Screening，Characterization, And Functional Investigation Of Lactic Acid Bacteria With Oxidative Stress-alleviating Activities

Development of oxidative stress and damage is a typically physiological and pathological progress insome important human systemic and metabolic diseases, which makes it to be as one of the most often-usedaction target and research model in drug discovery and functional foods development. The dietaryintervention program based on nutrition regulation is an effective strategy for controlling oxidative stress. Ahuge number of studies have demonstrated that the Lactic Acid Bacteria (LAB) may have the potential toalleviate oxidative stress induced by different factors. It possesses great value both in theoretical researchand in practical application to explore the intervening effect of LAB strains on the oxidative stress; screenand excavate specific LAB strains with prominent capacity to reduce the oxidative stress; develop specificfunctional LAB strains and related products based on the principle of regulating gut micro-ecology.According to the literature review on LAB intervening the oxidative stress, this paper conducted a series ofprograms including screening the specific LAB strains with antioxidation capacity in vitro; analyzing andevaluating the intervening effect of specific LAB strains on the oxidation stress induced by high-fat dietand lead-exposure diet; proposing its potential effect mechanisms; as well as study on the microbiologicalproperties and the basis of food processing and application of the specific LAB strains with prominentcapacity to relieve oxidative stress. The study results were as follows.1. Screening and identification of lactic acid bacteria with excellent antioxidant activities in vitro.The structure and constitution of the bacteria flora of Chinese traditional naturally-fermented vegetableswas analyzed by the molecular ecological method, PCR-DGGE. It has been proved that bacteria species,such as Leuconostoc mensenteroides, Lactobacillus plantarum, L. curvatus, L. sake, Lactococcus lactiswere detected in Chinese naturally-fermented vegetables. According to the indicator of acid and bile saltresistance, some strains resistant to bile salt and acid were screened from the naturally-fermentedvegetables. A novel two-step screen method, based on the peroxide stress model in vitro, was developed toscreen the specific LAB strains resistant to the oxidative stress in vitro. With the plate consisting ofthree-layer MRS media containing hydrogen peroxide as the first-step screen tool, together with the cellsurvival rate in the peroxide solution as the secondary-step screening indicator,6LAB isolates withprominent antioxidant activities in vitro were screened from the previous LAB isolates resistant to bile saltand acid, fermented food, and feace samples collected from healthy infants.Antioxidant effects of isolated LAB strains was evaluated by in vitro indicators including potassiumferricyanide reduction activity, lipid peroxidation, DPPH and hydroxyl free radical scavenging action, Fe2+and Cu2+chelating properties. The reducing activities of these two isolates CCFM8661and CCFM1566reached226.47±4.68,212.35±6.45μmol/L cysteine equivalent, respectively. In the liposome system,the IC50value of lipid peroxidation ranged between8.1and9.3（Log10cfu/mL）among different LABisolates tested. Of those isolates, the strain with the strongest capacity to inhibit lipid peroxidation wasCCFM8661(IC50＝8.1Log10cfu/mL). The strains whose scavenging action level of DPPH and hydroxylfree radical was more than60%(cell concentration was1010cfu/mL) were CCFM1106, CCFM8661,CCFM1566and the control strain ATCC53103. The strains CCFM8661, CCFM1566together withATCC53103possessed superior chelating ability to Fe2+and Cu2+whose metal chelating amount rangedfrom9.8to48.7mg/kg. Finally, two LAB strains L.plantarum CCFM8661and L.casei CCFM1566wereselected and identified for further study due to their preferable antioxidant activities in vitro. These twobacteria were submitted to be deposited in China General Microbiological Culture Collection (CGMCC),Beijing.2. Alleviation of high-fat diet-induced oxidative stress by specific LAB strains in mice model.The ability, of the two strains L. plantarum CCFM8661and L. casei CCFM1566, to alleviate theoxidative stress in vivo was assessed by mice model fed on high-fat diet. The results demonstrated that L.plantarum CCFM8661, L. casei CCFM1566and ATCC53103had the potential to alleviate oxidative stressin blood and liver induced by high fat diet. There was a dose-response relationship to some degree betweenthe relieving effect and administration dose.Ingestion of high-fat diet led to the alteration of microflora in mice gut with the amount of Enterobacter,Enterococcus as well as Clostridium perfringens significantly increasing while the level of Lactobacillusand Bifidobacteria are dramatically decreasing. When administrated with L. plantarum CCFM8661and L.casei CCFM1566, the gut microflora in the mice fed on the high fat diet was effectively reversed, and theendotoxin level in the mice plasma was reduced significantly (P<0.05). 3. Screening and characterization of lead-tolerating and binding lactic acid bacteria in vitro.The abilities, of different LAB strains, to tolerate and bind lead ions were evaluated by thelead-tolerating and absorption binding in vitro tests. Besides, the features and rules of lead binding by of L.plantarum CCFM8661were throughly analyzed. The results showed that the lead tolerance was strainspecific. Among strains tested, L. plantarum CCFM8661had the capacity to tolerate the highestconcentration of lead ion (150mg/L) where the bacteria can still grow well.The test of lead-binding ability indicated that L. plantarum CCFM8661had the strongest ability to bindlead which reached49.55mg/g dry bacteria weight (living cell) and53.02mg/g dry bacteria weight (deadcell). The study on the features and rules of lead-binding showed that the pH value has a great impact onbacterial lead-binding. The ability of lead-binding by L. plantarum CCFM8661was improved with theincreased pH value. When the pH value ranged between6.0and7.0, it reached to the maximum bindingamount. By contrast, L. plantarum CCFM8661had the weak capacity to bind little lead or even not anywhen the pH value was below2. The ability of lead-binding by L. plantarum CCFM8661was improvedwith the increased concentration of lead.4. Alleviation of lead exposure-induced oxidative stress and toxicity by L. plantarum CCFM8661.Alleviation effects of different oral administration of L. plantarum CCFM8661on lead-inducedoxidative stress and toxicity were investigated in an orally lead-exposed mice model. The mice fed on L.plantarum CCFM8661(cell concentration1.0×109cfu/mL;0.5mL/d) had significantly lower level of leadin the blood and kidney of the treatment and intervention group compared with the model group (P＜0.05)which indicated that L. plantarum CCFM8661possessed a prominent lead-excreting effect.L. plantarum CCFM8661recovered the activity of target enzyme, thereby avoiding the inhibition ofheme synthesis caused by lead exposure. In addition, L. plantarum CCFM8661reduced the reactive oxygenspecies level caused by lead exposure and restored the damaged antioxidation defense system throughimproving the SOD enzyme activity as well as the level of GSH. The means of intervention by L.plantarum CCFM8661(administrated with CCFM8661when the mice were exposed to lead) was moreeffective than the means of treatment by the same strain (administrated with CCFM8661after the micewere exposed to lead). The bacterial activity of the two intervention methods made no difference in theeffect of reversing the target enzyme activity and relieving the oxidative stress.5. Microbiological characterization of L. plantarum CCFM8661and its preliminary study for foodbioprocessingThe strain L. plantarum CCFM8661is a mesophilic bacteria; its optimal growth temperature is32℃;its growth temperature ranges from10-42℃; its optimal initial pH for growth is6.5; and the range of initialgrowth pH is3.5-8.5. Under the culture temperature of32℃in MRS liquid media, the growth of L.plantarum CCFM8661was corresponding to Boltzman logarithmic growth model: The bacterial growthreached logarithmic growth phase after6-hour’s culture; its growth arrived at stationary phase after18-hour’s culture; the maximum concentration of the live bacteria in MRS media reached4.8×109cfu/mL.L. plantarum CCFM8661grew in the reconstituted skim milk poorly. With the inoculation level of0.1%, the number of its bacterial cell increased by nearly one logarithm cycle in the reconstituted skim milkwithout any carbohydrate while increased by two logarithm cycles in the reconstituted skim milk withglucose. The result indicated that the cow’s milk was not the suitable media for the growth of L. plantarumCCFM8661. The maximum concentration of table salt and bile salt which L. plantarum CCFM8661cantolerate reached8.5%(W/W) and0.3%(tested in the MRS liquid media) respectively. In addition, L.plantarum CCFM8661possessed the preferable resistance to the artificial gastric juice and intestinal juice.L. plantarum CCFM8661presented aggregation as well as co-aggregation capability with pathogenicEscherichia coli to a certain degree. The hydrophobic test result proved that L. plantarum CCFM8661possessed superior hydrophobicity. L. plantarum CCFM8661had a prominent adhesion ability whoseadhesion level on HT29cell line reached1.8±0.3×106CFU/well approximating to the adhesion ability ofL. rhamnosus ATCC53103.pH value was one important factor influencing the viability of L. plantarum CCFM8661duringstorage. The strain L. plantarum CCFM8661maintained good stability when pH value ranged between4.0and6.0. The live bacterial number stayed above106cfu/mL all the time during24-day storage at4℃. As aresult, L. plantarum CCFM8661could be applied in fermented milk production as supplementary culturewhich was not detrimental either to other strains in fermented milk or the chemical and physical indicators.L. plantarum CCFM8661had a pretty good growth and acid production abilities in soybean milk whichmade it suitable as a starter culture for sour soyamilk production.