Intervention Effect Of Lactobacillus Brevis On Type ? Diabetes

During the past two decades,diabetes has become the third most serious threat to human health,following cancer and cardiovascular disease.Nowadays,food plays a vital role in inhibiting diseases and ensuring health.One of the most important challenges facing customers is the safety of high-quality healthy foods and the creation of a new diet that focuses on the prevention of chronic diseases and disorders.For instance;obesity;diabetes;atherosclerosis;heart disease;retinopathy;kidney toxicity;atherosclerosis;hypertension;diabetic foot ulcers,and cystic fibrosis.Lactobacillus brevis strains are a highly efficient cell factory that produces bioactive?-aminobutyric acid?GABA?through its gad operon-encoded glutamic acid decarboxylase?gad?system.Gamma-aminobutyric acid?GABA?is a biologically active ingredient in functional foods due to its health-enhancing properties as an anti-stress,antihypertensive,an anti-diabetic agent.GABA is the main central nervous system?CNS?inhibitory neurotransmitter and is significantly involved in regulating many physiological and psychological processes.The aim of the present study was investigated of potential probiotic characterization of Lactobacillus brevis?KLDS 1.0727 and KLDS 1.0373?strains in vitro as digestion models mimicking the conditions of gastrointestinal tract and different types of antibiotics.Further,large antagonistic activity against pathogeneses.Moreover,tolerance of Lactobacillus brevis strains to different pH levels or bile and intestinal juices.As well,the ventilation effect on viability of Lactobacillus brevis strains in aerobic or anaerobic conditions.In addition,investigate the administration of?-Aminobutyric acid?GABA?produced by KLDS 1.0727 and KLDS 1.0373 as a gad gene carrier to express GABA that have preventive effects against the development of diabetes in Streptozotocin?STZ?induced diabetic male C57BL/6 mice as diabetic models in vivo.The methodology was carried out as follows:gad Gene were genetically labeled as 16S-rRNA sequence analyses to identify gad gene.Moreover,free or freeze-dried of KLDS 1.0727 and KLDS 1.0373 strains were tested in vitro.Evaluation of antimicrobial activity against foodborne pathogens and antibiotic susceptibility was assessed as well.The in vivo experiments as a body weight and blood glucose levels were weekly assessment.After mice euthanasia,blood plasma was a collection for biochemical analysis.On the other hand,the mice organs?liver,pancreas,kidney,and spleen?were taken out External inspection histological.The obtained results show that:1)KLDS 1.0727 and KLDS 1.0373 have gad gene as 1,407 bp and biochemical analysis and genetic screening have confirmed the common existence of gad system in Lactobacillus brevis suggesting its species-specific characteristic of GABA production.2)meanwhile the effect of two types of pH?2,3?on the survival of free or freeze-dried Lactobacillus brevis strains at different cultivation time?0,30 and 60 minutes?showed values in 60minutes of free KLDS 1.0727 at pH 2 and 3 were?5.13±5.61×104,1.41±0.10 CFU×10⁴,respectively?.While KLDS1.0373 at pH 2 and 3?2.25±0.491,2.14±0.06 CFU×10⁴,respectively?.Conversely,freeze-dried KLDS 1.0727?3.40±0.43,1.76±0.11 CFU×10⁴,respectively?.Whereas,freeze-dried KLDS 1.0373?5.75±0.21,2.47±0.16 CFU×10⁴,respectively?.3)Bile salts resistance shows that the number of free KLDS 1.0727 strain is a high resistance to bile salts at 2%at 3 hours?8.50±0.28×10⁸ CFU?.While free KLDS 1.0373 was?1.06±0.08×10⁸CFU?.In contrast,freeze-dried KLDS 1.0727 was?8.00±0.57×10⁹ CFU?,while freeze-dried KLDS1.0373 was?1.15±0.11×10⁹ CFU?.the data disabled the highest growth in free or freeze-dried KLDS1.0727 at 2%concentration of bile.4)Resistance of KLDS 1.0727 and KLDS 1.0373 to different types of antibiotics showed a high significance?P<0.001?indicates that KLDS 1.0727 and KLDS 1.0373 show high resistance to antibiotics and low sensitivity to other antibiotics as free?KLDS 1.0727 and KLDS1.0373?or freeze-dried?KLDS 1.0727 and KLDS 1.0373?was sensitive to ampicillin;chloramphenicol;erythromycin,and gentamicin.While freeze-dried KLDS 1.0727 was resistant to kanamycin further KLDS 1.0727,KLDS 1.0373 was resistant to streptomycin.While free and dried freeze-dried KLDS 1.0373 were resistant to kanamycin.5)Tolerance of KLDS 1.0727 and KLDS 1.0373 to simulated intestinal juice?SIJ?with different pH levels under aerobic or anaerobically conditions expressed a very high significant value of<0.0001between different pH values under aerobic or anaerobic conditions with simulated intestinal juice?SIJ?.The capabilities of KLDS 1.0727 and KLDS 1.0373 were provided to carry the simulated intestinal juice?SIJ?The highest growth rate was 24 hours in control treated without any addition of simulated intestinal juice?SIJ??1.88±0.03 and 1.80±0.02,respectively?.The lowest growth rates were24 hours with pH 2.0?0.09±0.01 and 0.14,respectively?.In addition,the efficacy of KLDS 1.0727 and KLDS 1.0373 of the?SIJ?was shown to be anaerobic the validity of KLDS 1.0727 and KLDS 1.0373 of simulated intestinal juice?SIJ?,while the highest growth rates at 24 hours in control were treated?3.481±0.135 and 3.519±0.032,respectively?and the lowest growth rates were 24 hours with pH 2.0?0.166±0.011 and 0.140±0.001,respectively?.6)Survival of free or freeze-dried Lactobacillus brevis strains in simulated?GIJ?conditions showed a high significant different?P<0.01?between all test treated as follows?S1?and?FS1?were treated with artificial saliva?73.61 and 96.72%,respectively?and treated with gastric juice?96.59 and88.52%,respectively?,while simulated intestinal fluid treatment displayed?96.86 and 57.37%?.On the other hand,KLDS 1.0373?S2 and FS2?was shown the artificial saliva?101.77 and 98.83%,respectively?and gastric juice treatment?96.45 and 87.54,respectively?.Finally,treatment of artificial intestinal fluid was presented?395.78 and 54.47%,respectively?.By contrast,free strains have the highest survival rate for each treatment of simulated GIJ rather than freeze-dried strains.7)Inhibitory effect of free or freeze-dried KLDS 1.0727 and 1.0373 strains against foodborne pathogenic bacteria showed a highly significant degree of P<0.01.a strong activity of Lactobacillus brevis strains against all food-borne pathogens.Free KLDS 1.0727;KLDS 1.0373,and freeze-dried KLDS 1.0727;KLDS 1.0373 clear zone were showed Staphylococcus aureus?22.32±0.25,22.10±0.56,17.05±0.67,and 20.60±1.07 respectively?.While,E coli?22.35±0.92,23.37±1.12,17.17±0.68,and21.63±0.88,respectively?.In addition,it showed significant activity against Listeria monocytogenes?19.97±0.76,21.29±0.79,17.62±0.24 and 25.92±0.80,respectively?.However,Salmonella typhi?23.40±0.41,22.58±0.68 and 18.19±0.85,and 23.21±0.31,respectively?.8)Adhesion of KLDS 1.0727 and KLDS 1.0373 to Caco-2 cells showed the percentage of the KLDS 1.0727 and KLDS 1.0373 ranged between 55.6%and 95.2%with three different folds of the KLDS 1.0727 and KLDS 1.0373 strains as 3,4 and 5.9)Finally,it should mention the effect of KLDS 1.0727 and KLDS 1.0373 in vivo experiments of the male C57BL/6 diabetic model showed that due to using free or freeze-dried KLDS 1.0727 indicates to reduce the blood glucose levels weekly postprandial and after fasting.Hence,?S1;FS1,and S2?displayed a little convergence of glucose level.In contrary,?STZ+INS?showed a dramatic decrease in the weekly blood glucose levels in mice.Besides,the body weight was nonsignificant on the entire treated group but the difference varied between groups on the last week of the experiment.While the lowest body weight was?STZ and FS2?groups,were shown?19.78±2.26^a,19.06±2.15^a respectively?.As well as the biochemical analysis of blood plasma displayed the serum lipids concentrations as triglyceride?TG?;total cholesterol?CHOL?;high-density lipoprotein cholesterol?HDL?;low-density lipoprotein cholesterol?LDL?;glucose?GLU?,and magnesium(Mg⁺²).The results expressed?Cont?showed the highest level of?TG,CHOL,HDL,and LDL?,while,?STZ?showed the highest level of(Mg⁺²),Moreover,?STZ+INS?the lowest level of(CHOL and Mg⁺²)and the highest level of?GLU?as well.Conversely,?S1?expressed the lowest level of?TG,LDL,and GLU?further;?FS1?displayed a low level of?LDL?.The liver function parameter assessment showed?STZ?group was the highest level of?TP;AST;ALB,and GLUB?.Furthermore,?FS2?displayed the highest level of?TBA?.Whereas,?Cont,and S1?expressed the lowest level of Uric acid?URIC?as a kidney function.In Summary,the mild effect of type 1 diabetes on components of mice blood plasma might be due to the use of Lactobacillus brevis that one of the lactic acid bacteria group which,have a vital role in inhibiting the damage caused by type I diabetes.As a recommended,the use of Lactobacillus brevis strains should participate a wide range on the market as a natural source of GABA in pharmaceutical and food applications.