Screening Potential Probiotic Characteristics of ...

Research ArticleScreening Potential Probiotic Characteristics ofLactobacillus brevis Strains In Vitro and InterventionEffect on Type I Diabetes In Vivo

Amro Abdelazez 12 Heba Abdelmotaal 34 Smith Etareri Evivie15

Sherif Melak67 Fang-Fang Jia1 Mir Hassan Khoso4 Zong-Tao Zhu1

Lu-Ji Zhang1 Rokayya Sami 8 and Xiang-ChenMeng 1

1Key Laboratory of Dairy Science of Ministry of Education Northeast Agricultural University Harbin 150030 China2Department of Dairy Microbiology Animal Production Research Institute Agriculture Research Centre Dokki Giza 12618 Egypt3Department of Microbiology Soil Water Environment and Microbiology Research Institute Agriculture Research CenterGiza 12619 Egypt

4Department of Microbiology and Biotechnology College of Life Sciences Northeast Agricultural University Harbin 150030 China5Department of Animal Science Faculty of Agricultural University of Benin 1154 Benin City Edo State Nigeria6Department of Animal Science and Biotechnology College of Animal Science and TechnologyNanjing Agricultural University Nanjing 210095 China

7Department of Sheep and Goat Animal Production Research Institute Agriculture Research Centre Dokki Giza 12618 Egypt8Department of Food Science and Nutrition Taif University Taif Al-huwayah 888 Saudi Arabia

Correspondence should be addressed to Xiang-Chen Meng xchmenghotmailcom

Received 22 May 2018 Revised 21 July 2018 Accepted 8 August 2018 Published 19 September 2018

Academic Editor Kota V Ramana

Copyright copy 2018 AmroAbdelazez et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Diabetes has become the third most serious threat to human health after cancer and cardiovascular disease Notably Lactobacillusbrevis is the most common species of LAB that produces 120574-aminobutyric acid (GABA)The aim of this study is to clarify the effectof time strain types antibiotic concentrations different levels of pH and intestinal juices in aerobic or anaerobic conditions and theeffect of interactions between these factors on the potential properties of KLDS 10727 and KLDS 10373 furthermore antagonisticactivity against foodborne pathogens Moreover another aim is to study the capability of KLDS 10727 and KLDS 10373 strainsas gad gene carriers to express GABA that reduce the risk of type 1 diabetes in C57BL6 mice as diabetic models The obtainedresults exhibited the surprising tolerance of Lactobacillus brevis strains in vitro digestion models mimicking the conditions of thegastrointestinal tract further large antagonistic activity against foodborne pathogeneses In vivo results displayed the significanteffect on glucose level reduction blood plasma and histological assays of mice organs As recommended the use of Lactobacillusbrevis strains should be widely shared in the market as a natural source of GABA in pharmaceutical and food applications

1 Introduction

Nowadays food plays a vital role in inhibiting diseases andensuring health One of the most important challenges facingcustomers is the safety of high-quality healthy foods andthe creation of a new diet that focuses on the prevention ofchronic diseases and disorders [1] Functional food acts as

beneficial compounds or foods containing microorganismsexhibiting a pivotal role in strengthening and enrichinghealth well-being and suppressing some strict disease forinstance obesity diabetes atherosclerosis heart diseaseretinopathy kidney toxicity atherosclerosis hypertensiondiabetic foot ulcers and cystic fibrosis [2 3] The most vitalcomponents of new functional foods are called probiotics

HindawiBioMed Research InternationalVolume 2018 Article ID 7356173 20 pageshttpsdoiorg10115520187356173

2 BioMed Research International

which can increase host immunity and improve healthbenefits [4] Recently various conventional microorganismshave been used for probiotic cultures in fermented foodsFurthermore probiotics include a wide range of pharma-ceuticals or foods and special dietary applications suchas juices nutrition bars infant formulas and cosmetics[5]

The medical application of Lactobacillus brevis KB 290isolated from ldquoSugukirdquo is a traditional Japanese pickle thatplays a crucial role against people with influenza [6] In addi-tion it plays a vital role in suppressing inflammation causedby nitric oxide [7] Several studies have been conducted onseveral Lactobacillus brevis strains isolated from traditionalEgyptian dairy products which have been tested to confirmtheir antimicrobial effects These results show the extremeactivity of Lactobacillus brevis B23 among 38 species [8]Lactobacillus brevis is a vital biological organism to producebioactive 120574-aminobutyric acid biologically [9] Formerly 120574-aminobutyric acid (GABA) has been attracting attentionbecause it is the most abundant inhibitory neurotransmitterthat maintains the neurotransmitter functions of the humancentral nervous system Furthermore GABA may not havethe ability to penetrate the human blood-brain barrier [10]gad antibodies a symptom of type 1 diabetes can be detectedbefore the onset of clinical disease and the identification of asubset of patients with adult autoimmune disease [11]

Currently more than a thousand LAB strains have beensequenced and deposited in the NCBI GenBank databaseTherefore genetic assays were performed inmost continuousstrains in the presence or absence of gad operon and genesencoding glutamate decarboxylase in most of the sequencedstrains Moreover there are not only differences betweenmicrobial species from different sources in the gad enzymaticproperties but also differences between the subspecies as wellLactobacillus different sources of gad subunit compositionand molecular weight have a huge difference Lactobacillusbrevis gad has two subunits molecular weight of 57 60 kDa[12]120574-Aminobutyric acid (GABA) is a nonprotein four-

carbon free amino acid synthesized by the irreversible120572-decarboxylation reaction of L-glutamic acid or its saltsa pyridoxal-50-phosphate-dependent enzyme (PLP activevitamin B6)[13] GABA is permitted as a dietary ingredientand its nutrients have shown antihypertensive and antide-pressant activities as the two main functions of the host afteroral administration [9] However the GABA content in a fewnatural plants and animal products has been further studiedin the LABs because it also possesses the ability to produceGABA

Type 1 diabetes (T1D) is an autoimmune disease char-acterized by selective destruction of the pancreatic betacells [14] Furthermore it is a metabolic disease inducedby abnormal insulin secretion by damaged islet 120573 cellsThat damage accrues resulting in insulin insufficiency whichcan lead to life-threatening hyperglycemia that manifestsclinically as weight loss polyuria polyphagia and polydipsia[15] LAB could be utilized to grab foodborne diseasesresearchers used Salmonella typhimurium which causes

vomiting abdominal pain nausea and diarrhea [16] Listeriamonocytogenes which causes abortion and gastrointestinaldiseases [17] Staphylococcus comets which is involved inharmful gastroenteritis and E coli thereby exacerbating thesyndrome and colitis [18]

The main objective of the present study was to examinethe new Lactobacillus brevis probiotic strains that could effi-ciently produce high levels of bioactive 120574-Aminobutyric Acid(GABA) by exploring gad gene in addition study potentialprobiotic properties in vitro where they remain in severecase of simulation gastrointestinal juice and different typesof antibiotics and moreover antimicrobial activity againstfoodborne pathogens and finally control the development oftype 1 diabetes in vivo

2 Materials and Methods

21 Bacterial Strains and Cultivation Conditions Two strainsof Lactobacillus brevis KLDS 10727 and KLDS 10373 werefrozen stored at minus80∘C Further the foodborne pathogenicmicroorganisms used were Salmonella typhimurium (ATCC14028-MINI-PACK) (American Type Culture Collection)E coli IQCC 10126 Listeria monocytogenes IQCC 22221and Staphylococcus aureus IQCC 22035 (Inspection andQuarantine Culture Collection of the Chinese Academy ofInspection and Quarantine culture libraries IQCC) All teststrains were obtained from Key Laboratory of Dairy Science(KLDS) Heilongjiang Province Harbin China

Lactobacillus brevis KLDS 10727 and KLDS 10373 strainswere inoculated (1 vv) and grown in De Man RogosaSharpe medium (MRS Oxoid) while foodborne pathogenicbacteria were grown in Tryptic Soy Broth (TSB) (SigmaAldrich) All the strains were cultivated at 37∘C aerobicallyfor 24 hours The solid plates medium was cultivated for 48hours single colonies were picked after examination of thepure bacteria and they were then cultivated in an adequateliquid culture medium

KLDS 10727 and KLDS 103737 colonies were streakedon MRS agar plate and incubated at 37∘C for 24 hours underaerobic conditions Colonies were randomly picked from theplates and subculture two or three times on fresh MRS agarplates A single colony from the plate was transferred toMRSbroth and incubated for 18 hours at 37∘C The pure KLDS10727 and KLDS 10373 cultures were kept in MRS brothsupplemented with 30 (vv) glycerol and frozen at minus80∘Cuntil further analysis

22 Screening of Lactobacillus brevis KLDS 10727 and KLDS10373 Strains for the Presence of Glutamic Acid Decarboxylase(gad) Gene Genomic DNA was prepared using TIANampBacterial DNAKit (Tiangen Biotech Co Ltd Beijing China)and with some modification of manufacturerrsquos extractionsteps KLDS 10727 andKLDS 10373 were lysed with lysozymeDNA extracted

23 PCR Amplification of the 16S rDNA and Sequence Anal-ysis PCR amplification was performed using the universalprimers

BioMed Research International 3

Primer ID Sequences (51015840 997888rarr 31015840 ReferenceLb-PTC-F GCCAGAAACGCTCAAGAT

[19]Lb-PTC-R GGCTTCGTATAAGCCATACCLb-OTC-F GTGAAAGCAACTGGGAAGALb-OTC-R GTTATGGAAAGCAGGCAAACLb-TDC-F CGATCAAGCAGAGTCCATTACLb-TDC-R CGGCACCCTTCTCAAATAC

Genomic DNA was used as a template for PCR ampli-fication Fifty microliters of each PCR mixture containedDNA template 10 120583L two primer pairs (ComateBio CustomPrimers Jilin Changchun China) gad-FgadR (10 120583mol L) 20 120583L [19] DNA Polymerase (25 U 120583L) 05 120583L10times Taq Buffer 50 120583L 2rsquo-deoxynucleoside 5rsquo triphosphate(dNTPs) (25 mM) 40 120583L and ddH

2O 355 120583L Furthermore

PCR amplification was performed using the GeneAmp PCRSystem 9700 thermal cycler (Applied Biosystem USA) withthe thermal cycling parameters as follows denaturation at95∘C for 5 min 30 cycles of 95∘C for 30 s 55∘C for 130 minand 72∘C for 130 min with a final extension at 72∘C for 10min A 5-120583L volume of PCR product was electrophoresedon a 1 agarose gel (Gibco BRL France) at 90 V for15 hours and then stained with ethidium bromide andband patterns were visualized Sequence similarity analysiswas performed in the GenBank database using the BLASTprogram (httpblastncbinlmnihgovBlastcgi) (NCBI)

24 Evaluation of KLDS 10727 and KIDS 10373 In VitroDigestion Models Mimicking the Gastrointestinal Tract Invitro assessment has been tested for the viability of KLDS10727 and KLDS 10373 for example growth rate bile saltssimulated intestinal juice (SIJ) at different pH levels underaerobic or anaerobic conditions [20] and surviving in a widerange of different antibiotics types The antagonism of variousfoodborne pathogens and furthermore all experiments wereexpressed in triplicate using the MRS broth as a blank Inaddition the absorbance was measured with a wavelength of620 nm and the data were expressed as OD

241 Standard Curve of Growth Rate and pH for KLDS 10727and KLDS 10373 strains KLDS 10727 and KLDS 10373strains were inoculated (1 vv) and grown in MRS brothat 37∘C under aerobic conditions The growth bacteria weremonitored using a spectrophotometer (F-7000 Hitachi HighTechnologies 155 Shenzhen China) and pHwas determinedby using pH meter (MP 220 Mettler Toledo GreifenseeSwitzerland)

242 Resistance of KLDS 10727 and KLDS 10373 to DifferentBile Salts Concentration in MRS Incubated at 37∘C KLDS10727 and KLDS 10373 were tested for hydrolyzed bile saltactivity The strains were cultured in MRS broth added to03 1 and 2 (wv) of the bovine bile (Difco DB DiagnosticSystem reference 212820) and incubated at 37∘C underaerobic conditions then the tested media were obtained asa filtration solution through a 022 120583m filter (Critical SyringeFilters Critical Process Filtration Inc) The activated strainsin the MRS broth were taken without Ox gall powder as a

control [21] The following equation expressed the survivalpercentage of bile salts after 33 hours

Bile survival =logN1logN0times 100 (1)

N1 is absorbance of cultures in MRS broth containing 03 1and 2 bile salts (see [22]) log N0 is absorbance of culturesin MRS broth without bile salts

243 Resistance of KLDS 10727 and KLDS 10373 to DifferentTypes of Antibiotics The microdilution protocol was used toassess the resistance of probiotic bacteria to different types ofantibiotics [23] with some modifications according to EFSAGuidance [24] The antibiotics ampicillin chloramphenicolerythromycin gentamycin kanamycin (Sigma Aldrich StLouis MO) and streptomycin (MP Biomedicals Santa AnaCA) and the amounts of the active compound were placedinto the broth media tube ampicillin (10 120583g) chloram-phenicol (30 120583g) erythromycin (15 120583g) gentamycin (10 120583g)kanamycin (30 120583g) and streptomycin (10 120583g) Cultures wereinoculated in MRS broth filtered through a 022-120583m filterand incubating aerobically at 37∘C overnight Furthermoreantibiotic stock solutions were prepared according to thefollowing formula

W = 1000Ptimes V times C (2)

where P is potency given by the manufacturer (120583g mg) Vis volume required (ml) C is final concentration of solution(multiples of 1000 mgL) and W is weight of antibiotic (mg)to be dissolved in volume V (mL) The data was expressedafter 24 hours as the following formula

Survival =logN1logN0times 100 (3)

Sensitivity of antibiotic = 100 minus survival (4)

logN1 is absorbance of culture 620 nm in MRS broth withdifferent antibiotic types and logN0 is absorbance of culture620 nm in MRS broth as a control

244 Tolerance of KLDS 10727 and KLDS 10373 to Simu-lated Intestinal Juice (SIJ) with Different pH under Aerobicor Anaerobic Conditions Lactobacillus brevis strains are afacultative anaerobic LAB strains Therefore KLDS 10727andKLDS 10373 strainswere propagated in LactobacilliMRSbroth for 24 hours Simulated intestinal juice (SIJ) (grams100ml) was prepared as follows 01 of trypsin 18 of bile salts


11 of sodium bicarbonate 02 of sodium chloride followedby adjusting the final pH to 20 30 and 70 and sterilizingthe obtained solution by filtration through 022 120583m filter[25] Afterwards an appropriate amount (109 CFU mLminus1)of the freshly prepared inoculum of KLDS 10727 and KLDS10373 strains was inoculated into the prepared (SIJ) andincubated for 0 3 6 and 24 hours Moreover anaerobicconditions designed by anaerobic glove chamber (SheldonManufacturing Inc Shel LAB Cornelius OR USA) wereused with a gas mixture of (90 N

2 5 CO

2 5 H

2)

According to the comparison of these strains in the ability tosurvive after 24 hours


where logN1 is absorbance of culture 620 nm in MRS brothat pH 2 3 and 7 and logN0 is absorbance of culture 620 nmin MRS broth as a control

245 Screening Antagonistic Properties of KLDS 10727 andKLDS 10373 Strains Antagonistic activities were assessedby measuring clear zones mm from the edge as describedby Damaceno et al [26] Two single strains of KLDS10727 and KLDS 10373 and foodborne pathogens Salmonellatyphimurium ATCC 14028 E coli IQCC 10126 Listeriamonocytogenes IQCC22221 and Staphylococcus aureus IQCC22030 as indicator strains studied for antagonism were usedAgar slabs of 6-mm in diameter were aseptically cut off fromthe MRS agar overgrown with a lawn of KLDS 10727 andKLDS 10373 strains incubated for 24 hours at 37∘C underaerobic conditions and placed on plates with the Tryptic SoyMedium agar inoculated with the indicator strain (105ndash106CFU mL) The plates were kept at 4∘C for 3 hours to permitdiffusion on the assay material and incubated at 37∘C for24 hours of incubation and then the diameters of clearzones around the agar slabs were measured To compare theantagonistic activity of KLDS 10727 and KLDS 10373 strainsagainst the indicator cultures the clear zones were expressedas 14 mm 3 points 9ndash14 mm 2 points 1ndash89 mm 1 point

25 Caco-2 Cell Adhesion Assay The ability of KLDS 10727and KLDS 10373 strains was tested for adherence to humanepithelial cells using Caco-2 cells (human colonic adenocar-cinoma ATTC HTB-37) [27] Monolayers of Caco-2 cell lineobtained from China Cell Bank Shanghai were grown inDulbeccorsquos modified Eaglersquos Medium (Sigma Aldrich) highglucose supplemented with 10 (vv) fetal bovine serum(Gibco reference 12484-028) 1 (vv) nonessential aminoacid and 1 (vv) penicillin-streptomycin at 37∘C in ahumidified atmosphere of 95 air and 5 CO

2 The Caco-2

cell concentration was adjusted to 5times105 cellmL and seededinto well tissue culture plates and subsequently incubated at37∘C in a 5 CO

2incubator until the Caco-2 cells attained a

confluent differentiated monolayer state (15 plusmn 1 d)The Caco-2 monolayer was washed twice with sterile PBS to eliminatethe penicillin-streptomycin The KLDS 10727 and KLDS10373 strains were harvested by centrifugation (10000 g 5min 4∘C) and washed twice with sterile PBS Then density

was adjusted with high-glucose DMEM without antibioticsto 108 CFUmL Afterwards 1 mL of each KLDS 10727 andKLDS 10373 strains suspension were added to the wells andthe plates were incubated for 2 hours at 37∘C in a 5 CO

2

atmosphere At the end of the assay Caco-2 cells were washedthree times with sterile PBS to remove unadhered KLDS10727 and KLDS 10373 cells

The Caco-2 monolayers were lysed by treatment withEDTA-trypsin solution for 3ndash5 min at 37∘C for disruptingthe adherent cells The Caco-2 lysate and the attached KLDS10727 and KLDS 10373 cells were plated on MRS agar plateafter serial dilution and counted after 24 hours of incubationat 37∘CThe adhesion ability of KLDS 10727 and KLDS 10373was determined using the following formula

Adhesion =logNtlogN0times 100 (6)

where logNt is the number of KLDS 10727 and KLDS 10373strains that adhered to theCaco-2monolayers and log N0 is atotal number of KLDS 10727 and KLDS 10373 strains addedas a control blank

Moreover the direct microscopic examination methodwas used to disable the adhesion of KLDS 10727 and KLDS10373 with Caco2 cells after (15 plusmn 1 d) of cultivation asdescribed above The culture medium was replaced with anantibiotic-free medium one day prior to the adhesion assayAfterwards the cells were washed twice with phosphate-buffered saline (PBS) (pH = 72) KLDS 10727 and KLDS10373 strains were added to the prepared cell monolayersAfter 2 hours of incubation at 37∘C all monolayers werewashed 5 times with PBS to remove nonadherent bacteriaFinally binding between Lactobacillus brevis strains andCaco2 cells was examined by Gram-stained phase contrastmicroscopy (magnification fold 200 x) The adhered Lac-tobacillus brevis strains Caco2 cells were determined in 15randomly selected microscopic fields

26 In Vivo Experiments

261 Animal Experiments Design and Establishment of Dia-betic Model Mice Specific pathogen-free (SPF) male miceC57BL6 (6-8 weeks old) were purchased from Vital RiverLaboratory Animal Technology Co Ltd (Beijing China)housed in a roomunder controlled environmental conditionsat 23plusmn2∘C a relative humidity of 50plusmn20 with artificial lightcycle a 12-h lightdark Mice were raised in independentventilated cages and received pathogen-free food and waterThe mice were acclimatized for one week of the laboratoryconditions before beginning the experiments The experi-mental protocol was approved by the Institutional AnimalCare and Use Committee of the Northeast AgriculturalUniversity under the approved protocol number specificpathogen-free rodent management (SRM)-06

Table 1 shows the animal experimental designed theanimals were divided into five groups within four mice pergroup Afterwards the diabetic mice were treated with highdose of streptozotocin (STZ 180 mgkg) (Sigma Aldrich)freshly prepared in 50mM sodium citrate buffer (pH 45) andsubcutaneously injected within 10 to 15 min after dissolving


Table 1 Experiments design of diabetic model mice

Group Treatment Dose mouse Time of treated Methods of delivered(Cont) 250 120583L Saline Daily via gavage(STZ) 180 mgkg One time subcutaneously

(INS+STZ) 100 120583L Daily subcutaneously(S1) 250 120583L Daily via gavage

Male C57BL6 (S2) 250 120583L Daily via gavage(Cont) control group (STZ) streptozotocin (INS+STZ) insulin + streptozotocin (S1) KLDS10727+ streptozotocin and (S2) KLDS10373+ streptozotocin

for one time according to a previously described procedure[28]

The nondiabetic control group received an injection ofcitrate buffer only 3 days post-STZ-injection glucose levelswere measured using a glucometer (Yuwell Jiangsu China)The mice with glucose levels higher than ge7 mmoldl wereconsidered diabetic and STZ-induced mice that had a lowerglucose level were excluded

262Weekly Determination of Glucose Level and BodyWeightof Streptozotocin-Induced Diabetic Mice Following bloodglucose blood glucose level and body weight of overnightfasting for 12 hours and 2 hours postprandial were assessedweekly Glucose levels weremeasured using a glucometer andglucose expressed as mmol dl

Glucose level = Postprandial 2 hours

minus Fasting 12 hours(7)

263 Plasma Biochemical Analyses After feeding for fourweeks fasting blood samples were collected from the ocularvein of each group and allowed to clot at 4∘C and then cen-trifuged at 12000 times g for 10 min The plasma was transferredto a new microcentrifuge tube and stored in a -80∘C for bio-chemical measurements The blood plasma was determinedfor serum lipids concentrations as triglyceride (TG) totalcholesterol (CHOL) high-density lipoprotein cholesterol(HDL) low-density lipoprotein cholesterol (LDL) glucose(GLU) magnesium (Mg+2) which were measured Further-more liver functions were evaluated by assessing serum ala-nine aminotransferase (ALT) aspartate transaminase (AST)ASTALT total bile acid (TBA) albumin (ALB) globulin(GLUB) total protein (TP) Moreover determination ofkidney functions such as uric nitrogen (BUN) creatinine(CREA) uric acid (URIC) levels was assessed as well Alltest parameters were a determination by using a BeckmanCoulter UniCel DxC 800 (Beckman Coulter Miami FLUSA) analyzer

264 Serum Insulin Determination Mice were fed rodentchow for 4 weeks with injected daily with insulin (Sigma)using dose unit (05 units kg body weight) Insulin wasdiluted in acetic acid for a final injected volume of 100 120583lInsulin sensitivity was evaluated essentially as described bySurwit et al [29] Blood serum insulin was detected by

insulin determination ELISA kits (Meimian BiotechCo LtdYancheng China) The experimental process was accordingto the manufacturerrsquos instructions of insulin kits

265 Histological Evaluation The experimental proceduresused for routine histological examination of organs in micetissue were previously described [30] Briefly the mice organs(liver pancreas kidney and spleen) were removed andwashed by phosphate buffer (pH 72) and then fixed in 10 neutral formalin followed by dehydrating in gradientalcohol (75 85 95 and 100) and xylene (100) thenembedded in paraffin and sectioned at 5 mm thicknesssubsequently followed by staining in hematoxylin and eosinafter euthanasia tissues staining The sections were assessedby light microscopy (Olympus Japan) under 100 times magnifi-cations

27 Statistical Analysis All valueswere expressed as themeanplusmn standard deviation (SD) Aminimum of three independentexperiments was carried out for each assay The statisticalsignificance of data comparisons was determined using one-way analysis of variance (ANOVA) Values of p lt 005 wereconsidered statistically significant Statistical analysis usingSAS system software (version 91 SAS Institute Cary NCUSA) was used to calculate F values and compare betweenmeans byDuncanrsquos multiple range test Two statistical modelswere used to estimate phenotypic traits as follows

Yijk = 120583 + Ai + Bj + ABij + eijk (8a)

Yijkl = 120583 + Ai + Bj + Ck + ABij + ACik + BCjk

+ ABCijk + eijkl(8b)

where Yijk is phenotype traits 120583 is the overall mean Ai is theeffect of the ith strains Bj is the effect of the j

th time levelsCk is the effect of the k

th concentrations or antibiotic typesFurther ABij is the interaction between i

th strains and jth timelevels Moreover ACik is the interaction between strains andkth concentrations or antibiotic types BCjk is the interactionbetween jth time and kth concentrations or antibiotic typesABCijk is the interaction between ith strains jth time levelsand kth concentrations or antibiotic types and eijkl is the effectof the random error


bp M 1 2 3 4

Figure 1 PCR amplification of gad gene M marker 1 2 PCRproducts of Lactobacillus brevis KLDS 10727 and 3 4 of KLDS10373

3 Results

31 PCRAmplification of the 16S rDNAand Sequence AnalysisFigure 1 showed the PCR amplification results of the presencegad gene and the nucleotide sequence of gad displayed the1407 bp Further our previous study reported the HPLCchromatogram analysis for GABA produced by KLDS 10727and KLDS 10373 was 198plusmn007 and 005plusmn005 gL respec-tively (data no shown) Several studies have shown that theanalysis of the sequence of nucleotide gad showed clonedgenes consisting of 1407 bp and 468 amino acids wereencoded [31]These results were closely related to the findingsby Hiraga et al [32] who suggested that the gad gene ofLactobacillus brevis IFO 12005 was 1440 bp Moreover Wuet al [19] reported that biochemical analysis and geneticscreening have confirmed the common existence of gadsystem in Lactobacillus brevis suggesting its species-specificcharacteristic of GABA production

32 Assessment of Lactobacillus brevis KLDS 10727 and KLDS10373 In Vitro The in vitro results obtained in three mainTables 2 3 and 4 will be expressed to show the effect ofsome factors (strain types time level concentrations andinteraction of different parameters) as the equations of thestatistics model mentioned above

33 Determination of the Growth Rate and Bile Tolerance ofLactobacillus brevis KLDS 10727 and KLDS 10373 StrainsThe obtained results in Figure 2 showed the standard curveof growth rate and bile tolerant with the accumulated acidof KLDS 10727 and KLDS 10373 strains There was asignificant positive correlation between growth rate and pH(098) Duncan comparisons between strains showed highlysignificant differences betweenKLDS 10727 andKLDS 10373as shown in Table 2 KLDS 10727 showed a high growthrate and decreased pH values more than KLDS 10373 (124

plusmn 019 and 115 plusmn 021 respectively) where the pH is shown inTable 3 as follows (445 plusmn 020 and 459 plusmn 023 respectively)Meanwhile the comparison between the time levels of thegrowth rate in Table 2 shows highly significant differencesbetween all-time levelswith the highestmean time at 27 hours(197plusmn 001) and lowest at zero hours (022plusmn 003)whereas pHat 33 hours was the highest values (370 plusmn 001) and the lowestvalues at zero hours (556 plusmn 005)

Meanwhile Table 4 displayed the highest survival rateof strain KLDS 10373 at 3 hours and 03 of bile salts(197plusmn007b) Moreover the lowest survival rate in KLDS10727 after 9 hours and 2 of bile (178plusmn007) Otherwisethe effect of bile salts concentration on growth rate and pH (Plt0001) was observed and there was a significant correlationbetween the growth rate of strains and pH values (097) Inaddition according to Table 2 there was a little significantdifference between two strains KLDS 10727 showed highergrowth rate and pH values than KLDS 10373 079 plusmn 008 and076 plusmn 008 490 plusmn 010 and 492 plusmn 010 respectively

As Duncanrsquos multiple range test mentioned above thecomparison between time levels of growth rate showedhighly significant differences between all-time levels with thehighest pH 140 and the lowest 110 Furthermore Table 3showed the highest pH 411 and the lowest 580 Table 2showed the comparison between the concentration levels ofthe growth rate that explained high significant differencesbetween all categories of bile concentration with the highestmean in zero of the bile (108 plusmn 015) and was lower at 2(052 plusmn 007) while pH with the highest values in zero (460plusmn 016) and lowest in 2 of bile (521 plusmn 010)

34 Resistance of KLDS 10727 and KLDS 10373 to DifferentTypes of Antibiotics Sensitivity to antibiotics is the mostimportant factor in assessing the safety of probiotics andaffecting the growth rate of strains and pH Moreover it isa potential threat to vital applications [33] Figure 3 showedthe effect of different types of antibiotics on the viabilityof Lactobacillus brevis strains The obtained data in Table 2referring to all factors and all possible interactions werehighly significant (Plt0001) The correlation between bacte-rial growth rate and pH values (099) was highly significantas well KLDS 10373 showed high growth rate and pH thanKLDS 10727 045 plusmn 006 and 044 plusmn 006 for growth rate533 plusmn 006 and 532 plusmn 006 for pH respectively Time levelsshowed significant differences between all-time categorieswith the highest growth rate at 24 hours (010 plusmn 015) and thelowest values at 3 hours (005 plusmn 0003) Additionally Table 3showed the highest pH values at zero time (574 plusmn 001) andthe lowest values at 24 hours (455plusmn 014) Antibiotics showedhighly significant differences between all antibiotics with thehighest growth rate of control (080 plusmn 014) and the lowest inampicillin (005 plusmn 0003) and the same results for pHwith thehighest pH in chloramphenicol (571plusmn 001) and the lowest incontrol (497 plusmn 014) It is expected since when LAB did notgrow there is no lactic acid production and vice versa

Table 4 showed the factors affecting survival rate wassignificant as well as the interaction between the factors(P lt00001) KLDS 10373 showed the highest survival at 3hours of streptomycin (161 plusmn 001 275 plusmn 002 and 236


Table2Sign

ificancemeansand

stand

arderrors(SE)

offactorsaffectingtheg

rowth

rateof

KLDS10

727andKL

DS10

373str

ains

SOVExp

erim

ents

Growth

rate

Biletolerance

SIJw

ithdiffe

rent

pHaerobically

SIJw

ithdiffe

rent

pHan

aerobically

Antibiotic

resistan

ceAdh

esionto

Caco2

cell

High

Low

High

Low

High

Low

High

Low

High

Low

High

Low

Strain

(S1)

(S2)

(S1)

(S2)

(S1)

(S2)

(S2)

(S1)

(S2)

(S1)

(S2)

(S1)

Meanplusmn

SEof

strains

124plusmn

019

c115plusmn

021

079plusmn008

c076plusmn008

036plusmn007

d036plusmn007

051plusmn010

b049plusmn010

045plusmn006

c044plusmn006

245plusmn3651d

24467plusmn3331

Time

270

270

240

240

243

--

Meanplusmn

SEof

time

197plusmn001

c022plusmn003

140plusmn007

c110plusmn

001

056plusmn014

c014plusmn001

105plusmn029

c014plusmn001

111plusmn015c

005plusmn0003

--

Con

centratio

nof

diffe

rent

parameters

--

02

02

02

Con

tAmp

30

Meanplusmn

SE-

-108

c plusmn015

052plusmn007

100plusmn014

c012plusmn001

139plusmn023c

015plusmn001

080plusmn014

c005plusmn0003

3816

7plusmn4847c

1375

0plusmn626

R2lowast

9999

9996

9998

9988

9999

7236

CVlowast

067

211

264

671

183

3048

Inthes

ixexperim

ents

grow

thratethe

effecto

ftwo

strains

KLD

S10

727(S1)andKLD

S10

373(S2)and

fivelevelso

ftim

es03927and

33hon

grow

thrateandpH

bile

tolerancetheeffecto

ftwo

strainsfi

velevelsof

timesand

four

levelsof

bilesalts

concentrationCon

t031and2

ongrow

thrateandpH

sim

ulated

intestinaljuice(SIJ)w

ithdiffe

rent

pHaerobically

the

effecto

ftwo

strainsfi

velevelsof

times

0369and24and

simulated

intestinaljuice

with

different

four

levelsof

pHCon

t23and7on

grow

thratesim

ulated

intestinaljuice(SIJ)with

diffe

rent

pHan

aerobically

the

effecto

ftwo

strainsfi

velevelsof

times

0369and24and

simulated

intestinaljuice

with

different

four

levelsof

pHCon

t23and7an

tibiotic

resistan

cethe

effecto

ftwo

strainsfi

velevelsof

timesand

typeso

fantibioticsC

ontstr

eptomycin

ampicillin

chloramph

enicolerythromycingentamycinand

kanamycin

ongrow

thrateandpH

adh

esionwith

Caco2

cell

theeffecto

ftwo

strainsfou

rlevelso

fdilu

tionof

2str

ains

Con

t34and5adhesio

nof

Caco2cellSign

ificanceaPlt001bPlt000

1cPlt

000

01dn

onsig

nificantlowastR2

determinationcoeffi

cientandCV

coefficiento

fvariatio

nlowastSO

Vsource

ofvaria

nce


Table3Sign

ificancemeansand

stand

arderrors(SE)

offactorsaffectingpH

ofKL

DS10

727andKL

DS10

373str

ains

lowastSO

VExp

erim

ents

Growth

ratean

dpH

Biletolerance

Antibiotic

stoleran

ceHigh

Low

High

Low

High

Low

Strain

(S2)

(S1)

(S2)

(S1)

(S2)

(S1)

Meanplusmn

SEof

strains

459plusmn023

c445plusmn020

492plusmn010

a490plusmn010

533

b plusmn006

532plusmn006

Time

033

033

024

Meanplusmn

SEof

time

556plusmn005

c370plusmn001

581plusmn002

c411plusmn

007

574

c plusmn001

455plusmn014

Con

centratio

nof

bileor

type

ofan

tibiotic

--

20

Chloramph

enicol

Con

trol

Meanplusmn

SE-

-521plusmn010

c460plusmn016

572

c plusmn001

497plusmn014

R29996

9985

9992

CV

05

073

04

Seefoo

tnoteo

fTable2


Table4Sign

ificancemeansand

stand

arderrors(SE)

offactorsaffectingsurvivalor

adhesio

nof

KLDS10

727andKL

DS10

373str

ains

lowastSO

VExp

erim

ents

Biletolerance

(SIJ)w

ithdiffe

rent

pHaerobically

(SIJ)w

ithdiffe

rent

pHan

aerobically

Antibiotic

resistan

ceAdh

esionto

Caco2

cell

High

Low

High

Low

High

Low

High

Low

High

Low

Strain

(S2)

(S1)

(S1)

(S2)

(S2)

(S1)

(S2)

(S1)

(S1)

(S2)

Meanplusmn

SEof

strains

197plusmn007

b17

8plusmn007

116plusmn011

b113plusmn

010

117plusmn009

c112plusmn

01

160plusmn010

c14

1plusmn009

213plusmn019

d211plusmn020

Time

39

024

024

06

Meanplusmn

SEof

time

210plusmn014

c15

2plusmn009

208plusmn010

c056plusmn001

212plusmn010

c052plusmn002

275plusmn002

c12

9plusmn014

Con

centratio

nof

bileor

type

ofan

tibiotic

03

27

27

2Str

Erth

35

Meanplusmn

SEof

concentration

217plusmn009

c16

6plusmn009

138plusmn016

c094plusmn009

134plusmn014

c096plusmn009

236plusmn014

c083plusmn009

268plusmn015

c148plusmn

012

R2866

9971

9964

9648

078

CV

1204

387

394

1391

152

Seefoo

tnoteo

fTable2


3 9 27 330Time (h)

3 9 27 330Time (h)

40

45

50

55

pH

05

10

15

20O

D 6

20

(a)

03 1 20Bile salts concentration ()


40

45

50

55

60

pH

00

05

10

15

OD

620

(b) Bile concentration (0 03 1 2)

Figure 2 Determining a standard curve for growth ratehours and tolerant bile (0 03 1 2) with accumulated acid of KLDS 10727 andKLDS 10373 strains (a) Growth rate and pH (b) bile salts and pH

plusmn 014 respectively) Meanwhile erythromycin showed thelowest effect on strain KLDS 10727 at 6 hours (141 plusmn009 129 plusmn 014 and 083 plusmn 009 respectively) Notablythe sensitivity of KLDS 10727 after 24 hours to ampicillinchloramphenicol and erythromycin was 9746 9791 and9811 respectively Further KLDS 10373 was 9881 9838and 9861 respectively On the other hand the sensitivityof KLDS 10727 after 6 hours to streptomycin gentamicinand kanamycinwas 1218 6964 and 6622 respectively andKLDS 10373 showed 228 4182 and 1140 respectively

35 Tolerance KLDS 10727 and KLDS 10373 to SimulatedIntestinal Juice (SIJ) with Different pH under Aerobic orAnaerobic Conditions The tolerance of GIT conditions is animportant criterion for the selection of potential probioticsSeveral studies have reported that MRS broth with pH value(20 - 30) was used to determine Lactobacillus acid resistance

[34 35] Figure 4 shows the stander curve of the growthrate of KLDS 10727 and KLDS 10373 to tolerate (SIJ) withdifferent levels of pH under aerobic or anaerobic conditionThe data showed no significant effect (P lt 005) growth rateof strains However the time and different pH and theirinteraction were extremely high (P lt0001) Table 2 indicatesthat the highest growth rate at 24 hours aerobically in controlwithout any addition of (SIJ) was (056 plusmn 015 and 100 plusmn014 respectively) and the lowest growth rate at zero timewith pH 20 was (014 plusmn 001 and 012 plusmn 001 respectively)The obtained data in Table 4 showed that the growth rateand interaction between the factors were very significant (Plt00001) The Duncan groups showed the highest survivalrate forKLDS 10727 at zero time and pH70 (116plusmn 011 208plusmn010 and 138plusmn 016 respectively) Meanwhile KLDS 10373 atpH 20 showed lower survival rate (113 plusmn 010 056 plusmn 001 and094 plusmn 009 respectively) These results were consistent with


40

45

50

55

pH

chl cont erth gen kan strampTypes of antibiotic


00

05

10

15

20O

D 6

20

Figure 3 Resistance of KLDS 10727 and KLDS 10373 to different types of antibiotic as (OD)620

or pH (amp) ampicillin (chl)chloramphenicol (cont) control (erth) erythromycin (gen) gentamicin (kan) kanamycin and (str) streptomycin

00

05

10

15

20

25

30

35O

D 6

20

2 3 70Type of pH anaerobic

2 3 70 Type of pH aerobic

00

05

10

15

OD

620

Figure 4 Tolerance of KLDS 10727 and KLDS 10373 to simulated intestinal juice (SIJ) with different pH under aerobic or anaerobicconditions

those obtained by de Almeida Junior et al [36] On the otherhand Table 2 showed significant differences between strainsand time under anaerobic condition with different factors(P lt0001) and the significant interaction between them (Plt005) KLDS 10373 at 24 hours in pH control showed thehighest growth rate (051 plusmn 010 105 plusmn 029 and 139 plusmn 023respectively) However KLDS 10727 strain at zero time andpH 20 values were the lowest (049 plusmn 010 014 plusmn 001 and015 plusmn 001 respectively) Furthermore Table 4 showed thesurvival rate was influenced by the same factors (P lt00001)The highest survival rate of the KLDS 10373 at zero time andpH 70 was 117 plusmn 009 212 plusmn 010 and 134 plusmn 014 respectivelywhile the survival rate of KLDS 10727 was the lowest at 24hours and pH 20 (112 plusmn 010 052 plusmn 002 and 096 plusmn 009respectively)

36 Screening of the Antagonistic Properties of Lactobacillusbrevis KLDS 10727 and KLDS 10373 Strains against Food-borne Pathogenic Bacteria Due to the biological diversity

functions of probiotic bacteria there is a growing need fornew strains of the LAB to play an important role in identifyingdominant bacterial communities within intestinal ecosystem[35] In this study KLDS 10727 and KLDS 10373 have beentested against some foodborne pathogens namely Salmonellatyphimurium ATCC 14028 E coli IQCC 10126 Listeriamonocytogenes IQCC22221 and Staphylococcus aureus IQCC22035 Table 5 showed the strongest antimicrobial activity ofKLDS 10727 and KLDS 10373 strains against all the indicatorpathogens as expressed in terms of diameter of clear zones(mm) Therefore the clear zone ranging from (193 plusmn 007 to247 plusmn 003 mm) against all test indicators

37 Caco-2 Cell Adhesion Assay The most important con-siderations for the selection of probiotics are not only theability to survive and transit through the digestive systembut also the adhesion establishment or reproduction withinthe gastrointestinal tract The obtained results in Figure 5(A) showed that the adhesion of the KLDS 10727 and


Table5Antibacteria

lactivity

oftheK

LDS10

727andKL

DS10

373str

ains

againstfoo

dborne

pathogenicbacteriain

term

sofZ

DI

Testfo

odbo

rnes

trains

ZDI(mm)plusmn

SDof

IndicatorB

acteria

Salm

onellatyph

imurium

Ecoli

Liste

riamon

ocytogenes

Stap

hylococcus

aureus

KLD

S10

727

237plusmn003

+++

197plusmn

003

+++

217plusmn009

+++

220plusmn010

+++

KLD

S10

373

247plusmn003

+++

193plusmn

007

+++

193plusmn

007

+++

220plusmn010

+++

Sig

lt000

104226

00198

00572

R210

0000

0090

4762

0978495

0970588

+++refersto

clearz

one(14mm3

points

9ndash14mm2

points

1ndash99

mmone

point)


4 53Caco2 cell with dilution of strains

100

150

200

250

300

350

Adhe

sion

()

(a) Adhesion assay of KLDS 10727 and KLDS 10373to Caco-2 cell

(A)

(B) (C)

(b) Determination microscopy adhesion assay of KLDS 10727 and KLDS 10373 to Caco-2 cell (A) Caco2 cellscontrol (B) KLDS 10727+ Caco2 cells (C) KLDS 10373+ Caco2 cells

Figure 5

KLDS 10373 strains was (556 - 952) therefore using threedifferent dilutions KLDS 10727 and KLDS 10373 strains as103 104 and 105 Moreover Table 3 showed the effect of straintypes on the adhesion was nonsignificant (092) whilethe concentration effect was highly significant (plt00001)KLDS 10727 strain in the third dilution fold was the highestadhesion percentage (213 plusmn 019 and 268 plusmn 015 respectively)and KLDS 10373 strain in the dilution five was the lowestvalues (211 plusmn 020 and 148 plusmn 012 respectively) Moreover inFigure 5 (B) microscopic slides showed a high adherence of KLDS 10727 and KLDS 10373 strains

38 Intervention Effect of KLDS 10727 and KLDS 10373Strains on Type I Diabetes In Vivo

381 Hypoglycemic Activity and Body Weight of C57BL6Mice with Streptozotocin- (STZ-) Induced Diabetes duringFour Weeks Glucose levels and body weight were checked

weekly for five treated groups namely (Cont STZ STZ+INSS1 and S2)The obtained results in Figure 6 showed a chronicincrease in blood glucose level overnight fasting 12 hours and2 hours postprandial and body weight within four weeksThe data presented the glucose levels as equation mentionedabove for Cont STZ INS+STZ S1 S2 was 42675plusmn1116595plusmn163 -57375 358plusmn094 and 25925plusmn215 respectivelyIn addition the effect of STZ causing diabetes for C57BL6mice had a different effect on mice body weight It wasnonsignificant for all completely treated groups Briefly thelowest blood glucose and the highest body weight was in(INS+STZ) group (-57375 and 2112 plusmn 254a respectively)Notably the (INS + STZ) group showed the blood glucoselevels before eating or insulin injection were higher than theblood samples taken 2 hours postprandial In contrast toall other groups this may be due to insulin injection thatpromotes glucose uptake in blood


(INS+STZ)(Cont) (S2)

10

0

5

minus10

minus5

(S1)(STZ)

Glu

cose

leve

ls (w

)

(a)

10

15

20

25

0

5

(INS+STZ)(Cont) (S2)(S1)(STZ)

Wei

ght (

g)

(b)

Figure 6 Blood glucose mmoldl and body weightg of C57BL6 mice with streptozotocin- (STZ-) induced diabetes during four weeks (a)Average of blood glucose level (b) body weightg (Cont) control (STZ) streptozotocin (INS+STZ) insulin (S1) KLDS 10727 (S2) KLDS10373

39 Blood Plasma Assay

391 Serum Biochemistry Parameters Assay After overnightfasting mice have been killed and blood serum was col-lected and serum biochemistry parameters were measuredTable 6 displayed the serum lipids concentrations as triglyc-eride (TG) total cholesterol (CHOL) high-density lipopro-tein cholesterol (HDL) low-density lipoprotein cholesterol(LDL) glucose (GLU)magnesium (Mg+2) as well All treatedgroups were highly significant whereas Cont and STZshowed a high level of TG (254plusmn091 and 206plusmn084mmolLrespectively) Notably all groups displayed a high level ofMg+2 and low level of LDL Further Cont group showedthe highest level of CHOL (725plusmn093b) On the other handINS+STZ showed the highest level of glucose (940plusmn384b)Worthy of notice S1 falls within the reference range of TGCHOL HDL LDL and GLUTherefore it can conclude thatCont showed the highest level of TG CHOL HDL andLDL while STZ showed the highest level of Mg+2 MoreoverINS+STZ showed the lowest level of CHOL andMg+2 and thehighest level of GLU On the contrary S1 expressed the lowestlevel of TG LDL and GLU

310 Liver Functions Parameters Liver functions parametersevaluated were the serum alanine aminotransferase (ALT)aspartate transaminase (AST) ASTALT total bile acid(TBA) albumin (ALB) globulin (GLUB) total protein (TP)Table 6 showed (TP) total protein of all treated experi-ments falls in the average reference range except STZ thatwas (8490plusmn3466) (ALT) serum alanine aminotransferaseshowed a dramatic increase in all treated groups and outof average reference range except (S2) (3600plusmn1470) Inaddition (AST) aspartate aminotransferase showed a signif-icant increase in the levels of all treatment groups and alsoexceeded the mean of the reference range Further total bileacid (TBA) was expressed at a reasonable level for all treatedgroups while albumin (ALB) and globulin (GLUB) displayresults are coherent for all treated groups where it falls in

the average of the reference range Briefly STZ showed thehighest level of TP AST ALB and GLUB compared to alltreated groups

311 Kidney Function Parameter Table 6 determined thekidney functions parameters such as uric nitrogen (BUN)creatinine (CREA) and uric acid (URIC) levels The resultsdisplayed not alarming increase in uric nitrogen (BUN) andfurther creatinine (CREA) and uric acid (URIC) levels aswell All treated groups fall in the average of the referencerange except Cont and S1 which expressed the lowest level ofuric acid (URIC) (7650plusmn3123b and 12560plusmn5128ab respec-tively)

312 Insulin Blood Plasma Assay Table 6 exhibited that allthe treated groups fall within the reference range averageTherefore Cont and S1 displayed the heights of groupsin insulin levels as 1352plusmn023 and 1113plusmn028 respectivelyMeanwhile STZ and INS+STZ expressed the lowest levels as999plusmn 036 and 1060plusmn021 respectively

313 Histological Evaluation The slides in Figure 7 exhibitedthe histological mice organs (namely liver pancreas kidneyand spleen) The figure illustrates that the livers histology ofdifferent groups as follows no abnormal morphology in Contor STZ groups while INS+STZ showed a slight denaturedand a little fat Further S1 group was hepatic nucleus andshrunk as well S2 shows a combination of mild fatty degen-eration of the liver and inflammatory cells collected piles ofdiffuse inflammatory cells Moreover the pancreas histologyexhibited that there was no abnormal morphology in theCont while STZ was a significant atrophy in pancreatic isletscells with a huge vacuolar degeneration INS+STZ S1 and S2groups showed a mild integration The kidneys histologicalslides displayed that Cont has no abnormal morphology STZgroup was atrophy and glomerular shrinkage is evident inthe renal tube fused obviously Further the state of INS+STZand S1 glomerulus is normal renal tubule fused slightly


liver

(Cont) (STZ) (INS+STZ) (S1) (S2)




(Cont or STZ) No abnormal morphology (black arrow) (INS+STZ) show a slight denatured (orange arrow) and a little fat (yellow arrow) (S1) has hepatic nucleus and shrunk(blue arrow) (S2) Combination of mild fatty degeneration of the liver (yellow arrow) and inflammatory cells collected piles of diffuse inflammatory cells (black arrow)

pancreas

(Cont) no abnormal morphology (black arrow) (STZ) large atrophy (orange arrow) (INS+STZ S1 and S2) groups show a mild integration (black arrow)

liver

kidney

(Cont) no abnormal morphology (black arrow) (STZ) atrophy and glomerular shrinkage is evident in the renal tube fused obviously (orange arrow) (INS+STZ and S1) the state of glomerulus is normal (black arrow) renal tubule fused slightly (yellow arrow) and (S2) no abnormal morphology (black arrow) renal tubule fused slightly (yellow

arrow)Spleen

(Cont) no abnormal appearance (black arrow) (STZ) lymphocytes number were reduced and their structures are lost ( orange arrow) (INS+STZ) a trabecular increase (yellow arrow) and lymphatic decrease (orange arrow) (S1) the number of lymphocytes was reduced (yellow arrow) (S2) Spleen trabecular (white arrow) and Phagocyte was

increased (red arrow)

Figure 7 Histological examination of sacrifice mice organs (liver pancreas kidney and spleen) streptozotocin-induced diabetes (Cont)control (STZ) streptozotocin (INS+STZ) insulin (S1) KLDS 10727 and (S2) KLDS 10373


Table 6 Blood plasma assay (n=4 meanplusmnSE)

Ref ranges (Cont) (STZ) (INS+STZ) (S1) (S2)Plasma biochemical parameter

TG mmolLblowastlowastlowast 045 -17 254plusmn091 206plusmn084 102plusmn042 096plusmn039 134plusmn055CHOLmmolLlowastlowastlowast 285 ndash 57 725plusmn093b 489plusmn200bc 338plusmn138c 346plusmn141c 344plusmn140c

HDLmmolLlowast 093 ndash 181 420plusmn090a 155plusmn063b 226plusmn092b 239plusmn098b 222plusmn091b

LDL mmolLns 207 ndash 363 114plusmn047a 083plusmn034ab 049plusmn020b 038plusmn016b 048plusmn020b

Mg+2 mmolLblowast 07-11 150plusmn008 175plusmn071 148plusmn060 164plusmn067 164plusmn067GLUmmolLlowastlowast 357 ndash 612 44plusmn127bc 440plusmn180a 94plusmn271a 31plusmn089c 51plusmn147bc

Liver functions parametersTP gLblowastlowastlowast 600-800 6030plusmn090 8490plusmn3466 5810plusmn2372 6090plusmn2486 6490plusmn2650ALT IULblowastlowastlowast 10 - 400 12800plusmn082 9500plusmn3878 7700plusmn3144 8300plusmn3388 3600plusmn1470AST IULblowastlowastlowast 10 - 400 26300plusmn082 34100plusmn13921 17600plusmn7185 27400plusmn11186 18200plusmn7430ASTALTlowast 205plusmn001b 359plusmn001ab 229plusmn001b 330plusmn135ab 289plusmn118b

TBAmolLblowastlowastlowast 001-200 850plusmn090 490plusmn200 390plusmn159 260plusmn106 390plusmn159ALB gLbns 350-550 3450plusmn090a 3910plusmn1596ab 3370plusmn1376ab 3650plusmn1490ab 3750plusmn1531ab

GLUB gLblowastlowastlowast 250-400 2580plusmn090 4580plusmn1870 2440plusmn996 2440plusmn996 2740plusmn1119AGns 15 -25 134plusmn001a 085plusmn001ab 138plusmn001a 150plusmn061a 137plusmn056a

Kidney function parameterBUNmmolLblowastlowastlowast 107 ndash 714 860plusmn351 1050plusmn429 800plusmn327 1180plusmn482 1080plusmn441CREAmolLblowastlowastlowast 530 ndash 1320 4430plusmn1809 4050plusmn1653 3910plusmn1596 3740plusmn1527 4190plusmn1711URIC UmolLns 1420 ndash4010 7650plusmn3123b 17510plusmn7148ab 18060plusmn7373ab 12560plusmn5128ab 16490plusmn6732ab

Insulin blood plasmaInsulin uIUml 3-19 1352plusmn023 999plusmn 036 1060plusmn021 1113plusmn028 1093plusmn020TG triglyceride CHOL total cholesterol HDL high-density lipoprotein cholesterol LDL low-density lipoprotein cholesterol GLU glucose Mg+2 magnesium ALT serum alanine aminotransferase AST aspartate aminotransferase ASTALT presence of aspartate transaminase and serum alanineaminotransferase TBA total bile acid ALB albumin GLUB globulin TP total protein BUN uric nitrogen CREA creatinine URIC uric acid (Cont)control (STZ) streptozotocin (INS+STZ) insulin (S1) KLDS 10727 (S2) KLDS 10373

and S2 has no abnormalities Finally the spleens histologyexhibited that Cont has no abnormal appearanceMeanwhileSTZ group displayed lymphocytes number reduced and theirstructures are lost Obviously INS+STZ showed a trabecularincrease and lymphatic decrease and the number of lympho-cytes obviously reduced in S1 whereas S2 demonstrates thatthe spleen trabecular and phagocyte were increased

4 Discussion

One of the key terms of probiotics is that theymust withstandthe harsh conditions of gastrointestinal juices and furtherthe ability to survive in the presence of bile pancreatic juiceuntil the intestine is sufficient in number and provides healthbenefits for the host [37 38] In this study two strains ofLactobacillus breviswere genetically determined and tested invitro for some inappropriate simulated gastrointestinal con-ditions such as acidity bile tolerance antibiotic resistanceand antimicrobial activity Furthermore in vivo experimentssuch as blood glucose body weight blood plasma histologi-cal assay were tested to demonstrate the role of Lactobacillusbrevis strains in the intervention of type 1 diabetes

Lactobacillus brevis KLDS 10727 and KLDS 10373 isGram-positive of lactic acid bacteria which is a rod-typestrain with high GABA generating capacity due to the gad

gene To confirm the identification of KLDS 10727 andKLDS 10373 strains 16S rDNA was amplified and 540 bpof the DNA sequence was determined (unpublished data)The GenBank database was used to search for genes as the16S rDNA sequence that revealed the highest similarity inthe nucleotide chain was 99with L brevis (GenBank accessno AF515220) and L brevis (GenBank access no AF515219)Therefore the strains KLDS 10727 and KLDS 10373 are oneof L brevis strains

The basic logistics model of SAS software suitable forexperimental data showed that the factors (strains timeand interaction between them) had significant effects onthe growth rate of KLDS 10727 and KLDS 10373 strainsand pH values (P lt00001) Therefore the obtained datawere consistent with Liao et al [39] who reported thatpH values declined dramatically rapid in the first 9 hoursand then slowly declined attributed to strains that reachedthe stationary phase In general LAB growth reaches thestationary phase when the pH is less than 45 Zhang andYew [40] reported that the acid resistance of LAB strains isdependent on specific strains and species

Lactobacillus brevis strains were examined and tested forresistance and survival in acidic environment as well asfor growing in the presence of 03-2 bile salts a similarconcentration was found in the small intestine [41] Theobtained results showed Lactobacillus brevis strains were able


to grow in high bile concentrations (2) Similar results werepreviously reported by other researchers to study vital LABstrains from different environments [42] The resistance ofKLDS 10727 and KLDS 10373 strains of bile salts in MRSincubated at 37∘C with 03 concentration of bile salts wasclosely related to bile level as reported by Sahadeva et al[38] In the gastrointestinal tract observations were commonbetween comparing different strains and different concentra-tions of bile such as 03 1 and 2These concentrations wereclosely associated with Hyacinta et al [43] who explained thevitality of microorganisms affected by concentrations of bilesalts

Antibiotic is one of the most important growth deter-minants of most microbes and their resistance to growthis a criterion for selection of probiotic bacteria Thereforein the present study the obtained data were in agreementwith Klare et al [44] who confirmed that LAB is usuallysensitive to antibiotics such as chloramphenicol de AlmeidaJunior et al [36] studied that 96 of disabling strains weresensitive to chloramphenicol Furthermore our findingswerein agreement with Tulumoglu et al [35] who reported that90 of the tested Lactobacillus strains were resistant togentamicin meanwhile other studied strains were sensitiveto ampicillin and erythromycin

Lactobacillus brevis strains are a facultative anaerobic ormicroaerophilic LAB Therefore these explain our obtainedresults Wu and Shah [45] concluded that ventilation has abad effect on the feasibility and production of lactic acidand GABA by Lactobacillus brevis The obtained resultswere consistent with those of Zhang [46] who showed theperformance of antiacids of isolated strains In addition Wuet al [19] reported that pancreatic fluid had no significanteffect on survival of LAB

Lactic acid bacteria have extremely antagonists againstfoodborne pathogenic bacteria [47] Gautam and Sharma[48] report similar studies with Lactobacillus spicheri G2showing 60 of hostility against different test indicatorsFurther the obtained results were closely correlated withthe findings of Fossi et al [49] A number of studiesreported the potential antimicrobial mechanisms of specificmicroorganisms for the control pathogens are indicated as(i) production of antimicrobial compounds (ii) productionof bacteriocins (iii) competitive action on nutrients (iv)inhibition of binding due to competition and (v) formationof the immune system [50] Moreover these findings agreedwith our obtained results that show an extremely antagonismagainst common foodborne pathogenesis

The adhesion of the intestinal mucosa is one of themost important features of probiotic bacteria to reproductionand produce biological compounds in the host digestivesystem [51] LAB adhesion is a complex process involvingthe communication between the bacterial cell membraneand the interacting surfaces Ehrmann et al [52] reportedthat the ability of LAB to adhere to other cells is relatedto cell surface charge and hydrophobicity of bacteria Theability to hold hydrocarbons can be expressed as cell surfacehydrophobicity [53] Moreover Solieri et al [34] reportedthat the hydrophobicity above 70 is considered highlyhydrophobic The obtained results were consistent with the

study conducted by Zhang [46] who found that the highesthydrophobicity of the strains was 9215Our results showedthat KLDS 10727 and KLDS 10373 have a similar adhesionvalue to the recognized probiotic strain Lb rhamnosus GGsuggesting that it may be well-colonized in vivo

Recently diabetes has become the most common diseasein the world It causes many complications hence high bloodglucose levels may always damage tissues especially capillar-ies and glomerular mesenchymal cells [54] Streptozotocin(STZ) has been used clinically as a chemotherapeutic agentin the treatment of pancreatic cell 120573-cell carcinoma [55]Reference ranges for blood tests are sets of values used by ahealth professional to interpret a set of medical test resultsfrom blood samples In addition examples of referencedomains are given varying by age sex health and ethnicitya method of analysis and measurement units Individualresults should always be interpreted using the reference rangeprovided by the test laboratory

Glucose serum (GLU) was reported by Wasserman [56]The blood glucose test can measure the livers ability toproduce glucose usually the last function to be lost if theliver fails Glucose levels are usually the lowest in themorningbefore the first meal of the day and increase after meals forone to two hours Blood sugar levels outside the normalrange may be indicative of a medical condition The higherlevel systematically refers to the high level of blood sugarLow levels are known as hypoglycemia Among the mostobserved cases the obtained results are in close agreementwith American Diabetes Association and Sacks et al [57 58]that suggest diabetes diagnosis historically included fastingblood glucose more than 7 mmol dL (126 mg dL) to111 mmoldL (200 mg dL) or higher with hyperglycemiasymptoms or an abnormal oral glucose tolerance test of 2hours

High-density lipoprotein (HDL) is one of the fivemajor groups of lipoproteins HDL molecules are sometimesreferred to as ldquogood cholesterolrdquo because it can transfer fatmolecules from artery walls reduce pharyngeal accumula-tion and thus help to prevent or harden atherosclerosisTherefore the obtained results displayed that LactobacillusbrevisKLDS 10727 and KLDS 10373 strains have a slight sim-ilarity in the recommended ranges according to theAmericanHeart Association the National Institutes of Health and theNational Demining Program providing a set of guidelines onHDL levels and cardiovascular risk gt 155 mmol L [59]

Changes in ALT and AST levels are often used forliver pathological examination AST ALT is an importantbiochemical indicator [60] ALT is mainly present in the livercell plasmaWhen the liver cells are less damaged the changesin membrane permeability of the liver cells will elevate theALT blood levels On the other hand the increased levelsof AST blood are the result of severe damage to liver cellsIn other words the higher AST ALT ratio means moresevere damage to liver cells The findings results were inagreement with Prakasam et al [61] who reported that theuse of streptozotocin increases the level of AST ALT andALP in blood plasma Furthermore the obtained results ofKLDS 10727 and KLDS 10373 fall in the reference range andare in close similarity to total protein (TP) level 600-800


gmdl Besides the total serum albumin (ALB) level is 350-550 gLb Further the total plasma globulin (GLUB) level is250-400 gLb [62]

Renal dysfunction can be fatal for diabetic patients [63]Hyperuricemia and hyperglycemia are the main causes ofrenal failure [63] Diabetic nephropathy high blood pressureand high blood sugar are the main causes of renal failureKidney function in the kidney disease is an indicator of theactivity vitality and overall health of kidney The obtainedresults refer to the creatinine levels of all treated groupsfalling in the range of safe creatinine levels that is one ofthe most important kidney function tests and the resultsof this parameter are used to evaluate kidney functions[64]

Insulin is an anabolic hormone that promotes glucoseuptake glycogenesis lipogenesis and protein synthesis ofskeletal muscle and fat tissue through the tyrosine kinasereceptor pathway In addition insulin is the most impor-tant factor in the regulation of plasma glucose homeosta-sis as it counteracts glucagon and other catabolic hor-mones epinephrine glucocorticoid and growth hormoneThe obtained results are closely similar to Graham et al[65] who indicated that the insulin range in blood plasmafalls between 3 and 19 uIUml Finally based on previ-ous studies the role of probiotics in the development ofT1D demonstrated that oral probiotics inhibit the devel-opment of T1D in diabetic mice model Therefore pro-biotics showed the ability to the intervention of diabetes[15]

5 Conclusions

Lactobacillus brevis KLDS 10727 and KLDS 10373 strainswere exhibited to be extremely viable in in vitro experimentsand exhibited survival in in vitro digestionmodelsmimickingthe gastrointestinal conditions Moreover it shows significanthostility against foodborne pathogenic bacteria In additionKLDS 10727 and KLDS 10373 strains expressed adequateGABA that demonstrate a superb physiological effect in invivo experiment On the other hand it shows a significanteffect in lowering blood glucose levels or insulin in plasmaIn addition most functional liver and kidney tests fall withinthe reference range of plasma blood parameters Thereforewe recommended using Lactobacillus brevis KLDS 10727 andKLDS 10373 strains widely in the market as a natural sourceof GABA in pharmaceutical and food applications to reducethe threat of type 1 diabetes

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors would like to thank Dr Hamdi Ahmad(mrahmadhamdiyahoocom) from the University of Edin-burgh UK for revising the manuscript This research wasfunded byNational Key Research and Development Programof China under Grant no 2017YED0400304

References

[1] E Salvetti and P W OrsquoToole ldquoWhen regulation challengesinnovationThe case of the genus LactobacillusrdquoTrends in FoodScience amp Technology vol 66 pp 187ndash194 2017

[2] A Abdelazez Z Muhammad and Q X Zhang ldquoProduction ofa Functional FrozenYogurt Fortifiedwith Bifidobacterium spprdquoBioMed Research International vol 2017 Article ID 6438528 10pages 2017

[3] K Ji N Y Jang and Y T Kim ldquoIsolation of lactic acid bacteriashowing antioxidative and probiotic activities from kimchi andinfant fecesrdquo Journal of Microbiology and Biotechnology vol 25no 9 pp 1568ndash1577 2015

[4] J Xing G Wang Q Zhang et al ldquoDetermining AntioxidantActivities of Lactobacilli Cell-Free Supernatants by CellularAntioxidant Assay A Comparison with Traditional MethodsrdquoPLoS ONE vol 10 no 3 p e0119058 2015

[5] T Kuda M Kawahara M Nemoto H Takahashi and BKimura ldquoIn vitro antioxidant and anti-inflammation propertiesof lactic acid bacteria isolated from fish intestines and fer-mented fish from the Sanriku Satoumi region in Japanrdquo FoodResearch International vol 64 pp 248ndash255 2014

[6] S I Pavlova A O Kilic S S Kilic et al ldquoGenetic diversity ofvaginal lactobacilli from women in different countries based on16S rRNA gene sequencesrdquo Journal of AppliedMicrobiology vol92 no 3 pp 451ndash459 2002

[7] P Falck S Precha-Atsawanan C Grey et al ldquoXylooligosac-charides from hardwood and cereal xylans produced by athermostable xylanase as carbon sources for lactobacillus brevisand bifidobacterium adolescentisrdquo Journal of Agricultural andFood Chemistry vol 61 no 30 pp 7333ndash7340 2013

[8] Y Ma Y Deng Z Xu et al ldquoDevelopment of a propidiummonoazide-polymerase chain reaction assay for detection ofviable Lactobacillus brevis in beerrdquo Brazilian Journal of Micro-biology vol 48 no 4 pp 740ndash746 2017

[9] Q Wu and N P Shah ldquoHigh gamma-aminobutyric acidproduction from lactic acid bacteria Emphasis on Lactobacillusbrevis as a functional dairy starterrdquo Critical Reviews in FoodScience and Nutrition vol 57 no 17 pp 3661ndash3672 2017

[10] D N D Riccia F Bizzini M G Perilli et al ldquoAnti-inflammatory effects of Lactobacillus brevis (CD2) on peri-odontal diseaserdquoOral Diseases vol 13 no 4 pp 376ndash385 2007

[11] A A Rushdy and E Z Gomaa ldquoAntimicrobial compoundsproduced by probiotic Lactobacillus brevis isolated from dairyproductsrdquoAnnals of Microbiology vol 63 no 1 pp 81ndash90 2013

[12] Y Ueno K Hayakawa and S Takahashi ldquoPurification andcharacterizationof glutamate decarboxylase from Lactobacillusbrevis IFO 12005rdquo Biosci Biotechnol Biochem vol 61 no 7 pp1168-71 Jul 1997

[13] H Zhang H-Y Yao F Chen and X Wang ldquoPurification andcharacterization of glutamate decarboxylase from rice germrdquoFood Chemistry vol 101 no 4 pp 1670ndash1676 2007


[14] J S Skyler G L Bakris E Bonifacio et al ldquoDifferentiation ofdiabetes by pathophysiology natural history and prognosisrdquoDiabetes vol 66 no 2 pp 241ndash255 2017

[15] M A Atkinson G S Eisenbarth and A W Michels ldquoType 1diabetesrdquoThe Lancet vol 383 no 9911 pp 69ndash82 2014

[16] E Fan J Huang S Hu L Mei and K Yu ldquoCloning sequencingand expression of a glutamate decarboxylase gene from theGABA-producing strain Lactobacillus brevis CGMCC 1306rdquoAnnals of Microbiology vol 62 no 2 pp 689ndash698 2012

[17] E Boonstra R de Kleijn L S Colzato A Alkemade B UForstmann and S Nieuwenhuis ldquoNeurotransmitters as foodsupplements the effects of GABA on brain and behaviorrdquoFrontiers in Psychology vol 6 2015

[18] M Diana J Quılez and M Rafecas ldquoGamma-aminobutyricacid as a bioactive compound in foods A reviewrdquo Journal ofFunctional Foods vol 10 pp 407ndash420 2014

[19] Q Wu H M Tun Y-S Law E Khafipour and N P ShahldquoCommon distribution of gad operon in Lactobacillus brevisand its GadA contributes to efficient GABA synthesis towardcytosolic near-neutral pHrdquo Frontiers in Microbiology vol 82017

[20] D Dupont M Alric S Blanquet-Diot et al ldquoCan dynamicin vitro digestion systems mimic the physiological realityrdquoCritical Reviews in Food Science and Nutrition pp 1ndash17 2018

[21] P Chen Q Zhang H Dang et al ldquoScreening for potentialnew probiotic based on probiotic properties and 120572-glucosidaseinhibitory activityrdquo Food Control vol 35 no 1 pp 65ndash72 2014

[22] Y Bao Y Zhang Y Zhang et al ldquoScreening of potentialprobiotic properties of Lactobacillus fermentum isolated fromtraditional dairy productsrdquo Food Control vol 21 no 5 pp 695ndash701 2010

[23] M C Casado Munoz N Benomar L L Lerma A Galvez andH Abriouel ldquoAntibiotic resistance of Lactobacillus pentosusandLeuconostoc pseudomesenteroides isolated fromnaturally-fermented Alorena table olives throughout fermentation pro-cessrdquo International Journal of Food Microbiology vol 172 pp110ndash118 2014

[24] E F S A S Committee ldquoGuidance on selected default values tobe used by the EFSA Scientific Committee Scientific Panels andUnits in the absence of actualmeasured datardquoEFSA Journal vol10 no 3 2012

[25] W P Charteris P M Kelly L Morelli and J K CollinsldquoDevelopment and application of an in vitro methodologyto determine the transit tolerance of potentially probioticLactobacillus and Bifidobacterium species in the upper humangastrointestinal tractrdquo Journal of Applied Microbiology vol 84no 5 pp 759ndash768 1998

[26] Q S Damaceno J P Souza J R Nicoli et al ldquoEvalua-tion of Potential Probiotics Isolated from Human Milk andColostrumrdquo Probiotics and Antimicrobial Proteins vol 9 no 4pp 371ndash379 2017

[27] A M O Leite M A L Miguel R S Peixoto et al ldquoProbioticpotential of selected lactic acid bacteria strains isolated fromBrazilian kefir grainsrdquo Journal of Dairy Science vol 98 no 6pp 3622ndash3632 2015

[28] C-H Wu S-M Huang and J-A Lin ldquoInhibition of advancedglycation endproduct formation by foodstuffsrdquo Foodampfunctionvol 2 no 5 pp 224ndash234 2011

[29] R S Surwit C M Kuhn C Cochrane J A McCubbin andMN Feinglos ldquoDiet-induced type II diabetes in C57BL6J micerdquoDiabetes vol 37 no 9 pp 1163ndash1167 1988

[30] L Chen D J Magliano and P Z Zimmet ldquoThe worldwideepidemiology of type 2 diabetes mellitusmdashpresent and futureperspectivesrdquo Nature Reviews Endocrinology vol 8 no 4 pp228ndash236 2012

[31] NWakiMMatsumotoY Fukui andH Suganuma ldquoEffects ofprobiotic Lactobacillus brevis KB290 on incidence of influenzainfection among schoolchildren an open-label pilot studyrdquoLetters in Applied Microbiology vol 59 no 6 pp 565ndash571 2014

[32] K Hiraga Y Ueno and K Oda ldquoGlutamate decarboxylasefrom Lactobacillus brevis Activation by ammonium sulfaterdquoBioscience Biotechnology and Biochemistry vol 72 no 5 pp1299ndash1306 2008

[33] A Sharma and S Srivastava ldquoAnti-Candida activity of two-peptide bacteriocins plantaricins (Pln EF and JK) and theirmode of actionrdquo Fungal Biology vol 118 no 2 pp 264ndash2752014

[34] L Solieri A Bianchi G Mottolese F Lemmetti and P GiudicildquoTailoring the probiotic potential of non-starter Lactobacillusstrains from ripened Parmigiano Reggiano cheese by in vitroscreening and principal component analysisrdquo Food Microbiol-ogy vol 38 pp 240ndash249 2014

[35] S Tulumoglu Z N Yuksekdag Y Beyatli O Simsek B CinarandE Yasar ldquoProbiotic properties of lactobacilli species isolatedfrom childrenrsquos fecesrdquo Anaerobe vol 24 pp 36ndash42 2013

[36] W L G de Almeida Junior I D S Ferrari J V de Souza et alldquoCharacterization and evaluation of lactic acid bacteria isolatedfrom goat milkrdquo Food Control vol 53 pp 96ndash103 2015

[37] C-F Guo S Zhang Y-H Yuan et al ldquoComparison oflactobacilli isolated from Chinese suan-tsai and koumiss fortheir probiotic and functional propertiesrdquo Journal of FunctionalFoods vol 12 pp 294ndash302 2015

[38] R Sahadeva S Leong and K Chua ldquoSurvival of commercialprobiotic strains to pH and bilerdquo International Food ResearchJournal vol 18 no 4 2011

[39] Q Liao X Hang X Liu et al ldquoThe influence of pH on heatstress response by probiotic Lactobacillus plantarum LP-OnllyrdquoAnnals of microbiology vol 60 no 2 pp 341ndash348 2010

[40] Y Zhang and W Yew ldquoMechanisms of drug resistance inMycobacterium tuberculosis [State of the art series Drug-resistant tuberculosis Edited by CY Chiang Number 1 in theseries]rdquo The International Journal of Tuberculosis and LungDisease vol 13 no 11 pp 1320ndash1330 2009

[41] G Vinderola B Capellini F Villarreal et al ldquoUsefulness of aset of simple in vitro tests for the screening and identificationof probiotic candidate strains for dairy userdquo LWT-Food Scienceand Technology vol 41 no 9 pp 1678ndash1688 2008

[42] C L Ramos LThorsenR F Schwan and L Jespersen ldquoStrain-specific probiotics properties of Lactobacillus fermentum Lac-tobacillus plantarum and Lactobacillus brevis isolates fromBrazilian food productsrdquo Food Microbiology vol 36 no 1 pp22ndash29 2013

[43] M Hyacinta K S Hana B Andrea et al ldquoBile tolerance andits effect on antibiotic susceptibility of probiotic Lactobacilluscandidatesrdquo Folia Microbiol (Praha) vol 60 no 3 pp 253ndash2572015

[44] I Klare C Konstabel G Werner et al ldquoAntimicrobial suscep-tibilities of Lactobacillus Pediococcus and Lactococcushumanisolates and cultures intended for probiotic or nutritional userdquoJournal of Antimicrobial Chemotherapy vol 59 no 5 pp 900ndash912 2007

[45] Q Wu and N P Shah ldquoRestoration of GABA productionmachinery in Lactobacillus brevis by accessible carbohydrates


anaerobiosis and early acidificationrdquo FoodMicrobiology vol 69pp 151ndash158 2018

[46] L Zhang Evaluation of the Potential Probiotic Properties andImmune Regulation Function of Lactobacillus Strains Isolatedfrom Traditional Fermented Yak Milk Ph D Thesis GansuAgricultural University Lanzhou Gansu China 2011

[47] P AsurmendiMGarcıa L Pascual et al ldquoBiocontrol of Listeriamonocytogenes by lactic acid bacteria isolated from brewerrsquosgrains used as feedstuff in Argentinardquo Journal of Stored ProductsResearch vol 61 pp 27ndash31 2015

[48] N Gautam and N Sharma ldquoEvaluation of probiotic potentialof new bacterial strain Lactobacillus spicheri G2 isolated fromGundrukrdquo Proceedings of the National Academy of SciencesIndia Section B Biological Sciences vol 85 no 4 pp 979ndash9862015

[49] B T Fossi N B Ekue G T Nchanji et al ldquoProbiotic propertiesof lactic acid bacteria isolated from fermented sap of palm tree(Elaeis guineensis)rdquo Journal of Microbiology andAntimicrobialsvol 7 no 5 pp 42ndash52 2015

[50] M J Scourboutakos B Franco-Arellano S A Murphy et alldquoMismatch between Probiotic Benefits in Trials versus FoodProductsrdquo Nutrients vol 9 no 4 2017

[51] A BelicovaMMikulasova and R Dusinsky ldquoProbiotic poten-tial and safety properties of lactobacillus plantarum fromSlovakBryndza cheeserdquo BioMed Research International vol 2013 8pages 2013

[52] M A Ehrmann P Kurzak J Bauer and R F Vogel ldquoCharac-terization of lactobacilli towards their use as probiotic adjunctsin poultryrdquo Journal of Applied Microbiology vol 92 no 5 pp966ndash975 2002

[53] T Garcıa-Cayuela A M Korany I Bustos et al ldquoAdhesionabilities of dairy Lactobacillus plantarum strains showing anaggregation phenotyperdquo FoodResearch International vol 57 pp44ndash50 2014

[54] H Cheng and R C Harris ldquoRenal endothelial dysfunc-tion in diabetic nephropathyrdquo Cardiovascular amp Haematolog-ical Disorders-Drug Targets (Formerly Current Drug Targets-Cardiovascular amp Hematological Disorders) vol 14 no 1 pp22ndash33 2014

[55] S Lenzen ldquoThe mechanisms of alloxan-and streptozotocin-induced diabetesrdquoDiabetologia vol 51 no 2 pp 216ndash226 2008

[56] D H Wasserman ldquoFour grams of glucoserdquo American Journalof Physiology-Endocrinology andMetabolism vol 296 no 1 ppE11ndashE21 2009

[57] D B Sacks M Arnold G L Bakris et al ldquoGuidelines andrecommendations for laboratory analysis in the diagnosis andmanagement of diabetes mellitusrdquo Diabetes Care vol 34 no 6pp e61ndashe99 2011

[58] A D AssociationDiabetes and retinopathy (eye complications)vol 19 2009

[59] D Lloyd-Jones R J Adams T M Brown et al ldquoHeart diseaseand stroke statisticsmdash2010 updaterdquo Circulation vol 121 no 7pp e46ndashe215 2010

[60] S Agrahari and K Gopal Retracted Fluctuations of certainbiochemical constituents and markers enzymes as a consequenceof monocrotophos toxicity in the edible freshwater fish channapunctatus Elsevier 2009

[61] A Prakasam S Sethupathy and K V Pugalendi ldquoInfluenceof Casearia esculenta root extract on protein metabolism andmarker enzymes in streptozotocin-induceddiabetic ratsrdquoPolishJournal of Pharmacology vol 56 no 5 pp 587ndash593 2004

[62] P N Newsome R Cramb S M Davison et al ldquoGuidelines onthe management of abnormal liver blood testsrdquoGut vol 67 no1 pp 6ndash19 2018

[63] M Brownlee ldquoThe pathobiology of diabetic complications aunifying mechanismrdquo Diabetes vol 54 no 6 pp 1615ndash16252005

[64] P E Stevens and A Levin ldquoEvaluation and managementof chronic kidney disease synopsis of the kidney diseaseimproving global outcomes 2012 clinical practice guidelinerdquoAnnals of Internal Medicine vol 158 no 11 pp 825ndash830 2013

[65] T E Graham C J Wason M Bluher and B B Kahn ldquoShort-comings in methodology complicate measurements of serumretinol binding protein (RBP4) in insulin-resistant humansubjectsrdquo Diabetologia vol 50 no 4 pp 814ndash823 2007

Hindawiwwwhindawicom

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Submit your manuscripts atwwwhindawicom


which can increase host immunity and improve healthbenefits [4] Recently various conventional microorganismshave been used for probiotic cultures in fermented foodsFurthermore probiotics include a wide range of pharma-ceuticals or foods and special dietary applications suchas juices nutrition bars infant formulas and cosmetics[5]

The medical application of Lactobacillus brevis KB 290isolated from ldquoSugukirdquo is a traditional Japanese pickle thatplays a crucial role against people with influenza [6] In addi-tion it plays a vital role in suppressing inflammation causedby nitric oxide [7] Several studies have been conducted onseveral Lactobacillus brevis strains isolated from traditionalEgyptian dairy products which have been tested to confirmtheir antimicrobial effects These results show the extremeactivity of Lactobacillus brevis B23 among 38 species [8]Lactobacillus brevis is a vital biological organism to producebioactive 120574-aminobutyric acid biologically [9] Formerly 120574-aminobutyric acid (GABA) has been attracting attentionbecause it is the most abundant inhibitory neurotransmitterthat maintains the neurotransmitter functions of the humancentral nervous system Furthermore GABA may not havethe ability to penetrate the human blood-brain barrier [10]gad antibodies a symptom of type 1 diabetes can be detectedbefore the onset of clinical disease and the identification of asubset of patients with adult autoimmune disease [11]

Currently more than a thousand LAB strains have beensequenced and deposited in the NCBI GenBank databaseTherefore genetic assays were performed inmost continuousstrains in the presence or absence of gad operon and genesencoding glutamate decarboxylase in most of the sequencedstrains Moreover there are not only differences betweenmicrobial species from different sources in the gad enzymaticproperties but also differences between the subspecies as wellLactobacillus different sources of gad subunit compositionand molecular weight have a huge difference Lactobacillusbrevis gad has two subunits molecular weight of 57 60 kDa[12]120574-Aminobutyric acid (GABA) is a nonprotein four-

carbon free amino acid synthesized by the irreversible120572-decarboxylation reaction of L-glutamic acid or its saltsa pyridoxal-50-phosphate-dependent enzyme (PLP activevitamin B6)[13] GABA is permitted as a dietary ingredientand its nutrients have shown antihypertensive and antide-pressant activities as the two main functions of the host afteroral administration [9] However the GABA content in a fewnatural plants and animal products has been further studiedin the LABs because it also possesses the ability to produceGABA

Type 1 diabetes (T1D) is an autoimmune disease char-acterized by selective destruction of the pancreatic betacells [14] Furthermore it is a metabolic disease inducedby abnormal insulin secretion by damaged islet 120573 cellsThat damage accrues resulting in insulin insufficiency whichcan lead to life-threatening hyperglycemia that manifestsclinically as weight loss polyuria polyphagia and polydipsia[15] LAB could be utilized to grab foodborne diseasesresearchers used Salmonella typhimurium which causes

vomiting abdominal pain nausea and diarrhea [16] Listeriamonocytogenes which causes abortion and gastrointestinaldiseases [17] Staphylococcus comets which is involved inharmful gastroenteritis and E coli thereby exacerbating thesyndrome and colitis [18]

The main objective of the present study was to examinethe new Lactobacillus brevis probiotic strains that could effi-ciently produce high levels of bioactive 120574-Aminobutyric Acid(GABA) by exploring gad gene in addition study potentialprobiotic properties in vitro where they remain in severecase of simulation gastrointestinal juice and different typesof antibiotics and moreover antimicrobial activity againstfoodborne pathogens and finally control the development oftype 1 diabetes in vivo

2 Materials and Methods

21 Bacterial Strains and Cultivation Conditions Two strainsof Lactobacillus brevis KLDS 10727 and KLDS 10373 werefrozen stored at minus80∘C Further the foodborne pathogenicmicroorganisms used were Salmonella typhimurium (ATCC14028-MINI-PACK) (American Type Culture Collection)E coli IQCC 10126 Listeria monocytogenes IQCC 22221and Staphylococcus aureus IQCC 22035 (Inspection andQuarantine Culture Collection of the Chinese Academy ofInspection and Quarantine culture libraries IQCC) All teststrains were obtained from Key Laboratory of Dairy Science(KLDS) Heilongjiang Province Harbin China

Lactobacillus brevis KLDS 10727 and KLDS 10373 strainswere inoculated (1 vv) and grown in De Man RogosaSharpe medium (MRS Oxoid) while foodborne pathogenicbacteria were grown in Tryptic Soy Broth (TSB) (SigmaAldrich) All the strains were cultivated at 37∘C aerobicallyfor 24 hours The solid plates medium was cultivated for 48hours single colonies were picked after examination of thepure bacteria and they were then cultivated in an adequateliquid culture medium

KLDS 10727 and KLDS 103737 colonies were streakedon MRS agar plate and incubated at 37∘C for 24 hours underaerobic conditions Colonies were randomly picked from theplates and subculture two or three times on fresh MRS agarplates A single colony from the plate was transferred toMRSbroth and incubated for 18 hours at 37∘C The pure KLDS10727 and KLDS 10373 cultures were kept in MRS brothsupplemented with 30 (vv) glycerol and frozen at minus80∘Cuntil further analysis

22 Screening of Lactobacillus brevis KLDS 10727 and KLDS10373 Strains for the Presence of Glutamic Acid Decarboxylase(gad) Gene Genomic DNA was prepared using TIANampBacterial DNAKit (Tiangen Biotech Co Ltd Beijing China)and with some modification of manufacturerrsquos extractionsteps KLDS 10727 andKLDS 10373 were lysed with lysozymeDNA extracted

23 PCR Amplification of the 16S rDNA and Sequence Anal-ysis PCR amplification was performed using the universalprimers






















2 5 CO

2 5 H

2)






2 The Caco-2





2
































bp M 1 2 3 4


3 Results










Table2Sign

ificancemeansand

stand

arderrors(SE)


rowth

rateof

KLDS10

727andKL

DS10

373str

ains

SOVExp

erim

ents

Growth

rate

Biletolerance

SIJw

ithdiffe

rent

pHaerobically

SIJw

ithdiffe

rent

pHan

aerobically

Antibiotic

resistan

ceAdh

esionto

Caco2

cell

High

Low

High

Low

High

Low

High

Low

High

Low

High

Low

Strain

(S1)

(S2)

(S1)

(S2)

(S1)

(S2)

(S2)

(S1)

(S2)

(S1)

(S2)

(S1)

Meanplusmn

SEof

strains

124plusmn

019

c115plusmn

021

079plusmn008

c076plusmn008

036plusmn007

d036plusmn007

051plusmn010

b049plusmn010

045plusmn006

c044plusmn006

245plusmn3651d

24467plusmn3331

Time

270

270

240

240

243

--

Meanplusmn

SEof

time

197plusmn001

c022plusmn003

140plusmn007

c110plusmn

001

056plusmn014

c014plusmn001

105plusmn029

c014plusmn001

111plusmn015c

005plusmn0003

--

Con

centratio

nof

diffe

rent

parameters

--

02

02

02

Con

tAmp

30

Meanplusmn

SE-

-108

c plusmn015

052plusmn007

100plusmn014

c012plusmn001

139plusmn023c

015plusmn001

080plusmn014

c005plusmn0003

3816

7plusmn4847c

1375

0plusmn626

R2lowast

9999

9996

9998

9988

9999

7236

CVlowast

067

211

264

671

183

3048

Inthes

ixexperim

ents

grow

thratethe

effecto

ftwo

strains

KLD

S10

727(S1)andKLD

S10

373(S2)and

fivelevelso

ftim

es03927and

33hon

grow

thrateandpH

bile

tolerancetheeffecto

ftwo

strainsfi

velevelsof

timesand

four

levelsof

bilesalts

concentrationCon

t031and2

ongrow

thrateandpH

sim

ulated


ithdiffe

rent

pHaerobically

the

effecto

ftwo

strainsfi

velevelsof

times

0369and24and

simulated

intestinaljuice

with

different

four

levelsof

pHCon

t23and7on

grow

thratesim

ulated


diffe

rent

pHan

aerobically

the

effecto

ftwo

strainsfi

velevelsof

times

0369and24and

simulated

intestinaljuice

with

different

four

levelsof

pHCon

t23and7an

tibiotic

resistan

cethe

effecto

ftwo

strainsfi

velevelsof

timesand

typeso

fantibioticsC

ontstr

eptomycin

ampicillin

chloramph


kanamycin

ongrow

thrateandpH

adh

esionwith

Caco2

cell

theeffecto

ftwo

strainsfou

rlevelso

fdilu

tionof

2str

ains

Con

t34and5adhesio

nof

Caco2cellSign


1cPlt

000

01dn

onsig

nificantlowastR2

determinationcoeffi

cientandCV

coefficiento

fvariatio

nlowastSO

Vsource

ofvaria

nce


Table3Sign

ificancemeansand

stand

arderrors(SE)


ofKL

DS10

727andKL

DS10

373str

ains

lowastSO

VExp

erim

ents

Growth

ratean

dpH

Biletolerance

Antibiotic

stoleran

ceHigh

Low

High

Low

High

Low

Strain

(S2)

(S1)

(S2)

(S1)

(S2)

(S1)

Meanplusmn

SEof

strains

459plusmn023

c445plusmn020

492plusmn010

a490plusmn010

533

b plusmn006

532plusmn006

Time

033

033

024

Meanplusmn

SEof

time

556plusmn005

c370plusmn001

581plusmn002

c411plusmn

007

574

c plusmn001

455plusmn014

Con

centratio

nof

bileor

type

ofan

tibiotic

--

20

Chloramph

enicol

Con

trol

Meanplusmn

SE-

-521plusmn010

c460plusmn016

572

c plusmn001

497plusmn014

R29996

9985

9992

CV

05

073

04

Seefoo

tnoteo

fTable2


Table4Sign

ificancemeansand

stand

arderrors(SE)


adhesio

nof

KLDS10

727andKL

DS10

373str

ains

lowastSO

VExp

erim

ents

Biletolerance

(SIJ)w

ithdiffe

rent

pHaerobically

(SIJ)w

ithdiffe

rent

pHan

aerobically

Antibiotic

resistan

ceAdh

esionto

Caco2

cell

High

Low

High

Low

High

Low

High

Low

High

Low

Strain

(S2)

(S1)

(S1)

(S2)

(S2)

(S1)

(S2)

(S1)

(S1)

(S2)

Meanplusmn

SEof

strains

197plusmn007

b17

8plusmn007

116plusmn011

b113plusmn

010

117plusmn009

c112plusmn

01

160plusmn010

c14

1plusmn009

213plusmn019

d211plusmn020

Time

39

024

024

06

Meanplusmn

SEof

time

210plusmn014

c15

2plusmn009

208plusmn010

c056plusmn001

212plusmn010

c052plusmn002

275plusmn002

c12

9plusmn014

Con

centratio

nof

bileor

type

ofan

tibiotic

03

27

27

2Str

Erth

35

Meanplusmn

SEof

concentration

217plusmn009

c16

6plusmn009

138plusmn016

c094plusmn009

134plusmn014

c096plusmn009

236plusmn014

c083plusmn009

268plusmn015

c148plusmn

012

R2866

9971

9964

9648

078

CV

1204

387

394

1391

152

Seefoo

tnoteo

fTable2


3 9 27 330Time (h)

3 9 27 330Time (h)

40

45

50

55

pH

05

10

15

20O

D 6

20

(a)



40

45

50

55

60

pH

00

05

10

15

OD

620







40

45

50

55

pH



00

05

10

15

20O

D 6

20



00

05

10

15

20

25

30

35O

D 6

20



00

05

10

15

OD

620







Table5Antibacteria

lactivity

oftheK

LDS10

727andKL

DS10

373str

ains

againstfoo

dborne


term

sofZ

DI

Testfo

odbo

rnes

trains

ZDI(mm)plusmn

SDof

IndicatorB

acteria

Salm

onellatyph

imurium

Ecoli

Liste

riamon

ocytogenes

Stap

hylococcus

aureus

KLD

S10

727

237plusmn003

+++

197plusmn

003

+++

217plusmn009

+++

220plusmn010

+++

KLD

S10

373

247plusmn003

+++

193plusmn

007

+++

193plusmn

007

+++

220plusmn010

+++

Sig

lt000

104226

00198

00572

R210

0000

0090

4762

0978495

0970588

+++refersto

clearz

one(14mm3

points

9ndash14mm2

points

1ndash99

mmone

point)



100

150

200

250

300

350

Adhe

sion

()


(A)

(B) (C)


Figure 5







10

0

5

minus10

minus5

(S1)(STZ)

Glu

cose

leve

ls (w

)

(a)

10

15

20

25

0

5


Wei

ght (

g)

(b)










liver






pancreas


liver

kidney


arrow)Spleen

















4 Discussion





















5 Conclusions


Data Availability




Acknowledgments


References







































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018























2 5 CO

2 5 H

2)






2 The Caco-2





2
































bp M 1 2 3 4


3 Results










Table2Sign

ificancemeansand

stand

arderrors(SE)


rowth

rateof

KLDS10

727andKL

DS10

373str

ains

SOVExp

erim

ents

Growth

rate

Biletolerance

SIJw

ithdiffe

rent

pHaerobically

SIJw

ithdiffe

rent

pHan

aerobically

Antibiotic

resistan

ceAdh

esionto

Caco2

cell

High

Low

High

Low

High

Low

High

Low

High

Low

High

Low

Strain

(S1)

(S2)

(S1)

(S2)

(S1)

(S2)

(S2)

(S1)

(S2)

(S1)

(S2)

(S1)

Meanplusmn

SEof

strains

124plusmn

019

c115plusmn

021

079plusmn008

c076plusmn008

036plusmn007

d036plusmn007

051plusmn010

b049plusmn010

045plusmn006

c044plusmn006

245plusmn3651d

24467plusmn3331

Time

270

270

240

240

243

--

Meanplusmn

SEof

time

197plusmn001

c022plusmn003

140plusmn007

c110plusmn

001

056plusmn014

c014plusmn001

105plusmn029

c014plusmn001

111plusmn015c

005plusmn0003

--

Con

centratio

nof

diffe

rent

parameters

--

02

02

02

Con

tAmp

30

Meanplusmn

SE-

-108

c plusmn015

052plusmn007

100plusmn014

c012plusmn001

139plusmn023c

015plusmn001

080plusmn014

c005plusmn0003

3816

7plusmn4847c

1375

0plusmn626

R2lowast

9999

9996

9998

9988

9999

7236

CVlowast

067

211

264

671

183

3048

Inthes

ixexperim

ents

grow

thratethe

effecto

ftwo

strains

KLD

S10

727(S1)andKLD

S10

373(S2)and

fivelevelso

ftim

es03927and

33hon

grow

thrateandpH

bile

tolerancetheeffecto

ftwo

strainsfi

velevelsof

timesand

four

levelsof

bilesalts

concentrationCon

t031and2

ongrow

thrateandpH

sim

ulated


ithdiffe

rent

pHaerobically

the

effecto

ftwo

strainsfi

velevelsof

times

0369and24and

simulated

intestinaljuice

with

different

four

levelsof

pHCon

t23and7on

grow

thratesim

ulated


diffe

rent

pHan

aerobically

the

effecto

ftwo

strainsfi

velevelsof

times

0369and24and

simulated

intestinaljuice

with

different

four

levelsof

pHCon

t23and7an

tibiotic

resistan

cethe

effecto

ftwo

strainsfi

velevelsof

timesand

typeso

fantibioticsC

ontstr

eptomycin

ampicillin

chloramph


kanamycin

ongrow

thrateandpH

adh

esionwith

Caco2

cell

theeffecto

ftwo

strainsfou

rlevelso

fdilu

tionof

2str

ains

Con

t34and5adhesio

nof

Caco2cellSign


1cPlt

000

01dn

onsig

nificantlowastR2

determinationcoeffi

cientandCV

coefficiento

fvariatio

nlowastSO

Vsource

ofvaria

nce


Table3Sign

ificancemeansand

stand

arderrors(SE)


ofKL

DS10

727andKL

DS10

373str

ains

lowastSO

VExp

erim

ents

Growth

ratean

dpH

Biletolerance

Antibiotic

stoleran

ceHigh

Low

High

Low

High

Low

Strain

(S2)

(S1)

(S2)

(S1)

(S2)

(S1)

Meanplusmn

SEof

strains

459plusmn023

c445plusmn020

492plusmn010

a490plusmn010

533

b plusmn006

532plusmn006

Time

033

033

024

Meanplusmn

SEof

time

556plusmn005

c370plusmn001

581plusmn002

c411plusmn

007

574

c plusmn001

455plusmn014

Con

centratio

nof

bileor

type

ofan

tibiotic

--

20

Chloramph

enicol

Con

trol

Meanplusmn

SE-

-521plusmn010

c460plusmn016

572

c plusmn001

497plusmn014

R29996

9985

9992

CV

05

073

04

Seefoo

tnoteo

fTable2


Table4Sign

ificancemeansand

stand

arderrors(SE)


adhesio

nof

KLDS10

727andKL

DS10

373str

ains

lowastSO

VExp

erim

ents

Biletolerance

(SIJ)w

ithdiffe

rent

pHaerobically

(SIJ)w

ithdiffe

rent

pHan

aerobically

Antibiotic

resistan

ceAdh

esionto

Caco2

cell

High

Low

High

Low

High

Low

High

Low

High

Low

Strain

(S2)

(S1)

(S1)

(S2)

(S2)

(S1)

(S2)

(S1)

(S1)

(S2)

Meanplusmn

SEof

strains

197plusmn007

b17

8plusmn007

116plusmn011

b113plusmn

010

117plusmn009

c112plusmn

01

160plusmn010

c14

1plusmn009

213plusmn019

d211plusmn020

Time

39

024

024

06

Meanplusmn

SEof

time

210plusmn014

c15

2plusmn009

208plusmn010

c056plusmn001

212plusmn010

c052plusmn002

275plusmn002

c12

9plusmn014

Con

centratio

nof

bileor

type

ofan

tibiotic

03

27

27

2Str

Erth

35

Meanplusmn

SEof

concentration

217plusmn009

c16

6plusmn009

138plusmn016

c094plusmn009

134plusmn014

c096plusmn009

236plusmn014

c083plusmn009

268plusmn015

c148plusmn

012

R2866

9971

9964

9648

078

CV

1204

387

394

1391

152

Seefoo

tnoteo

fTable2


3 9 27 330Time (h)

3 9 27 330Time (h)

40

45

50

55

pH

05

10

15

20O

D 6

20

(a)



40

45

50

55

60

pH

00

05

10

15

OD

620







40

45

50

55

pH



00

05

10

15

20O

D 6

20



00

05

10

15

20

25

30

35O

D 6

20



00

05

10

15

OD

620







Table5Antibacteria

lactivity

oftheK

LDS10

727andKL

DS10

373str

ains

againstfoo

dborne


term

sofZ

DI

Testfo

odbo

rnes

trains

ZDI(mm)plusmn

SDof

IndicatorB

acteria

Salm

onellatyph

imurium

Ecoli

Liste

riamon

ocytogenes

Stap

hylococcus

aureus

KLD

S10

727

237plusmn003

+++

197plusmn

003

+++

217plusmn009

+++

220plusmn010

+++

KLD

S10

373

247plusmn003

+++

193plusmn

007

+++

193plusmn

007

+++

220plusmn010

+++

Sig

lt000

104226

00198

00572

R210

0000

0090

4762

0978495

0970588

+++refersto

clearz

one(14mm3

points

9ndash14mm2

points

1ndash99

mmone

point)



100

150

200

250

300

350

Adhe

sion

()


(A)

(B) (C)


Figure 5







10

0

5

minus10

minus5

(S1)(STZ)

Glu

cose

leve

ls (w

)

(a)

10

15

20

25

0

5


Wei

ght (

g)

(b)










liver






pancreas


liver

kidney


arrow)Spleen

















4 Discussion





















5 Conclusions


Data Availability




Acknowledgments


References







































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




2 5 CO

2 5 H

2)






2 The Caco-2





2
































bp M 1 2 3 4


3 Results










Table2Sign

ificancemeansand

stand

arderrors(SE)


rowth

rateof

KLDS10

727andKL

DS10

373str

ains

SOVExp

erim

ents

Growth

rate

Biletolerance

SIJw

ithdiffe

rent

pHaerobically

SIJw

ithdiffe

rent

pHan

aerobically

Antibiotic

resistan

ceAdh

esionto

Caco2

cell

High

Low

High

Low

High

Low

High

Low

High

Low

High

Low

Strain

(S1)

(S2)

(S1)

(S2)

(S1)

(S2)

(S2)

(S1)

(S2)

(S1)

(S2)

(S1)

Meanplusmn

SEof

strains

124plusmn

019

c115plusmn

021

079plusmn008

c076plusmn008

036plusmn007

d036plusmn007

051plusmn010

b049plusmn010

045plusmn006

c044plusmn006

245plusmn3651d

24467plusmn3331

Time

270

270

240

240

243

--

Meanplusmn

SEof

time

197plusmn001

c022plusmn003

140plusmn007

c110plusmn

001

056plusmn014

c014plusmn001

105plusmn029

c014plusmn001

111plusmn015c

005plusmn0003

--

Con

centratio

nof

diffe

rent

parameters

--

02

02

02

Con

tAmp

30

Meanplusmn

SE-

-108

c plusmn015

052plusmn007

100plusmn014

c012plusmn001

139plusmn023c

015plusmn001

080plusmn014

c005plusmn0003

3816

7plusmn4847c

1375

0plusmn626

R2lowast

9999

9996

9998

9988

9999

7236

CVlowast

067

211

264

671

183

3048

Inthes

ixexperim

ents

grow

thratethe

effecto

ftwo

strains

KLD

S10

727(S1)andKLD

S10

373(S2)and

fivelevelso

ftim

es03927and

33hon

grow

thrateandpH

bile

tolerancetheeffecto

ftwo

strainsfi

velevelsof

timesand

four

levelsof

bilesalts

concentrationCon

t031and2

ongrow

thrateandpH

sim

ulated


ithdiffe

rent

pHaerobically

the

effecto

ftwo

strainsfi

velevelsof

times

0369and24and

simulated

intestinaljuice

with

different

four

levelsof

pHCon

t23and7on

grow

thratesim

ulated


diffe

rent

pHan

aerobically

the

effecto

ftwo

strainsfi

velevelsof

times

0369and24and

simulated

intestinaljuice

with

different

four

levelsof

pHCon

t23and7an

tibiotic

resistan

cethe

effecto

ftwo

strainsfi

velevelsof

timesand

typeso

fantibioticsC

ontstr

eptomycin

ampicillin

chloramph


kanamycin

ongrow

thrateandpH

adh

esionwith

Caco2

cell

theeffecto

ftwo

strainsfou

rlevelso

fdilu

tionof

2str

ains

Con

t34and5adhesio

nof

Caco2cellSign


1cPlt

000

01dn

onsig

nificantlowastR2

determinationcoeffi

cientandCV

coefficiento

fvariatio

nlowastSO

Vsource

ofvaria

nce


Table3Sign

ificancemeansand

stand

arderrors(SE)


ofKL

DS10

727andKL

DS10

373str

ains

lowastSO

VExp

erim

ents

Growth

ratean

dpH

Biletolerance

Antibiotic

stoleran

ceHigh

Low

High

Low

High

Low

Strain

(S2)

(S1)

(S2)

(S1)

(S2)

(S1)

Meanplusmn

SEof

strains

459plusmn023

c445plusmn020

492plusmn010

a490plusmn010

533

b plusmn006

532plusmn006

Time

033

033

024

Meanplusmn

SEof

time

556plusmn005

c370plusmn001

581plusmn002

c411plusmn

007

574

c plusmn001

455plusmn014

Con

centratio

nof

bileor

type

ofan

tibiotic

--

20

Chloramph

enicol

Con

trol

Meanplusmn

SE-

-521plusmn010

c460plusmn016

572

c plusmn001

497plusmn014

R29996

9985

9992

CV

05

073

04

Seefoo

tnoteo

fTable2


Table4Sign

ificancemeansand

stand

arderrors(SE)


adhesio

nof

KLDS10

727andKL

DS10

373str

ains

lowastSO

VExp

erim

ents

Biletolerance

(SIJ)w

ithdiffe

rent

pHaerobically

(SIJ)w

ithdiffe

rent

pHan

aerobically

Antibiotic

resistan

ceAdh

esionto

Caco2

cell

High

Low

High

Low

High

Low

High

Low

High

Low

Strain

(S2)

(S1)

(S1)

(S2)

(S2)

(S1)

(S2)

(S1)

(S1)

(S2)

Meanplusmn

SEof

strains

197plusmn007

b17

8plusmn007

116plusmn011

b113plusmn

010

117plusmn009

c112plusmn

01

160plusmn010

c14

1plusmn009

213plusmn019

d211plusmn020

Time

39

024

024

06

Meanplusmn

SEof

time

210plusmn014

c15

2plusmn009

208plusmn010

c056plusmn001

212plusmn010

c052plusmn002

275plusmn002

c12

9plusmn014

Con

centratio

nof

bileor

type

ofan

tibiotic

03

27

27

2Str

Erth

35

Meanplusmn

SEof

concentration

217plusmn009

c16

6plusmn009

138plusmn016

c094plusmn009

134plusmn014

c096plusmn009

236plusmn014

c083plusmn009

268plusmn015

c148plusmn

012

R2866

9971

9964

9648

078

CV

1204

387

394

1391

152

Seefoo

tnoteo

fTable2


3 9 27 330Time (h)

3 9 27 330Time (h)

40

45

50

55

pH

05

10

15

20O

D 6

20

(a)



40

45

50

55

60

pH

00

05

10

15

OD

620







40

45

50

55

pH



00

05

10

15

20O

D 6

20



00

05

10

15

20

25

30

35O

D 6

20



00

05

10

15

OD

620







Table5Antibacteria

lactivity

oftheK

LDS10

727andKL

DS10

373str

ains

againstfoo

dborne


term

sofZ

DI

Testfo

odbo

rnes

trains

ZDI(mm)plusmn

SDof

IndicatorB

acteria

Salm

onellatyph

imurium

Ecoli

Liste

riamon

ocytogenes

Stap

hylococcus

aureus

KLD

S10

727

237plusmn003

+++

197plusmn

003

+++

217plusmn009

+++

220plusmn010

+++

KLD

S10

373

247plusmn003

+++

193plusmn

007

+++

193plusmn

007

+++

220plusmn010

+++

Sig

lt000

104226

00198

00572

R210

0000

0090

4762

0978495

0970588

+++refersto

clearz

one(14mm3

points

9ndash14mm2

points

1ndash99

mmone

point)



100

150

200

250

300

350

Adhe

sion

()


(A)

(B) (C)


Figure 5







10

0

5

minus10

minus5

(S1)(STZ)

Glu

cose

leve

ls (w

)

(a)

10

15

20

25

0

5


Wei

ght (

g)

(b)










liver






pancreas


liver

kidney


arrow)Spleen

















4 Discussion





















5 Conclusions


Data Availability




Acknowledgments


References







































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018

























bp M 1 2 3 4


3 Results










Table2Sign

ificancemeansand

stand

arderrors(SE)


rowth

rateof

KLDS10

727andKL

DS10

373str

ains

SOVExp

erim

ents

Growth

rate

Biletolerance

SIJw

ithdiffe

rent

pHaerobically

SIJw

ithdiffe

rent

pHan

aerobically

Antibiotic

resistan

ceAdh

esionto

Caco2

cell

High

Low

High

Low

High

Low

High

Low

High

Low

High

Low

Strain

(S1)

(S2)

(S1)

(S2)

(S1)

(S2)

(S2)

(S1)

(S2)

(S1)

(S2)

(S1)

Meanplusmn

SEof

strains

124plusmn

019

c115plusmn

021

079plusmn008

c076plusmn008

036plusmn007

d036plusmn007

051plusmn010

b049plusmn010

045plusmn006

c044plusmn006

245plusmn3651d

24467plusmn3331

Time

270

270

240

240

243

--

Meanplusmn

SEof

time

197plusmn001

c022plusmn003

140plusmn007

c110plusmn

001

056plusmn014

c014plusmn001

105plusmn029

c014plusmn001

111plusmn015c

005plusmn0003

--

Con

centratio

nof

diffe

rent

parameters

--

02

02

02

Con

tAmp

30

Meanplusmn

SE-

-108

c plusmn015

052plusmn007

100plusmn014

c012plusmn001

139plusmn023c

015plusmn001

080plusmn014

c005plusmn0003

3816

7plusmn4847c

1375

0plusmn626

R2lowast

9999

9996

9998

9988

9999

7236

CVlowast

067

211

264

671

183

3048

Inthes

ixexperim

ents

grow

thratethe

effecto

ftwo

strains

KLD

S10

727(S1)andKLD

S10

373(S2)and

fivelevelso

ftim

es03927and

33hon

grow

thrateandpH

bile

tolerancetheeffecto

ftwo

strainsfi

velevelsof

timesand

four

levelsof

bilesalts

concentrationCon

t031and2

ongrow

thrateandpH

sim

ulated


ithdiffe

rent

pHaerobically

the

effecto

ftwo

strainsfi

velevelsof

times

0369and24and

simulated

intestinaljuice

with

different

four

levelsof

pHCon

t23and7on

grow

thratesim

ulated


diffe

rent

pHan

aerobically

the

effecto

ftwo

strainsfi

velevelsof

times

0369and24and

simulated

intestinaljuice

with

different

four

levelsof

pHCon

t23and7an

tibiotic

resistan

cethe

effecto

ftwo

strainsfi

velevelsof

timesand

typeso

fantibioticsC

ontstr

eptomycin

ampicillin

chloramph


kanamycin

ongrow

thrateandpH

adh

esionwith

Caco2

cell

theeffecto

ftwo

strainsfou

rlevelso

fdilu

tionof

2str

ains

Con

t34and5adhesio

nof

Caco2cellSign


1cPlt

000

01dn

onsig

nificantlowastR2

determinationcoeffi

cientandCV

coefficiento

fvariatio

nlowastSO

Vsource

ofvaria

nce


Table3Sign

ificancemeansand

stand

arderrors(SE)


ofKL

DS10

727andKL

DS10

373str

ains

lowastSO

VExp

erim

ents

Growth

ratean

dpH

Biletolerance

Antibiotic

stoleran

ceHigh

Low

High

Low

High

Low

Strain

(S2)

(S1)

(S2)

(S1)

(S2)

(S1)

Meanplusmn

SEof

strains

459plusmn023

c445plusmn020

492plusmn010

a490plusmn010

533

b plusmn006

532plusmn006

Time

033

033

024

Meanplusmn

SEof

time

556plusmn005

c370plusmn001

581plusmn002

c411plusmn

007

574

c plusmn001

455plusmn014

Con

centratio

nof

bileor

type

ofan

tibiotic

--

20

Chloramph

enicol

Con

trol

Meanplusmn

SE-

-521plusmn010

c460plusmn016

572

c plusmn001

497plusmn014

R29996

9985

9992

CV

05

073

04

Seefoo

tnoteo

fTable2


Table4Sign

ificancemeansand

stand

arderrors(SE)


adhesio

nof

KLDS10

727andKL

DS10

373str

ains

lowastSO

VExp

erim

ents

Biletolerance

(SIJ)w

ithdiffe

rent

pHaerobically

(SIJ)w

ithdiffe

rent

pHan

aerobically

Antibiotic

resistan

ceAdh

esionto

Caco2

cell

High

Low

High

Low

High

Low

High

Low

High

Low

Strain

(S2)

(S1)

(S1)

(S2)

(S2)

(S1)

(S2)

(S1)

(S1)

(S2)

Meanplusmn

SEof

strains

197plusmn007

b17

8plusmn007

116plusmn011

b113plusmn

010

117plusmn009

c112plusmn

01

160plusmn010

c14

1plusmn009

213plusmn019

d211plusmn020

Time

39

024

024

06

Meanplusmn

SEof

time

210plusmn014

c15

2plusmn009

208plusmn010

c056plusmn001

212plusmn010

c052plusmn002

275plusmn002

c12

9plusmn014

Con

centratio

nof

bileor

type

ofan

tibiotic

03

27

27

2Str

Erth

35

Meanplusmn

SEof

concentration

217plusmn009

c16

6plusmn009

138plusmn016

c094plusmn009

134plusmn014

c096plusmn009

236plusmn014

c083plusmn009

268plusmn015

c148plusmn

012

R2866

9971

9964

9648

078

CV

1204

387

394

1391

152

Seefoo

tnoteo

fTable2


3 9 27 330Time (h)

3 9 27 330Time (h)

40

45

50

55

pH

05

10

15

20O

D 6

20

(a)



40

45

50

55

60

pH

00

05

10

15

OD

620







40

45

50

55

pH



00

05

10

15

20O

D 6

20



00

05

10

15

20

25

30

35O

D 6

20



00

05

10

15

OD

620







Table5Antibacteria

lactivity

oftheK

LDS10

727andKL

DS10

373str

ains

againstfoo

dborne


term

sofZ

DI

Testfo

odbo

rnes

trains

ZDI(mm)plusmn

SDof

IndicatorB

acteria

Salm

onellatyph

imurium

Ecoli

Liste

riamon

ocytogenes

Stap

hylococcus

aureus

KLD

S10

727

237plusmn003

+++

197plusmn

003

+++

217plusmn009

+++

220plusmn010

+++

KLD

S10

373

247plusmn003

+++

193plusmn

007

+++

193plusmn

007

+++

220plusmn010

+++

Sig

lt000

104226

00198

00572

R210

0000

0090

4762

0978495

0970588

+++refersto

clearz

one(14mm3

points

9ndash14mm2

points

1ndash99

mmone

point)



100

150

200

250

300

350

Adhe

sion

()


(A)

(B) (C)


Figure 5







10

0

5

minus10

minus5

(S1)(STZ)

Glu

cose

leve

ls (w

)

(a)

10

15

20

25

0

5


Wei

ght (

g)

(b)










liver






pancreas


liver

kidney


arrow)Spleen

















4 Discussion





















5 Conclusions


Data Availability




Acknowledgments


References







































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



bp M 1 2 3 4


3 Results










Table2Sign

ificancemeansand

stand

arderrors(SE)


rowth

rateof

KLDS10

727andKL

DS10

373str

ains

SOVExp

erim

ents

Growth

rate

Biletolerance

SIJw

ithdiffe

rent

pHaerobically

SIJw

ithdiffe

rent

pHan

aerobically

Antibiotic

resistan

ceAdh

esionto

Caco2

cell

High

Low

High

Low

High

Low

High

Low

High

Low

High

Low

Strain

(S1)

(S2)

(S1)

(S2)

(S1)

(S2)

(S2)

(S1)

(S2)

(S1)

(S2)

(S1)

Meanplusmn

SEof

strains

124plusmn

019

c115plusmn

021

079plusmn008

c076plusmn008

036plusmn007

d036plusmn007

051plusmn010

b049plusmn010

045plusmn006

c044plusmn006

245plusmn3651d

24467plusmn3331

Time

270

270

240

240

243

--

Meanplusmn

SEof

time

197plusmn001

c022plusmn003

140plusmn007

c110plusmn

001

056plusmn014

c014plusmn001

105plusmn029

c014plusmn001

111plusmn015c

005plusmn0003

--

Con

centratio

nof

diffe

rent

parameters

--

02

02

02

Con

tAmp

30

Meanplusmn

SE-

-108

c plusmn015

052plusmn007

100plusmn014

c012plusmn001

139plusmn023c

015plusmn001

080plusmn014

c005plusmn0003

3816

7plusmn4847c

1375

0plusmn626

R2lowast

9999

9996

9998

9988

9999

7236

CVlowast

067

211

264

671

183

3048

Inthes

ixexperim

ents

grow

thratethe

effecto

ftwo

strains

KLD

S10

727(S1)andKLD

S10

373(S2)and

fivelevelso

ftim

es03927and

33hon

grow

thrateandpH

bile

tolerancetheeffecto

ftwo

strainsfi

velevelsof

timesand

four

levelsof

bilesalts

concentrationCon

t031and2

ongrow

thrateandpH

sim

ulated


ithdiffe

rent

pHaerobically

the

effecto

ftwo

strainsfi

velevelsof

times

0369and24and

simulated

intestinaljuice

with

different

four

levelsof

pHCon

t23and7on

grow

thratesim

ulated


diffe

rent

pHan

aerobically

the

effecto

ftwo

strainsfi

velevelsof

times

0369and24and

simulated

intestinaljuice

with

different

four

levelsof

pHCon

t23and7an

tibiotic

resistan

cethe

effecto

ftwo

strainsfi

velevelsof

timesand

typeso

fantibioticsC

ontstr

eptomycin

ampicillin

chloramph


kanamycin

ongrow

thrateandpH

adh

esionwith

Caco2

cell

theeffecto

ftwo

strainsfou

rlevelso

fdilu

tionof

2str

ains

Con

t34and5adhesio

nof

Caco2cellSign


1cPlt

000

01dn

onsig

nificantlowastR2

determinationcoeffi

cientandCV

coefficiento

fvariatio

nlowastSO

Vsource

ofvaria

nce


Table3Sign

ificancemeansand

stand

arderrors(SE)


ofKL

DS10

727andKL

DS10

373str

ains

lowastSO

VExp

erim

ents

Growth

ratean

dpH

Biletolerance

Antibiotic

stoleran

ceHigh

Low

High

Low

High

Low

Strain

(S2)

(S1)

(S2)

(S1)

(S2)

(S1)

Meanplusmn

SEof

strains

459plusmn023

c445plusmn020

492plusmn010

a490plusmn010

533

b plusmn006

532plusmn006

Time

033

033

024

Meanplusmn

SEof

time

556plusmn005

c370plusmn001

581plusmn002

c411plusmn

007

574

c plusmn001

455plusmn014

Con

centratio

nof

bileor

type

ofan

tibiotic

--

20

Chloramph

enicol

Con

trol

Meanplusmn

SE-

-521plusmn010

c460plusmn016

572

c plusmn001

497plusmn014

R29996

9985

9992

CV

05

073

04

Seefoo

tnoteo

fTable2


Table4Sign

ificancemeansand

stand

arderrors(SE)


adhesio

nof

KLDS10

727andKL

DS10

373str

ains

lowastSO

VExp

erim

ents

Biletolerance

(SIJ)w

ithdiffe

rent

pHaerobically

(SIJ)w

ithdiffe

rent

pHan

aerobically

Antibiotic

resistan

ceAdh

esionto

Caco2

cell

High

Low

High

Low

High

Low

High

Low

High

Low

Strain

(S2)

(S1)

(S1)

(S2)

(S2)

(S1)

(S2)

(S1)

(S1)

(S2)

Meanplusmn

SEof

strains

197plusmn007

b17

8plusmn007

116plusmn011

b113plusmn

010

117plusmn009

c112plusmn

01

160plusmn010

c14

1plusmn009

213plusmn019

d211plusmn020

Time

39

024

024

06

Meanplusmn

SEof

time

210plusmn014

c15

2plusmn009

208plusmn010

c056plusmn001

212plusmn010

c052plusmn002

275plusmn002

c12

9plusmn014

Con

centratio

nof

bileor

type

ofan

tibiotic

03

27

27

2Str

Erth

35

Meanplusmn

SEof

concentration

217plusmn009

c16

6plusmn009

138plusmn016

c094plusmn009

134plusmn014

c096plusmn009

236plusmn014

c083plusmn009

268plusmn015

c148plusmn

012

R2866

9971

9964

9648

078

CV

1204

387

394

1391

152

Seefoo

tnoteo

fTable2


3 9 27 330Time (h)

3 9 27 330Time (h)

40

45

50

55

pH

05

10

15

20O

D 6

20

(a)



40

45

50

55

60

pH

00

05

10

15

OD

620







40

45

50

55

pH



00

05

10

15

20O

D 6

20



00

05

10

15

20

25

30

35O

D 6

20



00

05

10

15

OD

620







Table5Antibacteria

lactivity

oftheK

LDS10

727andKL

DS10

373str

ains

againstfoo

dborne


term

sofZ

DI

Testfo

odbo

rnes

trains

ZDI(mm)plusmn

SDof

IndicatorB

acteria

Salm

onellatyph

imurium

Ecoli

Liste

riamon

ocytogenes

Stap

hylococcus

aureus

KLD

S10

727

237plusmn003

+++

197plusmn

003

+++

217plusmn009

+++

220plusmn010

+++

KLD

S10

373

247plusmn003

+++

193plusmn

007

+++

193plusmn

007

+++

220plusmn010

+++

Sig

lt000

104226

00198

00572

R210

0000

0090

4762

0978495

0970588

+++refersto

clearz

one(14mm3

points

9ndash14mm2

points

1ndash99

mmone

point)



100

150

200

250

300

350

Adhe

sion

()


(A)

(B) (C)


Figure 5







10

0

5

minus10

minus5

(S1)(STZ)

Glu

cose

leve

ls (w

)

(a)

10

15

20

25

0

5


Wei

ght (

g)

(b)










liver






pancreas


liver

kidney


arrow)Spleen

















4 Discussion





















5 Conclusions


Data Availability




Acknowledgments


References







































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Table2Sign

ificancemeansand

stand

arderrors(SE)


rowth

rateof

KLDS10

727andKL

DS10

373str

ains

SOVExp

erim

ents

Growth

rate

Biletolerance

SIJw

ithdiffe

rent

pHaerobically

SIJw

ithdiffe

rent

pHan

aerobically

Antibiotic

resistan

ceAdh

esionto

Caco2

cell

High

Low

High

Low

High

Low

High

Low

High

Low

High

Low

Strain

(S1)

(S2)

(S1)

(S2)

(S1)

(S2)

(S2)

(S1)

(S2)

(S1)

(S2)

(S1)

Meanplusmn

SEof

strains

124plusmn

019

c115plusmn

021

079plusmn008

c076plusmn008

036plusmn007

d036plusmn007

051plusmn010

b049plusmn010

045plusmn006

c044plusmn006

245plusmn3651d

24467plusmn3331

Time

270

270

240

240

243

--

Meanplusmn

SEof

time

197plusmn001

c022plusmn003

140plusmn007

c110plusmn

001

056plusmn014

c014plusmn001

105plusmn029

c014plusmn001

111plusmn015c

005plusmn0003

--

Con

centratio

nof

diffe

rent

parameters

--

02

02

02

Con

tAmp

30

Meanplusmn

SE-

-108

c plusmn015

052plusmn007

100plusmn014

c012plusmn001

139plusmn023c

015plusmn001

080plusmn014

c005plusmn0003

3816

7plusmn4847c

1375

0plusmn626

R2lowast

9999

9996

9998

9988

9999

7236

CVlowast

067

211

264

671

183

3048

Inthes

ixexperim

ents

grow

thratethe

effecto

ftwo

strains

KLD

S10

727(S1)andKLD

S10

373(S2)and

fivelevelso

ftim

es03927and

33hon

grow

thrateandpH

bile

tolerancetheeffecto

ftwo

strainsfi

velevelsof

timesand

four

levelsof

bilesalts

concentrationCon

t031and2

ongrow

thrateandpH

sim

ulated


ithdiffe

rent

pHaerobically

the

effecto

ftwo

strainsfi

velevelsof

times

0369and24and

simulated

intestinaljuice

with

different

four

levelsof

pHCon

t23and7on

grow

thratesim

ulated


diffe

rent

pHan

aerobically

the

effecto

ftwo

strainsfi

velevelsof

times

0369and24and

simulated

intestinaljuice

with

different

four

levelsof

pHCon

t23and7an

tibiotic

resistan

cethe

effecto

ftwo

strainsfi

velevelsof

timesand

typeso

fantibioticsC

ontstr

eptomycin

ampicillin

chloramph


kanamycin

ongrow

thrateandpH

adh

esionwith

Caco2

cell

theeffecto

ftwo

strainsfou

rlevelso

fdilu

tionof

2str

ains

Con

t34and5adhesio

nof

Caco2cellSign


1cPlt

000

01dn

onsig

nificantlowastR2

determinationcoeffi

cientandCV

coefficiento

fvariatio

nlowastSO

Vsource

ofvaria

nce


Table3Sign

ificancemeansand

stand

arderrors(SE)


ofKL

DS10

727andKL

DS10

373str

ains

lowastSO

VExp

erim

ents

Growth

ratean

dpH

Biletolerance

Antibiotic

stoleran

ceHigh

Low

High

Low

High

Low

Strain

(S2)

(S1)

(S2)

(S1)

(S2)

(S1)

Meanplusmn

SEof

strains

459plusmn023

c445plusmn020

492plusmn010

a490plusmn010

533

b plusmn006

532plusmn006

Time

033

033

024

Meanplusmn

SEof

time

556plusmn005

c370plusmn001

581plusmn002

c411plusmn

007

574

c plusmn001

455plusmn014

Con

centratio

nof

bileor

type

ofan

tibiotic

--

20

Chloramph

enicol

Con

trol

Meanplusmn

SE-

-521plusmn010

c460plusmn016

572

c plusmn001

497plusmn014

R29996

9985

9992

CV

05

073

04

Seefoo

tnoteo

fTable2


Table4Sign

ificancemeansand

stand

arderrors(SE)


adhesio

nof

KLDS10

727andKL

DS10

373str

ains

lowastSO

VExp

erim

ents

Biletolerance

(SIJ)w

ithdiffe

rent

pHaerobically

(SIJ)w

ithdiffe

rent

pHan

aerobically

Antibiotic

resistan

ceAdh

esionto

Caco2

cell

High

Low

High

Low

High

Low

High

Low

High

Low

Strain

(S2)

(S1)

(S1)

(S2)

(S2)

(S1)

(S2)

(S1)

(S1)

(S2)

Meanplusmn

SEof

strains

197plusmn007

b17

8plusmn007

116plusmn011

b113plusmn

010

117plusmn009

c112plusmn

01

160plusmn010

c14

1plusmn009

213plusmn019

d211plusmn020

Time

39

024

024

06

Meanplusmn

SEof

time

210plusmn014

c15

2plusmn009

208plusmn010

c056plusmn001

212plusmn010

c052plusmn002

275plusmn002

c12

9plusmn014

Con

centratio

nof

bileor

type

ofan

tibiotic

03

27

27

2Str

Erth

35

Meanplusmn

SEof

concentration

217plusmn009

c16

6plusmn009

138plusmn016

c094plusmn009

134plusmn014

c096plusmn009

236plusmn014

c083plusmn009

268plusmn015

c148plusmn

012

R2866

9971

9964

9648

078

CV

1204

387

394

1391

152

Seefoo

tnoteo

fTable2


3 9 27 330Time (h)

3 9 27 330Time (h)

40

45

50

55

pH

05

10

15

20O

D 6

20

(a)



40

45

50

55

60

pH

00

05

10

15

OD

620







40

45

50

55

pH



00

05

10

15

20O

D 6

20



00

05

10

15

20

25

30

35O

D 6

20



00

05

10

15

OD

620







Table5Antibacteria

lactivity

oftheK

LDS10

727andKL

DS10

373str

ains

againstfoo

dborne


term

sofZ

DI

Testfo

odbo

rnes

trains

ZDI(mm)plusmn

SDof

IndicatorB

acteria

Salm

onellatyph

imurium

Ecoli

Liste

riamon

ocytogenes

Stap

hylococcus

aureus

KLD

S10

727

237plusmn003

+++

197plusmn

003

+++

217plusmn009

+++

220plusmn010

+++

KLD

S10

373

247plusmn003

+++

193plusmn

007

+++

193plusmn

007

+++

220plusmn010

+++

Sig

lt000

104226

00198

00572

R210

0000

0090

4762

0978495

0970588

+++refersto

clearz

one(14mm3

points

9ndash14mm2

points

1ndash99

mmone

point)



100

150

200

250

300

350

Adhe

sion

()


(A)

(B) (C)


Figure 5







10

0

5

minus10

minus5

(S1)(STZ)

Glu

cose

leve

ls (w

)

(a)

10

15

20

25

0

5


Wei

ght (

g)

(b)










liver






pancreas


liver

kidney


arrow)Spleen

















4 Discussion





















5 Conclusions


Data Availability




Acknowledgments


References







































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Table3Sign

ificancemeansand

stand

arderrors(SE)


ofKL

DS10

727andKL

DS10

373str

ains

lowastSO

VExp

erim

ents

Growth

ratean

dpH

Biletolerance

Antibiotic

stoleran

ceHigh

Low

High

Low

High

Low

Strain

(S2)

(S1)

(S2)

(S1)

(S2)

(S1)

Meanplusmn

SEof

strains

459plusmn023

c445plusmn020

492plusmn010

a490plusmn010

533

b plusmn006

532plusmn006

Time

033

033

024

Meanplusmn

SEof

time

556plusmn005

c370plusmn001

581plusmn002

c411plusmn

007

574

c plusmn001

455plusmn014

Con

centratio

nof

bileor

type

ofan

tibiotic

--

20

Chloramph

enicol

Con

trol

Meanplusmn

SE-

-521plusmn010

c460plusmn016

572

c plusmn001

497plusmn014

R29996

9985

9992

CV

05

073

04

Seefoo

tnoteo

fTable2


Table4Sign

ificancemeansand

stand

arderrors(SE)


adhesio

nof

KLDS10

727andKL

DS10

373str

ains

lowastSO

VExp

erim

ents

Biletolerance

(SIJ)w

ithdiffe

rent

pHaerobically

(SIJ)w

ithdiffe

rent

pHan

aerobically

Antibiotic

resistan

ceAdh

esionto

Caco2

cell

High

Low

High

Low

High

Low

High

Low

High

Low

Strain

(S2)

(S1)

(S1)

(S2)

(S2)

(S1)

(S2)

(S1)

(S1)

(S2)

Meanplusmn

SEof

strains

197plusmn007

b17

8plusmn007

116plusmn011

b113plusmn

010

117plusmn009

c112plusmn

01

160plusmn010

c14

1plusmn009

213plusmn019

d211plusmn020

Time

39

024

024

06

Meanplusmn

SEof

time

210plusmn014

c15

2plusmn009

208plusmn010

c056plusmn001

212plusmn010

c052plusmn002

275plusmn002

c12

9plusmn014

Con

centratio

nof

bileor

type

ofan

tibiotic

03

27

27

2Str

Erth

35

Meanplusmn

SEof

concentration

217plusmn009

c16

6plusmn009

138plusmn016

c094plusmn009

134plusmn014

c096plusmn009

236plusmn014

c083plusmn009

268plusmn015

c148plusmn

012

R2866

9971

9964

9648

078

CV

1204

387

394

1391

152

Seefoo

tnoteo

fTable2


3 9 27 330Time (h)

3 9 27 330Time (h)

40

45

50

55

pH

05

10

15

20O

D 6

20

(a)



40

45

50

55

60

pH

00

05

10

15

OD

620







40

45

50

55

pH



00

05

10

15

20O

D 6

20



00

05

10

15

20

25

30

35O

D 6

20



00

05

10

15

OD

620







Table5Antibacteria

lactivity

oftheK

LDS10

727andKL

DS10

373str

ains

againstfoo

dborne


term

sofZ

DI

Testfo

odbo

rnes

trains

ZDI(mm)plusmn

SDof

IndicatorB

acteria

Salm

onellatyph

imurium

Ecoli

Liste

riamon

ocytogenes

Stap

hylococcus

aureus

KLD

S10

727

237plusmn003

+++

197plusmn

003

+++

217plusmn009

+++

220plusmn010

+++

KLD

S10

373

247plusmn003

+++

193plusmn

007

+++

193plusmn

007

+++

220plusmn010

+++

Sig

lt000

104226

00198

00572

R210

0000

0090

4762

0978495

0970588

+++refersto

clearz

one(14mm3

points

9ndash14mm2

points

1ndash99

mmone

point)



100

150

200

250

300

350

Adhe

sion

()


(A)

(B) (C)


Figure 5







10

0

5

minus10

minus5

(S1)(STZ)

Glu

cose

leve

ls (w

)

(a)

10

15

20

25

0

5


Wei

ght (

g)

(b)










liver






pancreas


liver

kidney


arrow)Spleen

















4 Discussion





















5 Conclusions


Data Availability




Acknowledgments


References







































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Table4Sign

ificancemeansand

stand

arderrors(SE)


adhesio

nof

KLDS10

727andKL

DS10

373str

ains

lowastSO

VExp

erim

ents

Biletolerance

(SIJ)w

ithdiffe

rent

pHaerobically

(SIJ)w

ithdiffe

rent

pHan

aerobically

Antibiotic

resistan

ceAdh

esionto

Caco2

cell

High

Low

High

Low

High

Low

High

Low

High

Low

Strain

(S2)

(S1)

(S1)

(S2)

(S2)

(S1)

(S2)

(S1)

(S1)

(S2)

Meanplusmn

SEof

strains

197plusmn007

b17

8plusmn007

116plusmn011

b113plusmn

010

117plusmn009

c112plusmn

01

160plusmn010

c14

1plusmn009

213plusmn019

d211plusmn020

Time

39

024

024

06

Meanplusmn

SEof

time

210plusmn014

c15

2plusmn009

208plusmn010

c056plusmn001

212plusmn010

c052plusmn002

275plusmn002

c12

9plusmn014

Con

centratio

nof

bileor

type

ofan

tibiotic

03

27

27

2Str

Erth

35

Meanplusmn

SEof

concentration

217plusmn009

c16

6plusmn009

138plusmn016

c094plusmn009

134plusmn014

c096plusmn009

236plusmn014

c083plusmn009

268plusmn015

c148plusmn

012

R2866

9971

9964

9648

078

CV

1204

387

394

1391

152

Seefoo

tnoteo

fTable2


3 9 27 330Time (h)

3 9 27 330Time (h)

40

45

50

55

pH

05

10

15

20O

D 6

20

(a)



40

45

50

55

60

pH

00

05

10

15

OD

620







40

45

50

55

pH



00

05

10

15

20O

D 6

20



00

05

10

15

20

25

30

35O

D 6

20



00

05

10

15

OD

620







Table5Antibacteria

lactivity

oftheK

LDS10

727andKL

DS10

373str

ains

againstfoo

dborne


term

sofZ

DI

Testfo

odbo

rnes

trains

ZDI(mm)plusmn

SDof

IndicatorB

acteria

Salm

onellatyph

imurium

Ecoli

Liste

riamon

ocytogenes

Stap

hylococcus

aureus

KLD

S10

727

237plusmn003

+++

197plusmn

003

+++

217plusmn009

+++

220plusmn010

+++

KLD

S10

373

247plusmn003

+++

193plusmn

007

+++

193plusmn

007

+++

220plusmn010

+++

Sig

lt000

104226

00198

00572

R210

0000

0090

4762

0978495

0970588

+++refersto

clearz

one(14mm3

points

9ndash14mm2

points

1ndash99

mmone

point)



100

150

200

250

300

350

Adhe

sion

()


(A)

(B) (C)


Figure 5







10

0

5

minus10

minus5

(S1)(STZ)

Glu

cose

leve

ls (w

)

(a)

10

15

20

25

0

5


Wei

ght (

g)

(b)










liver






pancreas


liver

kidney


arrow)Spleen

















4 Discussion





















5 Conclusions


Data Availability




Acknowledgments


References







































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



3 9 27 330Time (h)

3 9 27 330Time (h)

40

45

50

55

pH

05

10

15

20O

D 6

20

(a)



40

45

50

55

60

pH

00

05

10

15

OD

620







40

45

50

55

pH



00

05

10

15

20O

D 6

20



00

05

10

15

20

25

30

35O

D 6

20



00

05

10

15

OD

620







Table5Antibacteria

lactivity

oftheK

LDS10

727andKL

DS10

373str

ains

againstfoo

dborne


term

sofZ

DI

Testfo

odbo

rnes

trains

ZDI(mm)plusmn

SDof

IndicatorB

acteria

Salm

onellatyph

imurium

Ecoli

Liste

riamon

ocytogenes

Stap

hylococcus

aureus

KLD

S10

727

237plusmn003

+++

197plusmn

003

+++

217plusmn009

+++

220plusmn010

+++

KLD

S10

373

247plusmn003

+++

193plusmn

007

+++

193plusmn

007

+++

220plusmn010

+++

Sig

lt000

104226

00198

00572

R210

0000

0090

4762

0978495

0970588

+++refersto

clearz

one(14mm3

points

9ndash14mm2

points

1ndash99

mmone

point)



100

150

200

250

300

350

Adhe

sion

()


(A)

(B) (C)


Figure 5







10

0

5

minus10

minus5

(S1)(STZ)

Glu

cose

leve

ls (w

)

(a)

10

15

20

25

0

5


Wei

ght (

g)

(b)










liver






pancreas


liver

kidney


arrow)Spleen

















4 Discussion





















5 Conclusions


Data Availability




Acknowledgments


References







































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



40

45

50

55

pH



00

05

10

15

20O

D 6

20



00

05

10

15

20

25

30

35O

D 6

20



00

05

10

15

OD

620







Table5Antibacteria

lactivity

oftheK

LDS10

727andKL

DS10

373str

ains

againstfoo

dborne


term

sofZ

DI

Testfo

odbo

rnes

trains

ZDI(mm)plusmn

SDof

IndicatorB

acteria

Salm

onellatyph

imurium

Ecoli

Liste

riamon

ocytogenes

Stap

hylococcus

aureus

KLD

S10

727

237plusmn003

+++

197plusmn

003

+++

217plusmn009

+++

220plusmn010

+++

KLD

S10

373

247plusmn003

+++

193plusmn

007

+++

193plusmn

007

+++

220plusmn010

+++

Sig

lt000

104226

00198

00572

R210

0000

0090

4762

0978495

0970588

+++refersto

clearz

one(14mm3

points

9ndash14mm2

points

1ndash99

mmone

point)



100

150

200

250

300

350

Adhe

sion

()


(A)

(B) (C)


Figure 5







10

0

5

minus10

minus5

(S1)(STZ)

Glu

cose

leve

ls (w

)

(a)

10

15

20

25

0

5


Wei

ght (

g)

(b)










liver






pancreas


liver

kidney


arrow)Spleen

















4 Discussion





















5 Conclusions


Data Availability




Acknowledgments


References







































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Table5Antibacteria

lactivity

oftheK

LDS10

727andKL

DS10

373str

ains

againstfoo

dborne


term

sofZ

DI

Testfo

odbo

rnes

trains

ZDI(mm)plusmn

SDof

IndicatorB

acteria

Salm

onellatyph

imurium

Ecoli

Liste

riamon

ocytogenes

Stap

hylococcus

aureus

KLD

S10

727

237plusmn003

+++

197plusmn

003

+++

217plusmn009

+++

220plusmn010

+++

KLD

S10

373

247plusmn003

+++

193plusmn

007

+++

193plusmn

007

+++

220plusmn010

+++

Sig

lt000

104226

00198

00572

R210

0000

0090

4762

0978495

0970588

+++refersto

clearz

one(14mm3

points

9ndash14mm2

points

1ndash99

mmone

point)



100

150

200

250

300

350

Adhe

sion

()


(A)

(B) (C)


Figure 5







10

0

5

minus10

minus5

(S1)(STZ)

Glu

cose

leve

ls (w

)

(a)

10

15

20

25

0

5


Wei

ght (

g)

(b)










liver






pancreas


liver

kidney


arrow)Spleen

















4 Discussion





















5 Conclusions


Data Availability




Acknowledgments


References







































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




100

150

200

250

300

350

Adhe

sion

()


(A)

(B) (C)


Figure 5







10

0

5

minus10

minus5

(S1)(STZ)

Glu

cose

leve

ls (w

)

(a)

10

15

20

25

0

5


Wei

ght (

g)

(b)










liver






pancreas


liver

kidney


arrow)Spleen

















4 Discussion





















5 Conclusions


Data Availability




Acknowledgments


References







































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




10

0

5

minus10

minus5

(S1)(STZ)

Glu

cose

leve

ls (w

)

(a)

10

15

20

25

0

5


Wei

ght (

g)

(b)










liver






pancreas


liver

kidney


arrow)Spleen

















4 Discussion





















5 Conclusions


Data Availability




Acknowledgments


References







































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



liver






pancreas


liver

kidney


arrow)Spleen

















4 Discussion





















5 Conclusions


Data Availability




Acknowledgments


References







































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018















4 Discussion





















5 Conclusions


Data Availability




Acknowledgments


References







































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018

















5 Conclusions


Data Availability




Acknowledgments


References







































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018






5 Conclusions


Data Availability




Acknowledgments


References







































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


























Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


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