Enrichment Of Trace Elements By Lactic Acid Bacteria And Evaluation Of Their Bioactivities

Lactic acid bacteria(LAB)as a class of GRAS(general regard as safety)edible germs were widely used in global food industry.It is a hotspot in the research field of microbiology due to its outstanding probiotic properties and healthcare function.Enrichment of trace elements by microorganism was a popular direction in functional food area recent decades.One of these most widely researched element-enriched microorganism is yeast.Recent researches indicated that most inorganic trace elements can also be enriched in LAB and transformed into organic forms,which is of great importance for the growth of strains and the demand of human nutrition element.Therefore,this kind of element-enriched LAB will be widely applied in the future.In this study,trace element(iron,zinc and selenium)enrichment ability,antioxidant activity,bile tolerance activity and antibacterial activity of trace element-enriched strains were carried out using Lactobacillus sakei.subsp.sakei WY1 and WY2,Lactobacillus plantarum WY3 and WY4,Lactobacillus brevis WY5,Oenococcus oeni WY6,Lactobacillus delbrueckii ssp.bulgaricus WY7 and Streptococcus thermophilus WY8 as test strains.Considering the massive demand of fermented dairy products in the market coupled with the results of preliminary test,Lactobacillus delbrueckii ssp.bulgaricus WY7 and Streptococcus thermophilus WY8,strains commonly found in yogurt,were further studied on the optimal technological conditions for elements enrichment,their maximum adsorption rate and antioxidant activities.The results of antibacterial activities demonstrated that selenium-enriched(Se-enriched)LAB have the excellent antibacterial activity.Therefore,antibacterial activities of WY7 and WY8 after selenium enrichment were also studied further.The main conclusions were as follows:(1)All LAB of different species in this study showed significant adsorptive abilities of inorganic iron,zinc and selenium with high enrichment ratios.Measured by atomic absorption spectrometry(AAS),it is observed that at the suitable mass concentration of iron,the enrichment rates of iron by different strains were WY6(85.36±1.85 %),WY1(73.74±1.64 %),WY8(73.01±1.25 %),WY5(62.42±0.79 %),WY7(42.69±0.89 %),WY2(39.18±0.94 %),WY3(35.56±1.69 %)and WY4(33.33±1.01 %),respectively.At the suitable mass concentration of zinc,the enrichment rate of zinc by WY6 was the highest(69.25±1.19 %),followed by WY7(59.03±0.90 %),WY1(54.54±1.38 %),WY5(43.42±0.88 %),WY8(36.35±1.02 %),WY4(32.73±1.02 %),WY2(25.78±0.94 %)and WY3(24.78±0.53 %).The 3,3'-diaminobenzidine(3,3'-DAB)method showed that the ratios of Se enrichment by different strains,in descending order,were WY12(79.19±3.01%),WY7(78.72±1.07%),WY6(73.42±1.61 %),WY5(67.65±3.24 %),WY1(64.27±3.31%),WY3(61.49±4.49%),WY2(56.29±3.01%)and WY4(34.98±1.46%),respectively.In vitro study demonstrated that Fe-,Zn-and Se-enriched strains showed improvement on scavenging 1,1-diphenyl-2-picrylhydrazyl(DPPH)and 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate)(ABTS)radical scavenging rates and reducing activities.Compared with the control(strains without elements),significant enrichment of 0.1% bile salts tolerance and antibacterial effect on Escherichia coli and Salmonella typhimurium by Fe-,Zn-and Se-enriched strains were also observed.(2)WY7 and WY8 were selected to test iron enrichment,the range of initial pH,inoculum dose and temperature were defined through single-factor experiment and the optimal iron enrichment conditions were ascertained by response surface methodology in DESIGN EXPERT ANOVA program.The results indicated that the iron enrichment ratio of WY7 reached 52.14 % in pH of 6.7,inoculum dose of 5.86 % and 35.55?.The optimized condition for WY8 was pH of 7.23,inoculum dose of 6.06 % and 40? in which the ratio of iron enrichment was 97.05%.(3)WY7 and WY8 were selected to test selenium enrichment.Based on the results of single factor experiment,the optimization of Se enrichment efficiency was conducted using response surface design.The results indicated that the optimum initial pH,inoculum dose,temperature and the ratio of selenium enrichment of WY7 and WY8 were 5.96,6.73%,33.24?,94.34% and 6.37,6%,40?,97.05%,respectively.FT-IR spectra showed similar or same absorption peaks from Se-enriched samples of both strains.Morphological observation by scanning electron microscope(SEM)equipped with energy dispersion X-ray spectrometry showed the existence of elemental selenium particles on the surface of the cells.(4)WY7 and WY8 were selected to prepare Se-enriched LAB,the accumulation amount of selenium by WY7 and WY8 reached 12.05±0.43 ?g·mL-1 and 11.56±0.25 ?g·mL-1 respectively when 80 ?g·mL-1 of selenite was added in the MRS broth(80 ?g·mL-1 was the suitable concentration in terms of plate colony-counting methods).Oxford cup double plate method was used in order to evaluate their in vitro inhibitory activities against pathogenic Salmonella typhimurium,Escherichia coli,Staphylococcus aureus and Listeria monocytogenes.The results indicated that the cell-free culture supernatant(CFCS)and bacterial culture solution from Se-enriched LAB exerted strong antibacterial activities.The logarithmic phase and stationary phase of the four pathogenic bacteria were delayed by the CFCS from non-Se strains and Se-enriched LAB,the bacteriostasis of Se-enriched LAB was more significant than that of non-Se strains through the measurement of OD values.Analysis by a scanning electron microscope(SEM)showed that the CFCS from Se-enriched LAB induced more serious morphological damage of the four pathogenic bacteria compared with non-Se LAB.