3,but the yield of PLA was higher than that of L.brevis P3.The equation parameter showed that L.plantarum ATCC8014 and L.brevis P3 were partially growth coupled.The yield of PLA could be increased by increasing the cell concentration and the substrate of PLA.Therefore,L.plantarum ATCC8014 was selected as the following experimental strain.The effects of phenylalanine?Phe?,phenylpyruvic acid?PPA?,yeast powder and soybean milk on the synthesis of PLA were compared.It was found that the synthesis of PLA with PPA was 8.01 times as much as that with Phe.Single factor and orthogonal experiments were carried out with PPA,yeast powder,inoculation amount,initial p H of fermentation broths and fermentation temperature The optimum conditions for the synthesis of PLA were 0.022 mol/L of PPA,8%of yeast powder,3%of inoculum,6.5of initial p H and 35?of fermentation temperature.