SE544508C2 - Lactobacillus plantarum strains - Google Patents

Abstract

The present invention relates to a probiotic bacterial Lactobacillus plantarum strain isolated from quinoa grains, said strain being selected from the group consisting of Lactobacillus plantarum ChB11 having accession number LMG P-31891, Lactobacillus plantarum ChG33 having accession number LMG P-31892, Lactobacillus plantarum ChR228 having accession number LMG P-31893 and Lactobacillus plantarum ChJ239 having accession number LMG P-31894. The invention relates to composition comprising such a strain and to a method for preparation of a food composition such as a beverage.

Description

LACTOBACILL US PLANTARUM STRAI NS Technical field of the invention The present invention relates to probiotic bacterial Lactobacillus p/antarumstrains isolated from quinoa, said strains being selected from Lactobacillusp/antarum ChB11 (LIVIG P-31891), Lactobacillus p/antarum ChG33 (LIVIG P-31892), Lactobacillus p/antarum ChFï228 (LIVIG P-31893) and Lactobacillusp/antarum ChJ239 (LIVIG P-31894). The invention also relates to acomposition such as a food or feed composition comprising said strains andto a method for preparing a food composition such as quinoa milk by usingsaid strains.

Background Art To the Bolivian indigenous people Quinoa (Chenopodium quinoa Willd.) hasbeen one of the most important crops for thousands of years. During the lasttwo decades quinoa has been playing an increasing role in human dietsworldwide. Quinoa is cultivated in the agricultural part of the vast Andeanregion in Bolivia, located 4000 meters above the sea level. The Andeanregion has harsh environmental conditions including intense UV radiation,seasonal drought and occasional frost. Consumption of seeds is the mostcommon use of quinoa and they have a high nutritional value containing,several vitamins, minerals, oils, high content of proteins, starch, and essentialfree amino acids. ln addition quinoa seeds are potential source of phenoliccompounds with antioxidative capacity. Because of all the health's benefitsthat quinoa has, this pseudo cereal is being consumed by the Europeanpopulation including people suffering from celiac disease.

Lactobacillus p/antarum and Lactobacillus brevis have been reported asdominant species bacteria isolated from spontaneous fermented quinoasourdough in Argentina, the quinoa grain was imported from Bolivia.However, it should be necessary to considering that on spontaneousfermentation the naturally proliferation of native bacteria is an unpredictableprocess, conditioned by the growth of the dominated genus present in thesubstrate. lnduced fermentation by Lactobacillus p/antarum CRL 778 was used for ferment quinoa slurries, increasing the bioavailability of the nutrients.The fermented quinoa slurry can be used in the bakery industry. The contentof probiotic bacteria on spontaneous or induced fermentation depends on thepH of the medium, and acidity in the substrate. Generally the pH should belower than 4 to guarantee that no pathogenic bacteria are present, and theacidity is reported as a function of percentage of |actic acid synthesized by the bacteria present in the substrate. ln 2015 a research article entitled "Biodiversity and technological-functionalpotential of Lactic acid bacteria isolated from spontaneously fermentedquinoa sourdough" by L. Ruiz Rodriguez et. al. was published in the Journalof Applied l\llicrobiology. L. Ruiz Rodriguez et. al. used back slopping for thefermentation process and isolated Lactobacillus plantarum, LactobacillusThe methodology used by the group can however change the microbiology brevis and Pediococcus pentosaceous from the quinoa grains. environment, since is not possible to ensure which species, strains and/orquantity of microorganism had been transferred. This mechanism cantherefore be interpreted as induced fermentation instead of spontaneousfermentation. The study supports the presence of |actic acid bacteria (LAB)and identified isolated species at genotypical level but not at phenotypicallevel.

Lactic acid bacteria are considered as GRAS (generally regarded as safe)and the microorganisms are commonly used by the Food and Pharmaceuticallndustry as starter cultures for fermentations and as probiotics.

The genus Lactobacillus consist of different species and subspecies, with acatalogue of 90 different Lactobacillus spp. so far sufficiently characterized.To simplify the catalogue of Lactobacillus spp. these have been divided intothree different subcategories depending on their fermentation abilities. Thefacultative heterofermentative (Group ll) subcategory has the ability toferment hexoses into |actic acid and in addition to this, pentoses and/orgluconate can also be fermented. Strains of the species Lactobacillusplantarum belong to either one of the two remaining subcategories: Obligate homofermentative (Group l) which can only ferment hexoses into |actic acidand obligate heterofermentative (Group lll) which ferments hexoses intolactic acid, acetic acid and/or ethanol and carbon dioxide. ln many ways Lactobacillus plantarum is separated from other Lactobacillusspp., e.g. in the unusual high tolerance towards environmental stress that thespecies displays when surviving the passage through the low pH environmentof the human stomach.

Lactobacillus plantarum is thought to have a higher adaptive ability towards avariety of different conditions than other Lactobacillus spp., something thatcan be credited its unusually large genome. Also, high levels of intracellularmanganese reducing oxygen free radicals to hydrogen peroxide andeventually oxygen and water make Lactobacillus plantarum more resistanttowards oxygen poisoning than other Lactobacillus spp. Lactobacillusplantarum has furthermore the ability to ferment a wide variety of differentcarbohydrates and is frequently found in the human Gl-tract. The observationis unsurprising since it is found and spontaneously multiply in most lactic acidfermented foods, with one example being sourdough. Adult humans acquireimmune system tolerance towards harmless, food-associated bacteria,revealing a close relationship between human immune reactions andconsumption of Lactobacillus plantarum. Strains belonging to the speciesLactobacillus plantarum is also generally regarded as safe for consumptionand the species does not contain any pathogenic strains.

There is a need on the planet for continuous development of healthy,vegetable and/or vegan alternative foods. For instance, the dietary vegeterianand vegan population is steadily growing throughout the world and there is agrowing demand to reduce the consumption of dairy and animal products.The European population including people suffering from celiac disease isalso increasing and demands new alternatives. Therefore, there is need tofind new healthy alternatives to this part of the growing population, which areboth healthy and have a pleasant taste.

Summary of the invention The present invention relates in one aspect to a probiotic bacterialLactobacillus plantarum strain isolated from quionoa grains selected from thegroup consisting of Lactobacillus plantarum ChB11 having accession numberLIVIG P-31891, Lactobacillus plantarum ChG33 having accession numberLIVIG P-31892, Lactobacillus plantarum ChF¶228 having accession numberLIVIG P-31893 and Lactobacillus plantarum ChJ239 having accession numberLIVIG P-31894.ln another aspect, the present invention relates to a composition or a foodcompositions comprising a probiotic bacterial Lactobacillus plantarum strainselected from the group consisting of Lactobacillus plantarum ChB11 havingaccession number LIVIG P-31891, Lactobacillus plantarum ChG33 havingaccession number LIVIG P-31892, Lactobacillus plantarum ChF¶228 havingaccession number LIVIG P-31893 and Lactobacillus plantarum ChJ239 havingaccession number LIVIG P-31894.ln another aspect, the present invention relates to a method for thepreparation of a quinoa milk comprising the steps:a) toasting quinoa grains;b) adding water to the toasted grains;c) mixing the water and the grains with a blender and filtering the mixture;andd) inoculation the mixture with at least one probiotic strain selected fromLactobacillus p|antrum ChB11 having accession number LIVIG P-31891, Lactoacillus plantarum ChG33 having accession number LIVIGP-31892, Lactobacillus plantarum Ch R228 having accession numberLIVIG P-31893 and Lactobacillus plantarum ChJ239 having accessionnumber LIVIG P-31894 at 30 °C for 48 h in anaerobic conditions.Description of the figures The microorganisms were isolated from Violet Red Bile Dextrose agar (red), Figure 1. I\llicrobiota characterization of quinoa sourdough.Tryptic Soy agar (orange), I\llalt Extract agar (green) and Fïogosa agar (blue).Definitions The term "probiotic strain" as used herein means a live microorganism (hereinLactobacillus plantarum stain) that provides health benefits when consumed by an individual, usually within the gastrointestinal tract.The term "cfu" means colony forming units and is a generally Lesed Lenit in microbiologyto estimate the number of viablebacteria cells in a. sample. The number of cfu En a fermented drink Es usually above 108 CPU.Detailed description of the invention As described above the present invention relates to a probiotic bacteriaiLactobacillus p/antarum strain selected from the group consisting ofLactobacillus p/antarum ChB11 having accession number LIVIG P-31891,Lactobacillus p/antarum ChG33 having accession number LIVIG P-31892,Lactobacillus p/antarum ChF¶228 having accession number LIVIG P-and Lactobacillus p/antarum ChJ239 having accession number LIVIG P-31894, wherein said strains have been isolated from quinoa. There is needfor alternative healthy products to the growing population of vegeterians andvegans. ln addition, the population with celiac disease also requires new andhealthy alternatives.

Based on the analyzes of the results as have been performed herein, it isconcluded that the 4 Lactobacillus p/antarum strains showed to have potentialto be used as probiotic based on their metabolic scope, the ability tocounteract potential pathogenic microorganisms and because Lactobacillusspp. are considered as safe bacteria. Additionally, the most remarkable factwas that two strains able to ferment Xylose are Lactobacillus p/antarum.Those were named as Lactobacillus p/antarum ChB11 and Lactobacillusp/antarum ChG33. Besides, the other two strains identified as Lactobacillusp/antarum ChJ239 and Lactobacillus p/antarum ChF¶228 are different fromeach other.

The Lactobacillus p/antarum strains isolated according to the presentinvention have been identified at genotypical and phenotypical level. lt hasbeen demonstrated that two of the Lactobacillus p/antarum strains(Lactobacillus p/antarum strains ChB11and Lactobacillus p/antarum ChG33)are capable to ferment xylose, a pentose carbohydrate that is otherwiseexclusively metabolized by Lactobacillus pentosus. The enzymatic action overxylose is used as a strong test to distinguish Lactobacillus pentosus from therest of the Lactobacillus spp.A composition comprising at least one bacterial strain is also provided herein,for instance a food composition comprising at least one bacterial strain is alsoprovided. With the growing demand for vegeterian alternatives a foodcomposition compring at least one of the Lactobacillus p/antarum strains asdisclosed herein may be provided. Said food composition may be a fermentedfood composition such as a fermented vegetable-based beverage, functionalfood, food additive, dietary supplement, or nutritional product. Said fermentedfood composition may be fermented with at least one of the strains selectedfrom the group Lactobacillus p/antarum ChB11 having accession numberLIVIG P-31891, Lactobacillus p/antarum ChG33 having accession numberLIVIG P-31892, Lactobacillus p/antarum ChF¶228 having accession numberLIVIG P-31893 and Lactobacillus p/antarum ChJ239 having accession numberLIVIG P-Said fermented vegetable-based beverage may for instance be afermented quinoa milk. lt is understood that the Lactobacillus p/antarumstrains as disclosed herein may be used for fermenting any other vegetarianbeverage based on any other vegetables such as amaranth, rice, oat orwheat. Since two of the isolated strains have the capability of fermentingxylose, it would particularly suitable to ferment vegetables crops, eg quinoa,containing contents of xylose which otherwise may be difficult to ferment byother strains of Lactobacillus p/antarum. Thus, healthy fermented productscontaining nutrients released during fermentation may be provided. lt hasbeen shown in the experimental part herein that Lactobacillus plantaruntChBt t, Lactobacillus plantarum Chiíšíšß, Lactobacillus plantarurn Chti239showed high inhibition against potential pathogenic bacteria ensuring that thefinal fermented quinoa milk was free of those microorganisnt after 48 hours offermentation. Besides, the amount ot i.. piantanim rernained stable in qtiantityduring storage tirne. ln comparison to the fermentation of quinoa milk with theoornmercialiy available Lactobacillus plantarumâšäêv strain, thernicroorganisrn Ešnterocoooiis inudtii was still present in a oonsiderableamount cornpromising the hygiene quality oi the linal otiiona rniik product.The Lactobacillus strains Chßt t, ChGEB, Ghtlâêåi and Chlïš 228 totally inhibitthe growth of Enteroccccus rnudtii safeguarding the hygiene quaiity of theforrtiuiated fermented quinoa miik.

Said composition or food composition as disclosed herein may comprisesaid at least one strain present in the composition in an amount of from about1x1O6 CFU/day to about 1x 1014 CFU/day, for instance 1x1O8 CFU/day toabout 1x 1012 CFU/day, e.g. about 1x1O9 CFU/day, 1x1O1° CFU/day or 1x1O11CFU/day. The at least one strain may be present in the composition asattenuated, inactivated, alive or dead.

A feed composition is also provided herein, which composition is suitablefor animals to consume. For instance, at least one of the two bacterial strainsLactobacillus plantarum strains ChB11and Lactobacillus plantarum ChG33which are capable to ferment xylose would be suitable to add to any xylosecontaining cereal or vegetable. The strains could ferment such xylosecontaining cereal or vegetable to metabolize the xylose present in the cerealor vegetables.

The present invention also relates to a method for the preparation of aquinoa milk comprising the steps: a) toasting quinoa grains, for instance in an oven or on a stove at above145 °C;b) adding water to the toasted grains in a proportion of about 1:8;c) mixing the water and the grains with a blender to provide a mixture andfiltering the mixture; andd) inoculation the mixture with at least one probiotic strain selected fromLactobacillus plantrum ChB11 having accession number LIVIG P-31891, Lactoacillus plantarum ChG33 having accession number LIVIGP-31892, Lactobacillus plantarum Ch R228 having accession numberLIVIG P-31893 and Lactobacillus plantarum ChJ239 having accessionnumber LIVIG P-31894 at 30 °C for 48 h in anaerobic conditions.A benefit of the method of the preparation of the milk is that there is no needto add any sugar such as carbohydrates to the mixture. There already iscarbohydrates available from the avaiable quiona grains. ln addition, stabilizers are not needed to add either. ln addition, the quinoa milk asprepared has the advantages of providing a good taste, i.e. the previous bittertaste has been reduced. This is because eg that saponins are removed by thewashing step. The toasting step also assists in providing a good taste.Deposited strains All strains were deposited at BCCl\ll/Ll\llG (Beigian Coordinated Collections ofl\llicro-organisms/Laboratorium voor l\llicrobiologie, Universiteit Gent (UGent)),Gent, Belgium on July 2nd 2020. The depositor is Äsa Håkansson. Theaccession numbers are as follows; Lactobacillus p/antarum ChB11 havingaccession number Ll\llG P-31891 ; Lactobacillus p/antarum ChG33 havingaccession number Ll\llG P-31892; Lactobacillus p/antarum ChF¶228 havingaccession number Ll\llG P-31893; Lactobacillus p/antarum ChJ239 havingaccession number Ll\llG P-Other objectives, features and advantages of the present invention willappear from the following detailed disclosure, from the attached claims, aswell as from the figures. lt is noted that the invention relates to all possiblecombination of features.

Generally, all terms used in the claims are to be interpreted accordingto their ordinary meaning in the technical field, unless explicitly definedotherwise herein. All references to "a/an/the [element, device, component,means, step, etc.]" are to be interpreted openly as referring to at least oneinstance of said element, device, component, means, step, etc., unlessexplicitly stated otherwise. The steps of any method disclosed herein do nothave to be performed in the exact order disclosed, unless explicitly stated.

As used herein, the term "comprising" and variations of that term arenot intended to exclude other additives, components, integers or steps.Experimental partAn aim of the experimental part is to report the isolation and identification of 4autochthonous Lactobacillus p/antarum strains from the Bolivian white quinoagrains. The method used were planned and applicated in order to distinguishbetween Lactobacillus p/antarum and Lactobacillus pentosus, two closelyrelated strains. l\llethods such as, polymorphic chain reaction (PCR), geneticsequencing by Eurofins Genomics in Germany, and edition of the resultsusing Sequence Alignment Editor (BioEdit) was used to identify the strains.Fïandomly applied polymorphic DNA (RAPD) was applied for identification atgenotypical level, PCR reactions with specific primers for identification ofLactobacillus pentosus were tested, API 50CHL was used to identify thebacteria at phenotypical level besides the tannase activity test and theidentification of phenolic compounds released from the quinoa grains afterfermentation. The interpretation of the results were analyzed and used toconclude the identity between Lactobacillus species.

Examples By way of examples, and not limitation, the following examples identifyembodiments of the present invention. l\/laterials and l\/lethods Quinoa sourdouoh preparation Six samples were prepared mixing the quinoa grains with demineralizedwater (1 :2.5 v/v) using a blender (Electrolux, Great blending TruFlowTl\/lblades, ESB5400BK) to obtain a homogenous semiliquid dough distributedinto a 500 mL glass flask (Reagent bottle with Screw Cap, clear). Thepresence of oxygen was minimized by filling the glass flasks until maximumcapacity and sealing them hermetically. The incubation was set up at 30°C for8 days. Samples were withdrawn daily, and the pH was monitored at thesame time using a l\/letrohm 744 pHmeter (l\/letrohm Ltd, Herisau,Switzerland), previously calibrated according to the manufacturerrecommendations. The demineralized water and the glass bottles wereautoclaved at 121 °C for 15 min and stored at 4°C. The blender was washedwith tap water and rinsed thoroughly with ethanol 70%, before to be used. l\/licrobiota characterization bv plate count The samples were collected, promptly tenfold diluted and plated by duplicate.ln general, 10 grams, per bottle, were withdrawn every day and transferredinto 90 mL sterile bacteriological peptone water and homogenized by vortex.Serial dilutions were made and obtained samples were plated (100 uL) on:Fiogosa agar (Oxoid) incubated anaerobically (Gas Pack Anaerobic system,BBI, Becton Dickinson and company, USA) at 37°C for 72 h for Lactobacillaceae, Violet Red Bile Dextrose Agar (VRBD, Merck, Germany)was incubated aerobically at 37°C for 24 h for Enterobacteriaceae, TrypticSoy Agar (TSA, Fluka, Missouri, USA) was incubated aerobically at 30°C for72 h for total aerobic count, and Malt Soy Agar (Sigma Aldrich, lndia) wasincubated at 26°C for 7 d for yeast and fungus. The number of viable cellswere recorded, and from the appropriated dilution (20 2 200), ten colonieswere randomly picked each day. The cells were streaked onto clean platesand incubated following the procedure previously explained for each agar.Following purification, the isolates were re-cultured into 5 mL Man Fïogosaand Sharpe Broth (MFïS-Broth, Merck, Germany) and incubated at 37°Covernight (max. 18 h) for the multiplication of Lactobacillaceae. The viablecells from TSA and VRBD agar were re-cultured in Tryptic Soy broth (TSB,Sigma Aldrich, India) incubated at 30°C for 48 h, finally Malt Broth (Fluka,Germany) was incubated at 26°C for 5 d. The cells were washed twice with aO.85% sterile NaCl solution (8263 > glycerol) at -80°C. (Fåket al., 2012).

Type strains Three type strains used as reference: Lactobacillus pentosus CCUG 33455T(also register as ATCC 8041 by the American Type Culture Collection),Lactobacillus paraplantarum CCUG 35983T, and Lactobacillus p/antarumATCC 14917 (also register as CCUG 30503T) were purchased from theCulture Collection University of Gothenburg, Sweden.

DNA extraction Fresh cells multiplied in broth were used to extract the DNA applying theglass bead beating method. Briefly: 200 uL was transferred into 1.5 mL tubesand centrifuged at 20.8> beads (0.2mm diameter) were added. The cell wall was broken using anEppendorf Mixer 5432 (Eppendorf, Hamburg, Germany) for 45 min at 4°C.The DNA was separated from the pellet by centrifugation (20.8> The 168 rRNA genes were amplified using ENV1 (5"- AGA GTT TGA TllTGG CTG AG -3", Escherichia Coli, 8-27 bp) and ENV2 (5'- CGG ITA CCTTGT TAC GAC TT -3", Escherichia coli, 1511-1492 bp) as forward andreverse primers. A total volume of 25 uL containing 2.5 uL template DNA and22.5 uL of PCR master mix (0.2 ml\/I of both primers, 2.5 uL of 168 PCRreaction buffer with 1.5 ml\/I l\/lgCl2 [Roche Diagnostic GmbH, Mannheim,Germany], 200 ml\/I of each deoxyribonucleotide triphosphate [dNTP, Qiagen,Germany], and 2.5 U of Taq DNA polymerase [Roche Diagnostic, l\/lannheim,Germany] and nuclease free water [Promega, 8weden] to completed the finalvolume). Amplification was achieved in a PCR l\/lastercycle 5333 (Eppendorf)according to the following profile: incubation at 94°C for 3 min, denaturation at94 °C for 60 s; annealing temperature at 50 °C for 45 s and elongation at 72°Cfor 120 s for 1 cycle. A total of 30 cycles were performed with an extensionstep at 72°C for 10 min, thereafter, cooled down to 4°C. To confirm theamplification products, 1.5 uL of PCR product mixed with 1 uL dye (6XOrange DNA Loading Dye) were gel electrophoresed on 15%, (w/v) agarosegel (8igma) in TAE buffer (50X, VWR Chemicals, U8A, pH 8.3) using asmolecular weight markers 100 bp (GelPilot 100bp Plus Ladder, Qiagen). Thegels were stained on GelRedTl\/l bath (30 uL [Biotium, U8A] dissolved in 100mL distilled water) for 15 min. The DNA bands were observed through UVchamber (Transilluminator UVP, U8A). The PCR products were sequenced atEurofins Genomics (Ebersberg, Germany) on an ABl 3130xl Genetic analyser(Applied biosystems, Foster City. CA, U8A) using ENV1 as sequencingprimer. The sequenced genes were edited by BioEdit 8equence AlignmentEditor (l\/lichigan 8tate University, U8A) (Hall, 1999) and the resulted geneswere submitted to RDP data base (http://rdp.cme.msu.edu.) in order to obtainthe identity of the microorganism confirmed by the percentage of similarity.Randomlv Amplified Polvmorphic DNA (RAPD) P73 (ACGCGCCCT) containing 80% of G+C was chosen as primer and waspreviously described by Johansson and Quednau. A total volume of 50 uLsolution composed by 2 uL templated DNA and 48 uL of master mix (0.2 ml\/Iof each deoxyribonucleotide triphosphate [dNTP, Qiagen, Germany], 2.5 uLreaction buffer with 1.5 ml\/I l\/lgCl2 [Roche Diagnostic GmbH, l\/lannheim,Germany], 0.8 ml\/l primer [Eurofins Genomics, Germany] and 2.5 U of TaqDNA polymerase [Roche Diagnostic, l\/lannheim, Germany] and nuclease freewater [Promega, Sweden]) were centrifuged and covered with mineral oil.Four cycles at the temperature profile: 94 °C, 45 s; 30 °C, 120 s; 72 °C, 60s;followed by 26 cycles at 94 °C, 5s; 36 °C, 30s; 72 °C, 30s. The extension stepwas at 75 °C for 10 min and cooled down to 4 °C. The amplified productswere electrophoresed on 1.5 °/> (w/v) agarose gel using DNA l\/lolecularWeight l\/larker Vi 0.15-2.1 kbp (Roche Diagnostic Gl\/lbH l\/lannhem,Germany) as ladder. The gels were photographed using Panasonic DMC-LX100 camera, and the pictures were transferred to the computer byPanasonic imagen app.

PCR specific primers for Lactobacillus pentosus spp. gene amplification Two pair primers specific to distinguish Lactobacillus pentosus were usedseparately on a final volume of 25 uL master mix containing 2.5 uL oftemplate DNA and 22.5uL PCR master mix (0.25 ml\/I of each primer [Eurofinsgenomic], 2.5 uL of 168 PCR reaction buffer with 1.5 ml\/l l\/lgCl2 [RocheDiagnostic, l\/lannheim, Germany], 1,5 uL of l\/lgCl2 [25ml\/l, QiagenGermany], 200 ml\/I of each deoxyribonucleotide triphosphate [dNTP, Qiagen,Germany], 2.5 U of Taq DNA polymerase [Roche Diagnostic, l\/lannheim,Germany] and nuclease free water to complete the final volume). The recAgene was amplified according to the method developed by Torriani et al.,using the primers pentF (5"- CAG TGG CGC GGT TGA TAT C -3", forward)and pREV (5"- TCG GGA TTA CCA AAC ATC AC - 3", reverse) and atemperature gradient settled in a PCR l\/lastercycler 5333 (Eppendorf),described as follows: incubation at 94 °C for 3 min, denaturation at 94 °C, 30s; annealing at 56 °C, 10 s; and elongation at 72 °C, 30s; final extension at°C for 5 min during 30 cycles and cooled down to 4 °C (Torriani and Felis,2001). The 168 rRNA region was amplified using the base pair primer 168(5"- GCT GGA ATC ACC TCC TTT C - 3', forward) and Lpe (5'- GTA TTCAAC TTA TTA CAA CG - 3", reverse) designed by Berthier et al. and, thePCR reaction was settled in a PCR l\/lastercycler gradient (Eppendorf) during30 cycles starting with an incubation at 94 °C, 5 min; denaturation at 94 °C, 1min; annealing at 53 °C, 1 min; and eiongation at 72 °C, 1 min and cooleddown to 4 °C (Berthier and Ehrlich, 1998). The amplified regions wereobserved on 2% (w/v) using 100bp plus molecular weight. The gels werestained on GelRedTlVl bath for 15 min, as previously described on section3.5., and the gels were photographed as described on section 3.6.Taxonomic identification usinq API 50 CHL Fermentation Assav The capacity of Lactobacillus spp., strains to ferment different carbohydrateswas evaluated using API 50CH strips and API 50CHL medium as inoculum(API System, biol\/lérieux lVlarcy-FEtoile, France) according to themanufacturer instructions. Overnight cultures of lactobacilli isolates weregrown in 10 ml IVIRS broth at 37oC. The multiplied cells were washed withsterile physiological saline solution (0.9 °/> w/v of NaCl) and centrifuged(20.8xg, 5 min), and the pellets were suspended on API 50 CHL medium andvortexed prior to transferring the mixture to the 50 wells on the API 50CHstrips (the well 0 served as a control). To generate anaerobic conditions allwells were overlaid with sterile paraffin oil (Merck, KGaA, Germany) and toenhance humidity the strips were moistened with water, previouslyautoclaved, covered and incubated at 37°C (Boyd et al., 2005). Changes incolor from violet to yellow (positive) or blue (negative) were monitored after 1,2 and 7 days. The results were graded as complete change to yellow by 1 orblue by 0. The well 25 (Esculin Ferric Citrate) is considered as positive if turnsblack. The results were analyzed using APIWEBTM(https://apiweb.biomerieux.com).

Lactobacillus spp., tannase enzvmatic capacity The capacity to degrade tannins and decarboxylate gallic acid was evaluatedbased on the colorimetric method developed by Osawa et al. (Osawa et al.,2000) with minor"s modifications described below. The colors of the solutionswere judged visually and confirmed spectrophotometrically on the UV/VISrange. The reactions were conducted in darkness and negative controlscontaining all the elements, but the bacterium was added to the analysis.lvlethiloallate dedradation The cells were re-culture on Rogosa agar (anaerobically, 37 °C, 72 h). Thefresh cells were harvested using a 10 uL loop and transferred into 1 mLnutrient solution with pH = 4.6 (33 l\/lm/L of NaH2PO4 [l\/lerck, Darmstadt, F.Ft., Germany] and 20ml\/I/L of methilgallate [Sigma Aldrich, USA]) andincubated at 37 °C for 24 h. Aften/vards, the samples were alkalinized adding1 mL of a saturated solution at pH 8.6 of NaHCO3 (Merck, Darmstadt,Germany) and expos oxygen for 1 h at room temperature. The color wasvisually evaluated before and after alkalinisation. Additionally, the maximumabsorbance at 440nm using a microplate reader (SPECTROstartNano, BMGLABTECH, Germany) was measured by triplicate using 360 uL of sample perwell. The color of the solution of the negative control was uncolored.

Gallic acid decarboxvlation From the freezing media, 50 uL were transferred into 5 mL of IVIRS Broth andincubated overnight (12-18 h, 37 °C). After cell multiplication, the tubes werevortexed and 50 uL were transferred into 10 ml\/I/L final concentration of gallicacid (Sigma Aldrich, 3,4,5-Trihydroxybenzoic acid monohydrated) in 10 mL ofMRS broth and incubated at 37°C for 72 h. Thereafter, the tubes werevortexed, and 2 mL aliquot were alkalinized with equal amount of saturatedsolution of NaCHO3 (pH 8.57). The samples were incubated at 37°C for 1 haerobically. The change in color to orange or brown were judged as positivereaction otherwise the solution became dark blue or dark green, the same asthe negative control.

Statistical calculations SigmaPlot version 14.0 (SYSTAT Software, Point Richmond, USA) was usedfor the statistical analysis. The number of viable count and pH betweensamples were evaluated by Kruskal Wallis One Way Analysis of Variance(ANOVA) on Ranks or a l\/lann-Whitney Ranks Sum test when required.

Results are presented as median and interquartile range and p-values s 0.05were considered significant.

Results Quinoa sourdough preparation The appearance of the liquid sourdough did not present sedimentation but incontrary the viscosity of the mixture increased. Resistance to flow wasobserved compared to before fermentation. The pH decreased from 6.20(6.19 - 6.22) to 4.34 (4.21 - 4.36) after the first 24 h of incubation and this isthe first change on the pH values statistically significant (p = 0.002).Thereafter, a continuous decrease without a significant statistical differencewas shown until the fourth day (pH = 4.06; 4.04 - 4.08) and fifth day (pH =3.98; 3.93 - 4.18) were the pH dropped down slightly below 4, becomingevident a second interval of time were the change on the pH was statisticallysignificant (p = 0.002). Consecutively, the upcoming days the pH reminedstatistically stable. See tablel\/licrobiota characterization bv plate count The samples were incubated at the same time and samples were withdrawnevery 24 hours and plated on Rogosa, TSA and VRBD agar immediately afterbeing tenfold diluted. The initial pH was 6.42 and decreased to 4.10 after 192hours (8 days). No presence of mold or yeast growing over the quinoa grainwas observed. The odor of the samples can be described as bitter and thecolor of the medium changed from uncolored to light yellow. Theautochthonous microorganisms increased in amount while the pH decreasedduring the time elapses. Before fermentation the number ofEnterobacteriaceae and Lactobacillaceae were below the limit of detection (<1). Nevertheless, the number on total aerobic count was 6.09 (6.04-6.17) logCFU/g, and for yeast and mold was 4.55 (4.39-4.60) log CFU/g. The colonieswere diverse in shape, size, and color, varying between white and yellow.After 24 hours of fermentation a sharp increase of the number of viable cellswere registered for all the agars compared with time zero. The number ofviable cells of Enterobacteriaceae was 7.24 (7.13-7.34) log of CFU/g, totalaerobic count was 9.35 (9.24 -9.40) log CFU/g, Lactobacillaceae was 6.(6.05-6.30) log CFU/g, and for yeast and mold it was 9.22 (9.08-9.44) logCFU/g. Through the fermentation time, the native microbiota communitycontinued to increase and changed gradually. lt became noticeable, that onTryptic soy agar and l\/lalt agar there was a loss of the microbial diversity. Theshape, size, and color of the colonies were mostly homogeneous.Furthermore, an equilibrium point between the number of log CFU/g onTryptic soy agar, Rogosa and l\/lalt agar was registered after 96 hours offermentation with p = lVlalt-Rogosa = 0.987; p = l\/lalt-TSA = 0.908; p = TSA -Fïogosa = 0.908, and no statistical difference was found between the threepairs pursued for a dominance presence by the Lactobacillaceae family at thesame time as a decreased number of VRBD viable cells was registered.Enterobacteriaceae became undetectable (below the limit of detection) after120 hours of fermentation, 0.00 (0.00-1 _23) log CFU/g (table 1).

Table 1. Daily progression of pH and microorganisms through the incubationtime of quinoa semiliquid sourdough Viable count CFU/g Time pH Enterobacteriaceae Total Lactobacillaceae Yeast and in Aerobic mold hours Count l\/ledian l\/ledian l\/ledian l\/ledian l\/ledian 0 6.20 <1 - 7.24 6.09 - 9.35 0.00 - 6.22 4.55 -9.24 4.34 7.24-5.19 9.35-9.10 6.22-8.12 9.22-9.48 4.21 5.19-5.05 9.10-8.97 8.12-9.02 9.12-8.72 4.17 5.05-3.13 8.97-8.94 9.02-8.93 8.86-8.96 4.06 3.13-<1 8.94-9.04 8.93-9.11 8.90-9.120 3.98 <1 - <1 9.04 - 9.04 9.11 - 9.24 9.08 -9.144 3.91 <1 - <1 9.04-9.21 9.24-9.33 9.04- 192 3.94 8.Detection limit A total of 1400 viable cells were isolated and divided as 420 isolates fromTryptic Soy agar, l\llalt Extract agar and Rogosa and 240 viable cells isolatedfrom Violet Red Bile Dextrose agar. The percentage of similarity to considerthe identity of the microorganism was between 0.999 - 1 percent forLactobacillaceae and between 0.997 - 1 percent for the rest of themicroorganisms. The presence of the same bacterial strains was found onmore than one agar and their detection depended on time and sampledilution. For example, Stenotropomonas maltophilia was isolated from VioletRed Bile Dextrose agar for a period of four consecutive days while from l\llaltextract agar they were detectable for two days. However, a correlation of timeoccurred between the first 24 and 48 hours of fermentation for Violet Red BileDextrose agar and l\llalt Extract agar. Violet Red Bile Dextrose agar platedsamples were less diluted compared to l\llalt agar as the fermentation processproceeded. Klebsiella michiganensis and Klebsiella oxytoca were able togrow on Tryptic Soy agar, l\llalt extract and Violet Red Bile Dextrose agar withan overlapping time of one day. A similar correlation was observed for theLactobacillaceae family. Those lactic acid bacteria were isolated fromRogosa, Tryptic Soy agar and l\llalt Extract agar at the highest dilution. Theidentified microorganism were Pediococcus pentosaceous, Lactobacillusp/antarum/Lactobacillus pentosus, Lactobacillus brevis to mention the mostfrequent isolated species (Figure 1).

Randomlv Amplified Polvmorphic DNA The closely related Lactobacillus pentosus CCUG 33455T, Lactobacillusplantarum ATCC 14917T and Lactobacillus paraplantarum CCUG 35983Twere clearly discriminated using the primer P73. The band patterns are characteristic for each type strain. Those patterns can be compared tofingerprints and were used as reference to compare and differentiate theundefined bacterium Lactobacillus plantarum/Lactobacillus pentosus due tothe 0.999 percentage of similarity according to the 168 rRNA genesequencing results in our study. Therefore, according to the band pattern andsize of the bands the iso|ated microorganisms from quinoa sourdough,compared to the type strains, belonged to the genera Lactobacillusplantarum.

Randomly Amplified Polymorphic DNA bands patterns present forLactobacillus ChB11, Lactobacillus ChG33, Lactobacillus ChJ239 andLactobacillus ChFï228. Electrophoresed gel showing the amplified bandspatterns for the microorganisms identified as Lactobacillusplantarum/Lactobacillus pentosus spp., iso|ated from quinoa sourdoughcompared to the bands patterns of the type strains Lactobacillus pentosusCCUG 33455T (L5), Lactobacillus plantarum ATCC 14917 (Lp), andLactobacillus paraplantarum CCUG 35983T (Lpp). The I\l|o|ecu|ar WeightI\l|arker Vl (Ld) used as size reference expressed in kpb.

PCR specific primers for Lactobacillus pentosus spp. dene amplification The developed method and the designed primers by Torriani et.al. specific forLactobacillus pentosus were used and evaluated on the iso|ated cells fromquinoa sourdough. Unfortunately, the pair primers pentF-pREV also attachedand amplified regions belonging to Lactobacillus plantarum and Lactobacillusparaplantarum, apart from Lactobacillus pentosus, the strain of interest. Thesize of the amplicons from the used type strains correlates with the sizesreported by Torriani et.al. 218 bp for Lactobacillus pentosus CCUG 33455Tand 318 bp for Lactobacillus paraplantarum CCUG 35983T and with doublebands at 218 bp and 118 bp for Lactobacillus plantarum ATCC 14917T. Thedouble band generated during the electrophoresis was also observed for thecells iso|ated from quinoa sourdough. The results are not consistent regardingthe specificity of the primers. However, for the purpose and scope of thisstudy the results can be interpreted as the Lactobacillus spp., iso|ated fromquinoa belongs to the specie Lactobacillus plantarum. Fïegarding the methodand designed primers by Berthier et.al., no bands were observed on the gelsexcept for Lactobacillus pentosus CCUG 33455T. The size of the ampliconswere approximately at 220 bp which could correlate to the amplicons sizereported by Berthier et.al., at 205 bp for the type strain Lactobacillus pentosusATCC 8041T, an homologous strain to Lactobacillus pentosus CCUG 33455Tstrain used in this study. Neither Lactobacillus paraplantarum CCUG 35983T,Lactobacillus plantarum ATCC 14917T or the Lactobacillus spp. isolated fromquinoa sourdough showed amplicons on the gel after the electrophoresis. Theresults confirmed the specificity of the primers pair straightening the identity ofthe Lactobacillus spp., at species level from quinoa dough as Lactobacillusplantarum.

Taxonomic identification using API 5OCHL The 4 Lactobacillus plantarum spp. strains identified by analytical methodspreviously described were phenotypically characterized using API 5OCH. Thetest was repeated twice to confirm the reactions. None of the bacteria wereable to fermented erythritol (2), D-arabinose (3), L-xylose (7), D-adonitol (8),methyl-ßD-xylopyranoside (9), L-sorbose (14), dulcitol (16), inositol (17), inulin(33), amidon (starch,36), glycogen (37), xylitol (38), D-lyxose (41), D-fucose(43), L-fucose (44), D-arabitol (45), L-arabitol (46), potassium 2-ketogluconate(48) or potassium 5-ketogluconate (49). The 4 strains have shown to befacultatively heterofermentative lactobacilli based on their ability to fermentpentoses, such as L-arabinose (4) and D-ribose (5), as well as their ability togrowth in presence of oxygen. The 2 strains were able to ferment D-xylose (6)and in the case of glycerol (1), the status changed from (?) to (T) meaningthat the reaction was considered as positive after interpretation of the resultsregarding glycerol. The change in solution color was not so noticeable;however, if the color of the solution change by 25% to green it should beconsidered as positive. For potassium gluconate (47), it changed from (?) to(T) for the two strains able to ferment xylose (6). Additionally, as the resultswere analyzed and compared with the reference (Lp.5*), the identity of themicroorganisms was more precisely confirmed as Lactobacillus pentosus forthe Lactobacillus ChB11 and ChG33 (see table 2). ln the case of the other 2strains Lactobacillus ChJ239 and ChF¶228 the fermentation of potassium gluconate (47) was interpreted as negative (i). The carbohydrate utilizationpatterns by the isolated lactobacilli are listed on table 2. The results werecompared to the apiwebTll data base. and viable cells were grouped accordingto their capacity to ferment the carbohydrates. From the 4 bacteria tested, 2belong to Lactobacillus pentosus and 2 to Lactobacillus plantarum. All thestrains were able to fermented L-arabinose (4), D-ribose (5), D-galactose(10), D-glucose (11), D-fructose (12), D-mannose (13), D- mannitol (18), D-(19),amygdaline (23), arbutin (24), esculin ferric citrate (25), salicin (26), D- sorbitol methyl-oiD-mannopyranoside (20), acetylglucosamine (22),cellobiose (27), D-maltose (28), D-lactose (bovine origen) (29), D- sacharose(sucrose) (31), D-trehalose (32), D- melezitose (34), gentibiose (39), D-turanose (40).

Table 2. Phenotypical characterization of Lactobacillus ChB11, ChG33,ChJ239 and ChF¶228 evaluating the metabolic capacity on a variety ofcarbohydrates.

LactobacillusChB11 ChG33 Lp.5* ChJ239 ChFi228 Lp.1* Lp.2* Carbohydrate 1 Glycerol ? ? 0 04 L-arabinose 15 D-ribose 16 D-xylose 110 D-galactose 11 1 D-glucose 112 D-fructose 113 D-mannose 115 L-rhamnose 018 D-mannitol 119 D-sorbitol 120 Methyl-dD-l\/lannopyranoside21 Methyl-dD-Glucopyranoside22 N-Acetylglucosamine23 Amygdalin24 Arbbutin25 Esculin ferric citrate26 Salicin27 D-cellobiose28 D-maltose 1111111?_i._i._i._|._i._i._i._i._i._i._i.+|..|_|..|_|._i._|._i._i._i._i.o_i..|+oO-l-HO-I-I-I-I-O-I-OO üo -----o------o---o o---o------o----l-O _|._|._|._|._|._L_|._|._|._|._|._|._L_|._L_L_L_L_L_L_L_|._|._|._|._|._L_|.29 D-Iactose (bovineorigin) D-melibiose 31 D-sacharose (sucrose)32 D-Trehalose 34 D-Melezitose D-raffinose 39 Gentibiose 40 D-Turanose 42 D-tagatose _;_;_;_;_;_;_; -o-------o-oo--o-----oo-»fl-o-ß-L-ß-L--o---o-----o-I-l-O-I-l-l-K-K-K-K-KOOO-H-OO-K-l-O 47 Potassium Gluconate >|< Lactobacillus pentosus (Lp.5*) and Lactobacillus plantarum 1 and 2 (Lp.1*,Lp.2*) are type strains from apiwebTll (API® 5OCHL V5.2) database. i I\lleans that the microorganisms are more than 50% capable to ferment thecarbohydrate in question but less than 100%.

T The microorganism is less than 50% able to ferment the carbohydrate inquestion but more than 0%. if The microorganism is 50% capable to ferment the carbohydrate inquestion.

Lactobacillus sbb. tannase enzvmatic activitv The four Lactobacillus ChB11, ChG33, ChJ239 and ChF¶228 strains showedto possess tannase enzymatic activity and decarboxylate gallic acid. Amaximum absorbance over 2, in general, was detected on a wavelengthrange of 380 to 440 nm for the species that could degrade methilgallate.(Table 3).

Table 3. Tannase activity evaluation of Lactobacillus strains isolated from quinoa grains.

I\llethilgallate Gallic acid SDGCÜGS COÖG Colour +/- Colour +/-Negative control Cleara - Dark greenf* -Ferment xylose ChB11 Orange + Dark orange + ChG33 Orange + Dark orange + Unable to ChJ239 Orange + Dark orange +ferment ChFï228 Orange + Dark orange +Xylose +/- Positive or negative result on methilgallate degradation and gallic aciddecarboxylation *** The negative control color solution for methilgallate was uncolored anddark green for gallic acid.Results The experiments report the Characterization of the microbiota of commercialwhite quinoa grains imported from Bolivia to Sweden for Kung I\/|arkatta. Thepresence or not of oxygen besides the temperature during fermentation arefactors that could affect the proliferation of some autochthonous bacteria. Tofind suitable conditions to characterize the microbiota on quinoa grainsspontaneous fermentation was controlled aerobically and anaerobically at30°C for 192 hours.

The value of the pH was used as a reference to control the fermentationprocess of the quinoa grains. At optimum fermentation, the pH should gobelow 4. Lactic acid bacteria can resist acid environments compared to otherstrains. Also, it was observed that during fermentation the percentage of theacidity increased. The number of Enterobacteriaceae viable cells decreaseduntil being undetectable and lactic acid bacteria displaced the number of totalaerobic bacteria dominating, assuring an optimum fermentation. ln general 168 rRNA gene sequencing was not enough to distinguishbetween Lactobacillus p/antarum and Lactobacillus pentosus. For thatreason, it was necessary to do deeper tests to distinguish between bothstrains. RAPD electrophoretic band profiles for the 4 lactic acid bacteriaisolated from Fïogosa agar gave clear distinct patterns for all the strains incomparison to the type strains. According to the type strain Lactobacillusp/antarum ATCC 14917 compared with the 4 strains isolated from quinoagrains, all had similar band patterns.

To distinguish between the species Lactobacillus p/antarum and Lactobacilluspentosus the selected isolates were evaluated for their capacity to fermentcarbohydrates using API 5OCHL test. The main point to identify some bacteriaas Lactobacillus pentosus was the ability of those to fermented D-Xylose(DXYL). However, lVlethyl-oiD-l\llannopyranoside (l\llDl\ll), D-l\llelezitose (l\llLZ),D-Tagatose (TAG), D-Raffinose (RAF) and D-l\llelibiose (l\llEL) were alsotaken i consideration. Glycerol (GLY) was partially fermented by lactobacillistrains isolated from quinoa grains in this research work hence, glycerol wasalso suitable to discriminate between L. plantarum and L. pentosus.Lactobacillus plantarum ChB11 and Lactobacillus plantarum ChGTwo different Lactobacillus plantarum strains ChB11 and Lactobacillusplantarum ChG33 were identified as dominating species isolated from whitequinoa grains imported from Bolivia. lt is the first time that a Lactobacillusplantarum can ferment D-Xylose, a carbohydrate that only the Lactobacilluspentosus species should be able to use as source. This enzymatic action canbe used to identify Lactobacillus pentosus from the rest of the LAB group. Thestrains are facultative heterofermentative (Group ll) and they grow under bothaerobic and anaerobic conditions. They were isolated from Fïogosa agar after72 hours of incubation at 37°C. However, Lactobacillus plantarum ChB11and Lactobacillus plantarum ChG33 were also isolated from l\llalt agar,incubated aerobically at 26°C for 7 days. l\llalt agar consists of a mixture ofmaltose, glycerol, and peptone. The capacity to grow on malt agar isuncommon for the species showing that Lactobacillus plantarum ChBB1 andLactobacillus plantarum ChG33 were able to adapt to the environment, usethe available nutrients and grow in presence or not of oxygen.

Lactobacillus plantarum ChB1The bacteria Lactobacillus plantarum ChB11 was identified to phenotypicallevel using the API 5OCHL test, showing 91 .3°/> of similarity. The strain is ableto fermented L-arabinose, D-ribose, D-galactose, D-glucose, D-fructose,D-mannose, D-mannitol, D-sorbitol, methyl-oiD-mannopyranoside, N-acetylglucosamine, amygdaline, arbutin, esculin ferric citrate, salicin, D-D-l\llelibiose, D-sacharose (sucrose), D-trehalose, D-melezitose, D-Raffinose, gentibiose, cellobiose, D-maltose, D-lactose (bovine origen),D-turanose, D-Tagatose and partially ferment L-Rhamnose and PotassiumThe the Enterobacteriaceae, including potential pathogenic bacteria expressing pro- Gluconate. growth of bacteria belonging to family inflammatory lipopolysaccharides on their cell wall, are inhibited by Lactobacillus plantarum ChB11 due to their possibilities of decreasing pH.The bacteria decrease the pH below 4 after 48 hours of fermentation (pH =3.45 i 0.013). Also, Lactobacillus p/antarum ChB11 express DL-Iactateracemase activity which catalyzes the conversion of D-Lactate to L-Iactateincreasing the percentage of the last one through time. ln general, DL- lactate racemase enzymes expression have been found in theLactobacillus p/antarum spp. species.

Lactobacillus p/antarum ChB11 increases the concentration of polyphenolssuch as rutin, vanillic acid, quercetine, kampherol, and luteolin increase at the the Liquid Chromatography-HPLC, on a mixture of acetonitrile:methanol (formic acid same time as the antioxidant capacity improve. The identification of phenolic compounds was done using High Performance1%):water as mobile phase, 20pl of sample was injected and the UV spectraobtained for each molecule was compared against the correspondingstandard. The phytase enzymatic activity will be evaluated and probablyLactobacillus p/antarum ChB11 increases the availability of minerals bydegradation of phytate through the fermentation process. The productionvitamins, including vitamin B and folates, essential for humans, are alsoexpected to increase in amount during growth of the Lactobacillus p/antarumChB11. lt is also believed that the strain can survive the gastrointestinalpassage, which will be demonstrated through re-isolation from humansamples after 2 weeks of consumption. The possibility to re-isolateLactobacillus p/antarum ChB11 from humans will then, to our knowledge, bereported for the first time.

Lactobacillus plantarum ChGLactobacillus p/antarum ChG33 was identified to phenotypical level using theAPI 5OCHL test, showing 93.1°/> of similarity. The strain is able to fermentedL-arabinose, D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D- mannitol, D-sorbitol, methyl-dD-mannopyranoside, acetylglucosamine,amygdaline, arbutin, esculin ferric citrate, salicin, D-cellobiose, D-maltose, D-lactose (bovine origen), D-l\llelibiose, D-sacharose(sucrose), D-trehalose, D-melezitose, D-Raffinose, gentibiose, D- turanose, D-Tagatose and partially ferment L-Rhamnose and PotassiumGluconate. Lactobacillus p/antarum ChG33 cannot be solely identified byrRNA gene sequencing but using phenotypical identification the species canbe identified and from other strains of the same species. Lactobacillusp/antarum ChG33 can be distinguished from Lactobacillus p/antarum ChB11in their ability to ferment methyl-oiD-glucopyranoside. Lactobacillus p/antarumChG33 is able to decrease the pH to values below 4 after 48 hours of3.56 i 0.012). Also for this strain and during thefermentation process the concentration of phenol compounds as well as the fermentation (pH antioxidant capacity will probably increase. The resistance to oxidative stresswas evaluated using the tannase test, and Lactobacillus p/antarum ChG33was capable to degrade methilgallate and gallic acid to an acceptableamount.

Lactobacillus plantarum ChJ239 and Lactobacillus p/antarum Ch RThe strains Lactobacillus p/antarum ChJ239 and Lactobacillus p/antarumChF¶228 were identified as dominating species isolated from white quinoagrains from Bolivia. Lactobacillus spp. are commonly isolated from plantmaterial. However, the phenotypical identification between strains from thesame species is overestimated for Lactobacillus p/antarum, indicating thenecessity of identification also based on PCR-based methods. Lactobacillusp/antarum ChJ239 and Lactobacillus p/antarum ChF¶228 belongs to group l(obligate homofermentative) and they grow under both aerobic and anaerobic72 hours of incubation anaerobically at 37 °C. Both microorganisms were also isolated conditions. They were isolated from Fïogosa agar afterfrom I\llalt agar, incubated aerobically at 26 °C for 7 days. I\llalt agar consist ofa mixture of maltose, glycerol, and peptone. The capacity of growing on |\llaltagar has not previously been reported for Lactobacillus spp. This adaptabilityof the strains can be explained by the ecological niche from where they havebeen isolated. The quinoa plant grows under extreme environmentallimited. The autochthonous flora is therefore highly adaptable to new environmental conditions, where the supply of oxygen and water is conditions such as different mediums, variable temperature, and times forgrowing. lt is believed that the strains can survive the gastrointestinalpassage, which will be demonstrated through re-isolation from human salivaand fecal samples after 2 weeks of consumption. lt is also believed that thestrains will be able to affect the population size and expression ofactivation-, homing, regulation, and memory markers as well as receptors forgram-positive and gram-negative bacterial cells on B-cells, T-cells,macrophages, and dendritic cells in mesenteric lymph nodes and Peyer'spatches.

Lactobacillus plantarum ChJLactobacillus p/antarum ChJ239 was identified at phenotypical level usingAPI 5OCHL, where 999% belongs to similarity confirms that the microorganism the species Lactobacillus p/antarum. The strain is able toferment L-arabinose, D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, D-sorbitol, methyl-oiD-mannopyranoside, N-acetylglucosamine, amygdaline, arbutin, esculin ferric citrate, salicin, D-cellobiose, D-maltose, D-lactose (bovine origen), D-melibiose, D-saccharodise, D-trehalose, D-melezitose, gentiobiose, D-turanose andundefined potassium gluconate (assuming a partial positive reaction of50%, represented by a light green color on the indicator color change).Lactobacillus p/antarum ChJ239 expresses DL-lactate racemase activitywhich catalyzes the conversion between enantiomers. Lactobacillusp/antarum ChJ239 synthesize L-lactate and D-lactate because of thefermentation and after 48 hours the pH decreases to 3.87. At this low pHvalue, the growth of Enterobacteriaceae is inhibited. The tannase test wasused for evaluated the tolerance on oxidative stress. Lactobacillus p/antarumChJ239 was able to resist the stress showing an acceptable percentage ofdecarboxylation of methilgallate and reduction of gallic acid. Duringfermentation, the concentration of phenolic compounds such as rutin,vanillic acid, quercetine, kampherol, and caffeic acid increased; however,the amount of saponins seemed to decrease since it was not possibleto find compounds belonging to this group. Nevertheless, at the sametime the antioxidant capacity increased. The precision of the identification ofphenolic compounds by HPLC improved after the enzymatic action of the bacteria. The production of vitamins, mainly vitamin group B, folates andphytase activity will be evaluated and the hypothesis is that Lactobacillusp/antarum ChJ239 increases the bioavailability of minerals.

Lactobacillus plantarum Ch F¶ln general, the characteristics of Lactobacillus p/antarum ChR228 are likeLactobacillus p/antarum ChJ239; however, Lactobacillus p/antarum ChJ239can ferment D-melibiose and methyl-dD-IVlannopyranoside whileLactobacillus p/antarum ChFï228 is not. Additionally, Lactobacillus p/antarumChF¶228 can ferment potassium gluconate.

Conclusion The present experiments demonstrated that the microbiota of quinoa grains ismainly constituted by the autochthonous bacteria Lactobacillus pentosus,Lactococcus lactis, Enterococcus mudtii, Enterococcus hirae andPediococcus pentosaceous. lt is important to highlight that it is the first timethat a species of Lactobacillus p/antarum is reported as part of the dominatingautochthonous bacteria on quinoa grains capable to ferment Xylose. Alsobecause of the metabolic capabilities, the microbiota of quinoa grainsincreases the nutritional properties and benefits on consuming this grain.

The general procedure was based on taking samples before fermentation,after fermentation and every 7 days until to completed 28 days of storagetime. The sampling times were, zero time (before fermentation), at 48 hours(fermentation time) and 7, 14 and 28 days. The samples were withdrawing atthe same time in each occasion, with a variance of 30 min.

Preparation of Fermented Quinoa Milk Quinoa milk The fermented quinoa milk is prepared using white quinoa grains, importedfrom Bolivia, and lactic acid bacteria as discloseed herein Lactobacillusp/antarum ChB11 (LIVIG P-31891), Lactobacillus p/antarum ChG33 (LIVIG P-31892), Lactobacillus p/antarum ChF¶228 (LIVIG P-31893) and Lactobacillusp/antarum ChJ239 (LIVIG P-31894),categorized as probiotic. isolated from the quinoa grains, 2300 grams of quinoa grains was washed with water in a proportion of 1:(V:V) for 30 minutes. The mixture was agitated few times and the water wasreplaced after 15 minutes in contact with the quinoa grains. The procedurewas repeated twice. The water was discarded and the quinoa grains werewashed down under running water. The procedure was repeated 3 timesobserving the discarded water looks clear and without foam. The washedquinoa grains were ready to be toasted.

The quinoa grains, still humid, were dried over a pant at 225 °C on the stove(level 7, Elektro SH Helios) for at least 10 to 15 minutes until dryness. Thenthe temperature was decreased to 195 °C (level 5, Elektro SH Helios) and thequinoa grains were toasted for 20 to 25 minutes. The temperature during thetoasting was measured (l\llulti-Thermometer) on the quinoa grains between140 to 145 °C.

The toasted quinoa grains were cooled down, at room temperature for at least30 minutes, spreading the toasted quinoa grains over a table. The table wascovered with a new clean baking paper to protect the quinoa grains from anycontamination coming from the surface of the table.

The quinoa milk was prepared using 100 grams of toasted quinoa grains with800 ml of water. The water was autoclaved at 121 °C for 15 minutes andstored at 4°C, overnight, before to be used. The toasted quinoa grains weremixed using a blender (Electrolux, blender ESB5400BK, 5 speeds setting) atmaximum speed, for 15 minutes. The mixture was filtrated through a juicestrainer cloth (l\llenuett, plastic stand). Total volume after filtration 900 ml ofquinoa milk. lnoculation of Quinoa milk The quinoa milk, 1000ml, was inoculated by the 4 Lactobacillus p/antarumChB11 (LIVIG P-31891), Lactobacillus p/antarum ChG33 (LIVIG P-31892),Lactobacillus p/antarum Ch R228 (LIVIG P-31893) and Lactobacillus p/antarumChJ239 (LIVIG P-31894), obtaining 4 formulations of fermented quinoa milk.The quinoa milk was inoculated with 0.2% of concentrated bacteria andfermented at 30°C during 48 hours at anaerobic conditions. The probioticbacteria, used to induce the fermentation, were isolated from white quinoagrain. Those strains were identified at genotypical and phenotypical level.Phvsicochemical propertiesThe physicochemical properties, such as color and viscosity, was measuredfor the four strains. l Konika Minolta (United Kingdom) colorimeter was used for measured thecolor of the quinoa milk at room temperature. The samples were analyzed bytriplicated. The parameters luminosity or lightness (L), redness (a) andyellowness (b) were used as a reference for determine the color of theproduct. The results were analyzed using Spectra Magic software and, theresults were classified according to the whiteness index (Wl), and colordifference defined as AE.

The mathematic equations used for classified the color of the quinoa milk are:ist-tr = :ïoo - ~ï~ (1) = 1:» a» (2) The total color difference (AE), which represents the overall difference in colorof the quinoa milk was calculated using equation 2. The results should beclassified, according to Cserhalmi et al., as not noticeable (0 to 0.5), slightlynoticeable (0.5 to 1.5) and noticeable (>1.5).

Viscosity The viscosity of the quinoa milk was measured using a rheometer with asensor system of coaxial cylinders were the shear stress was measured as afunction of the shear rate between 0.001 to 50 pa, for 900 seconds at 18 °C oftemperature. pH of the guinoa milk The pH of the fermented quinoa milk was measured using a digital pH meter(744pHmeter/l\/letrohom-Kebolab). The pH-meter was calibrated before to beused according to the manufacturer recommendations using buffer solutionsat pH 7 andLili! The lactic acid produced during fermentation was measurement usingEnzytech D/L lactic acid kit (Fï-Biopharm Darmstadt, Germany) following theinstructions recommended for the manufacturer. However, the volume of the samples was changed from ml to ul. Briefly, 15 ml of samples (n=6) were centrifuged at 6000 rpm for 5 minutes. Aliquots of the supernatant wasextracted and diluted 100 times with Milli-Q water. A volume of 224ul for D-lactic acid and 226 ul for L-lactic acid is required. The solution content 100ulglycylglycine buffer, 20ul of NAD solution, 2ul of GPT suspension, 10ul ofsample or 10ul l\/lilli-Q water (blank) and, 90ul of IVlilli-Q water was mixed andthe solution was stained for 5 minutes. The absorbance (A1) of the mixturewas measured. After, 2ul of the enzyme D-lactate dehydrogenase solutionwas added and incubated for 30 minutes and the absorbance (A2) wasmeasured. Finally, 2ul of the enzyme L-lactate dehydrogenase solution wasadded and incubated for 30 minutes more and, the last absorbance (A3) wasmeasured. The incubation of the samples was done at room temperature andthe absorbance of the samples was measured at 340nm using a SpectrostarNano l\/lultiplate Reader (BMG Labtech, Germany) by triplicate. Theconcentration of D-lactic acid or L-lactic acid was calculated applying thefollowing equation: First calculate the absorbance difference.

AAD-iactic acid = (A2 - A1) AAL-iactic acid = (As- A2) Use the absorbance difference from previous equation, and used on the question 3:v ...satta-f C = sfäfi.1fiv;>~í'ífit_šfi Where: V= final volume (ml) v = sample volume (ml) l\/IW = molecular weight of the substance to be assayed (g/mol) d = light path (cm) a = extinction coefficient of NADH at 340 nm = 6.3 [lxmmofixcml] ÅA = ÅAD-lactic acid OI' AAL-lactic acidNutritional propertiesß The ash content was determined incinerating the samples at 550 °C during 16to 20 hours in a Haraeus burning chamber. The melting pot was dried andweighted before transferring the samples into them. The samples wereweighted before and after incineration. The ash content was calculatedfollowing the equations below: °/> Ash (dry basis) = ( |V|AsH/|V|DRY)*1OO (4) °/> Ash (wet basis) = (|V|AsH/|V|wET)*1OO (5) l\/loisture The moisture content is used to analyze the water content. The water wasevaporated from the samples during approximately 2 hours followed byheating in a dryng oven (Termaks drying oven) at 105 °C for 24 hours. Thesamples were weighted before and after elimination of water. The equationused to determine the moisture content, expressed as percentage (°/>) is: °/> moisture = (Minmai - Mdried)/|V|inifla| *100 (6) Protein.

The protein analyzer (Flashea 112 series) was used to determine the proteincontent on the fermented quinoa milk. 1 ml of sample, by triplicated, was usedto be analyzed following the protocol for liquids where the nitrogen content ismeasured and the content of protein can be calculated using a conversionfactor of 6.25. This conversion factor is stablished by defect for the equipmentand the protein analyzer is based on the Kjeldhal method, were the totalcontent of nitrogen from the sample is detected by the equipment.

Lipids or Fat Approximately, 15 ml of sample were collected on a 50ml tube (sterilizedEppendorf tube) and centrifuged at 13600 rpm for 10 minutes at 4°C. Thesupernatant was discarded and between one or two ml of ethanol was addedto the solid portion. Then, the samples were dried at 105 °C overnight.Between 2.5 or 3 grams of dry sample (S) was weighted on a porous thimble. 80 ml of petroleum benzene, was used per sample to extract the fat, andtransferred to metal containers (W1). The equipment used was a Soxtechs2055 and has a capacity for 6 samples.The content of fat is calculated according to equation 7: - tft-gt cruitïe == 'J >< 'lå-Li Where Wi = weight of empty flask in (g).

Wz = weight of the flask and the extracted fat (g).

S = weight of the sample (g) Total Carbohydrates The calculation of the total carbohydrates depends on the result of previousdetermination. The Total carbohydrates can be calculated according toequation 8: °/> Carbohydrates= 100 - °/> of moisture - °/>protein - °/>lipids - °/> ashes (8)lVlicrobiolodical Characterization of the Fermented Quinoa Milk ln order to evaluate the safeties and hygiene quality of the fermented quinoamilk, two agars medium were used. Violet Red Bile Dextrose (VRBD) agar isuse for evaluate the presence of Enterobactericeae, the plates wereincubated at 37 °C for 24 hours and, Tryptic Soy Agar (TSA) was used fordetermine the total bacterial count, the plates were incubated at 30 °C for 72hours. Samples of quinoa milk were withdrawing and mixed with peptonewater (1 :9, v:v). Serial dilutions were made and from the proper dilution thesamples were plated on VRBD, TSA and Rogosa. The survivability of theprobiotic bacteria, used for induce the fermentation, was evaluated controlledevery seven days, starting after the 48 hours of fermentation. The Rogosaplates were incubated on anaerobic conditions at 37°C for 72 hours.

The viable colonies were recorded from each agar and used to calculate thenumber of colony forming units (CFU).

Statistical analyses The statistical analyses was performed using SigmaPlot software. The datawere analyzed comparing the values for each group in each samplingoccasion, meaning before fermentation, after fermentation and during storageusing ANOVE on rank basis. Whitney rank sum test was applied to comparebetween different groups. The statistical value was presented as mediam, 25-75 percentages.

Preparation of Fermented Quinoa Milk The quinoa grains were toasted with constant movement of the grains until anutty smell can be perceived and the color of the grains change from white togolden. The quinoa milk was inoculated immediately after being filtrated andcollected into 1 liter bottles. The concentration of viable bacteria found in thefermented quinoa milk is listed in tableTable 4. Content of probiotic bacteria after 48 hours of fermentation.

Fermented Quinoa Lactobacillus p/antarum ChB11 9.22 48Milk Lactobacillus p/antarum ChG33 9.24 48Lactobacillus p/antarum ChJ239 10.29Lactobacillus p/antarum Ch Ft228 1 0.28Lactobacillus plantarum 299v 8.5299v in comparison, for Lactobacillus piantarum299v, the number of viaoie ceifsexpressed as Log Ciïiiifmi is tower compared to the strairis Laotobaciiluspiantarum ChBfi, Lactobacillus piarrtarum Chêíšíš, Lactobacillus planiarumChti239andChiïtPhvsicochemicai properties .Galet The rrieasurement in coior is directly correiated with the production of iacticacid on fermented products. A major production of iactic acid iriay or is theprobabiiity that the product became whitishfihe amount of ili~or L-iactic acid isiisted on tabfe 4. For the formuiartion of fermented pianot base beverage isimportant because if the coior of the drink is sirniiar or ciose to bewhiteiooking iike miik, heiping on the acceptabiiity during consumption.Additionaiiy, ii; is easier to change the ooior oi the drink, ii the iirtai product iswhiiish. The use of the Laciobeciíius piantarunt ChBi i, Lactobacilluspiantarurn ChGíšS, Laotobacíiius piantarurn Chti239 and Lactobaoiiiuspiantarum Chift228 isoiated irorn the tvhite ouinoa grains improved the ooior5 oi the drink with a notioeaoie change to white atter and during ierrnentaiion.According *to a ciassiiioaticn systern it is ncticeahie ii a change oi *the ooior isgrater than i,5. (En the case oi our tour sirains the change oi the eoior isnotioibie. The vaiue on the AE Was ranged between 2,01 and 4.95. Thewhiteness index (Wi) can he considered extrenieiy good ii the vaiue is over10 50, meaning that the coior is oioseiy correiated to whitefihe whiteness index(Wi) is stiii siabie during the ioiiowing days, 14 and 28, meaning that the 48hours oi ierrnentation are essentiai to ensure the iinai coior oi the product.

Tahie ößifiíhiteness index ior the 4 iaetobaoiiius etrains aiter 48 hours oi ierrnentation.

SttttfittLtttvtoštrtoišlitts Aoitfzrtrttratrrr Cfitšií tft rzišifn-iirttrfisirit tfiíiíiiïiíiïlfiš .Li-*titfit-.iišttttfiíštts' g-.ii-*i-.triiiitfttairtt(lfizfïïšiši Lrttfiiašrotfiššrts' gišfrtzfiiiitrirtitst -ßišít ' Table 6. pH before iermentation (cero time) after iermentation and during20 storage time Lactobacillus p/antarumC h B 16.57 3.74 3.41 3.37 3.Lactobacillus plantarum 6.42 3.83 3.80 3.47 3.48ChG33Lactobacillus plantarum 6.35 3.78 3.80 3.85 3.82ChJ239Lactobacillus plantarum 6.46 3.45 3.48 3.42 **Ch F¶228Lactobacillus plantarum 6.21 3.97 3.92 3.76 3.63299V o *The decreasing of the pH reach to this value after 72 hours (3 days) offermentation.

**These values will be completed ones the samples are collected.aeiaiíiaiVicst Lactic acid bacteria, ihaihiy Lactobacillus species has the pctentiai ipproduce iZi-ei' L-iaciie acid inean whiie the enzynies are active in equaiprcpcrticns. Additipriaiiy, scrne ct thern have the capacity te traiisfcrih Ü»iactic acid inte L-iaetic acid er vice versa. ii vvas chserved that Lactobacilluspiantaruihíšhßii iahdiuactcbaciiius piantaruinChG33 had the capacity tetransfcrin L~iactic acid iiitc D-iactic acid in scihe peini; between the 14 daysand 28 days cf fetrneniaticn. As eah he chserved en Tahie 7,, the arncunt cfD-Lactic acid decrease drasticaiiy at 28 days, were the ariicunt cf Dmiacticacid decrease and the amount ci L-iaetic acid ieinained etahie. This capacitywas het tipsen/ed fcr Lactcbaciiius piantaruin GhiJ239 and Lactcbaciiiusplantarum ChR228. i-iewevet, these iaet-rtienticned strains eynihesize iïi~and L-iactic acid in eduai arhcurit during ierrrieritatipri.

Tahie ílccncentratien ef Dflarid L~Eaciie acid prcduetien during ferrnentatien Icatifsišsfffirišiits xtiiï-riiiitzietsiis. Acidšttf tfg.«-'§l.} CBE 1 š. (Éštiiiš ilïtilljšš LJ CihREL-f. SL» U i, »ia ifiïi- in. 1.3 ïš-»ša Vis» ia D» Låt 513%? 8115 5 _ Išii) i 5.5? .till Åšâfš itä? St? 3 _ i 6 Läs: imci 13» .sattes ti: iLi-ieictít: :acid istid Lil-itzctic iicrid.The quantity of iactic acid produced during ferrnentattion and the decreaee ofthe bi-i are the tvvo niainiy parameters considered and controiied duringferrnentation. The production of iactic acid, both L-and iI3~, and the change ofthe ett during ciuinoa rniik ferrnentation with the oornrnerciai probiotio bacteria5 Lactcbaciiius pIantarUmZQQV ie shown in tabie 7. it was obeerved that the production of iactic acid and the change of the phi are significantiy differentregardiess of the Lactooaciiius btanrarumChBf f, Lactcbaciiíuspiantarumfšiifiíšíš, Lactobacillus piantarumíšhdâíštäand LactobacilluspIantartrrnChF-âââß isoiated frorn the quinoa graine. The production of each iaotic acid, both L-and iii, ie higher in ccrrioarison with the iactic acid producedby Lactobacillus oiantarurn .fàšišâvffhe vaiues can be observed in tabieNutritional grogerties15 Table 8. Physicochemical properties before, after fermentation and during storgage time Fšttaitt Tiota- Moiatttte Ptotcíu Fat Ash C atbohtfïiratcI) h 915 íf-.ïí 9,36 9.44 5,49, _, 43 h 92.56 113.6? 1.3 *Jc-itä 4.54Lp LhBH Ci 92.63 0.61? (ÉLQÉ (L-'iífil 5.35 (i §É.S(i ilöš? G29 ÛÄÅ Ü it 915 LÉ 9.36 9.44 5.49T Ci f., w 48 Åh ÛÅS? 0.43 L? ' I m e 9324 0.66 (135 (ma CHF? 9.34 Û i; 91.5 Lfší ílšfå 0.44 5.49T I nu 'h å* š *ff 34 3'-Lp C-hïaååï* h: ä. *_ *_ I E96 (H59 "ïåšš(i it ÉhLí 1 5.49 _ , h Ûf-'Si låtL? LhRnß iii eat' o er ................................................................ ftiiicrobibiooicai Characterization of the ferrnented duinoa rniiit.20 Quinoa grains contains, as a bart of their autochthonoue rnierobiota, beneiiciai and oathogenic bacteria. The rnaiority of, thern are inactive andundetectahte. i-iowever, a change on the environmentai conditions, such astemperature, can active them and encourage their rnuitipiication. Some ot thepathogenic oacteria identified during termentation was Pantoea aggiorrierans,Kiehsieiia rnichiganensis, Pseudornonas spp., Staphyiococcus warnery,tššaciiius suotiiis and Ešnterococcus rrtundttiand Enterococcusspp. "thepresence ot those potentiai pathogenic bacteria can oomprornise the ouaiityot the product and, rnost important, the human heaith it they are consurned.Lactobacillus plantarumChBt t, Lactobacillus oiantarurnílhíšßíš, LactobacillusplantarumChJQSQ had shown the potentiai to growth and inhihit thosepotentiai pathogenic bacteria ensuring the ouaiity and sateness ot thetermented ouinoa rniik.

Gomoarison ot terrriented ouinoa rniiir with the 4 strains and LactobacillusQlantarum 299V Lactobacillus planraruin Chtšt t , Lactobacillus planrarum ChGEB,Lactobacillus plantarum ChiJ239 showed to have highest inhioition againstpotentiai pathogenic hacteria ensuring that the termented quinoa rniik wastree ot those microorganism atter 48 hours ot terrnentaticn. Besides, theyrernained stahie in ouantity during storage time. in comparison to theterrnentaticn ot ouinoa miik with Lactobacillus plantarurnâaây thernicroorganism Eriterococcus rnudtii tuas stiii present in a considerahieamount cornpromising the hygiene ouaiity ot the product. The Lactooaciiiusstrains ChBt t, ChGBS, Chii239 and Chi-t 228 totaiiy inhioit the growth otEnterococcus mudtii sateguarding the hygiene ouaiity ot the torrriuiatedterrnented quinoa rnitk.

Ftegarding Lactobacillus plantarurnßhttâââ, it has shown to inhihit the growthot the potentiai pathogenic bacteria present at the beginning in the duinoamiik, out not against Baciiius suhtiiis, a rnicroorganisrn that yvas tound in aconcentration heiow the iimit ot acoeptance, out in comparison toLactobacillus plantarumChBt t, Lactobacillus planiarumChGSS, Lactobacillusplantarurnílhiiâæ wherer it was cornpieteiy inhihited.Discussion The general procedure for preparing vegetable beverages, mainly known asvegetable milk, is based on cooking by boiling the cereal or grains. Thecooked grains are mixed with water and then the mixture is filtered and, aslast step, the none soluble particles are discarded. This procedure wasapplied on the preparation of vegetable milks such as oat, rice or wheatincluding quinoa. However, in the special case of quinoa, the describedprocedure above was consistency for the color, obtaining a whitish colorsimilar to cow milk, but regarding the flavor and aroma of the product it wasnot well accepted. The aroma and flavor are mainly influenced by thesaponins molecules, contained on the surface of the grain, bestowingbitterness to the vegetable drink. ln order to improve the characteristics of the formulated quinoa milk, thegrains were washed to remove saponins. lt is highly recommended to washthe grains, to decrease the saponins content and also to decrease the risk ofbacterial contamination that could occur at any point. As a result, the aromaof the vegetable milk improved. The taste of the vegetable milk was improvedsince the bitter flavor decreased until to be undetectable.

The decreasing of the temperature between the drying procedure and thetoasting of the grains was for avoiding the burning of the quinoa grainsAccording to the FDA (Food and Drog Administration) and WHO (WorldHealth Organization) to consider a fermented drink as functional the minimumamount of viable bacteria, expressed as CFU, cannot be below 108. Theincrease of the bacteria concentration during fermentation time (48 hours) isover the minimum required.

Lactobacillus plarilarumChBf t, Lactobacillus piantarurti Chíšßí-š, Lactobacillusplantarum ChJZSQ and Lactobacillus plahiarum Chitëââ also improved thecolor of the fermented quinoa drink. The formulated fermented quinoa drink iswhitish, safe and hygienic to consume. Based on those parameters, it ispossible to state that Lactobacillus plaritarum Chtši t, Lactobacillus plaritarumChíšßíš, Lactobacillus piantarurti Chtlâíšíäand Lactobacillus plaittarulti ChRQQEšimprove the nutritionai properties ot the dtiinoa rniik transtorrriing the theproduct to tunotionai toooi drink. i-ieaith benefits oonsuntino Lactobacillus plantarurrr strains isoiated trornwhite guinoa grains The Lactooaciiius strains identitied as Lactobacillus plarrtarurn ChBt t,Lactobacillus plantarum Chtšíšß, Lactobacillus plantartlm ChJESQandLactobacillus plantarum Chtäiïíâß were isoiated 'irorn quinoa grains. 2G heaithy voiunteers were asked to participate in a hurnan studyconsorning 250 deciiitres ot ouinoa rniik terrnented with Lactooaclllusplantartrrn ChBtt during Zweeks. The veiunteers aiso were asked to havea wash out period ot two weeks, consisting in the not consumption ot anyproduct containing tive bacteria or prohiotio bacteria, previous the studystarted, to avoid taise positives resuits. Faeces and saiiva sarnpies werecoiiected one day ioetore the study started and atter 2 weeks consumptionot the terrnented heveragefthe toiiowing preiiniiriary resutts wereohservedïi) The santpies were anaiysed using T-FtLFP, next q~FiCFt andgeneration seduencing. 2) Using o-PCR it was showed that theconsumption ot Lactobacillus plarttarurri ChBtt increased the arnount otLactooaciiiaceae atter consumption ot the terniented otiinoa drinit. SiiheLactobacillus plantarum ChBtt can siirvive the gastrointestinai track it theincreasing ot the arriorint ot Laotohaoiiiaceae corresponds to Lactobacillusplantarum ChBt t. 4)"i"he heaithy voiunteers were asked to answer aquestionnaire regarding to syrnptorns. it during consumption ot terrnentedquinoa drink, they oouid observe or teit any change on the consistenoy otthe stooi. Lactobacillus plantartrrn ChBi t can survive the passage ot thegastrointestinai track. Theretore the strain can be categorized as orohiotic.

Claims (1)

1. 1. CLAIMS A probiotic bacterial Lactobacillus plantarum strain isolated from quinoagrains, wherein said strain is selected from the group consisting ofLactobacillus plantarum ChB11 having accession number LIVIG P-31891,Lactobacillus plantarum ChG33 having accession number LIVIG P-31892,Lactobacillus plantarum ChF¶228 having accession number LIVIG P-31893 andLactobacillus plantarum ChJ239 having accession number LIVIG P-A composition comprising at least one bacterial strain according to c|aimA food composition comprising at least one bacterial strain according to c|aimA feed composition comprising at least one bacterial strain according to c|aimA food composition according to c|aim 3, wherein said food composition is afermented vegetable-based beverage, functional food, dietary supplement, food additive or a nutritional product. A food composition according to c|aim 3, wherein a fermented vegetable-based beverage has been fermented with at least one of the strains selectedfrom the group Lactobacillus plantarum ChB11 having accession number LIVIGP-31891, Lactobacillus plantarum ChG33 having accession number LIVIG P-31892, Lactobacillus plantarum ChF¶228 having accession number LIVIG P-31893 and Lactobacillus plantarum ChJ239 having accession number LIVIG P-A food composition according to c|aim 5 or 6, wherein said fermentedvegetable-based beverage is a quinoa milk. 8. A composition according to any one of claims 2-7, wherein said at least onestrain is present in the composition in an amount of from about 1x1O6 CFU toabout 1x 1014 CFU. 9. A composition according to any one of claims 2-8, wherein said strain hasbeen inactivated, attenuated, dead or is live. 10.A method for the preparation of a quinoa milk comprising the steps:a) toasting quinoa grains; b) c) mixing the water and the grains with a blender and filtering the mixture;d) adding water to the toasted grains; inoculation the mixture with at least one probiotic strain selected fromLactobacillus plantrum ChB11 having accession number LIVIG P-31891,Lactoacillus plantarum ChG33 having accession number LIVIG P-31892,Lactobacillus plantarum ChF¶228 having accession number LIVIG P-31893 andLactobacillus plantarum ChJ239 having accession number LIVIG P-31894 at30°C for 48 h in anaerobic conditions.