1. ANDEAN COUNTRIES Andean region of Argentina

2. Activities - Sampling (Argentina) - Isolation of LAB (Argentina) - Characterization by their ISR-PCR and RAPD-PCR profiles (Argentina) - Identification by 16S rRNA gene sequencing and - Analysis of LAB diversity - DGGE (Argentina) In progress…. Quinoa /amaranth grains Involved people Jenni Korhonen Luciana Ruiz Rodriguez Lorena Carrizo Monserrat Nácher Vázquez

3. Analyzed varieties Quinoa Amaranth (Chenopodium quinoa) (Amaranthus caudatus) CauquevaCauqueva Real Cauqueva Cica Hornillos Real Hornillos Pseudocereals (quinoa/amaranth grains)

4. Varieties SeedsPseudocerealtypeSourceProcesssing 1 Chenopodium quinoa Cica hornillos INTA Hornillos, Maimará, Jujuy Ninguno 2 Chenopodium quinoa Real cauqueva Coop. Cauqueva, Maimará, Jujuy Tamizado 3 Amaranthus caudatus Kiwicha INTA Hornillos, Maimará, Jujuy Ninguno 4Chenopodium quinoa Real hornillos INTA Hornillos, Maimará, Jujuy Ninguno Pseudocereals (quinoa/amaranth grains) - Isolation of LAB (Argentina)

5. RAPD-PCR (M13b-XD9) Sequencing 16S rDNA 2 strains Enterococcus casseliflavus 4 strains Enterococcus durans - Isolation from grains (Argentina) Pseudocereals (quinoa/amaranth grains)

6. Broth: 2.5 g seeds + 22.5 ml MRS-5 + cycloheximide Homogeneized Incubation 30°C - Anaerobiosis Sampling days 1, 3 y 7 Measurements: pH and cell counts on MRS- 5, PCA, BHI and McConkey Slurry: 10 g ground seeds + 20 ml NaCl 0.85% + 0.2 g Glucose Homogeneized Incubation 30°C - Anaerobiosis Sampling days 1, 3 y 7 Measurements: pH and cell counts on MRS- 5, PCA, BHI and McConkey - Isolation from grains (Argentina) Pseudocereals (quinoa/amaranth grains)

7. Cell Counts Samples Seed 1 (broth) Seed 1 (slurry) Seed 2 (broth) Seed2 (slurry) Seed 3 (broth) Seed 3 (slurry) Seed 4 (broth) Seed 4 (slurry) PCA 2,7x10 4 ufc/ml 1,8x10 4 ufc/ml 9,4x10 3 ufc/ml 2,8x10 4 ufc/ml 1,9x10 6 ufc/ml 2,8x10 6 ufc/ml 1,9x10 5 ufc/ml 8,5x10 5 ufc/ml H y L 1x10 4 ufc/ml 4,7x10 3 ufc/ml 0 ufc/ml 7,7x10 3 ufc/ml 2,3, x10 5 ufc/ml 6,7x10 3 ufc/ml 1,9x10 4 ufc/ml Mc Conkey 1,7x10 2 ufc/ml 1,4x10 3 ufc/ml 0 ufc/ml 4,9x10 4 ufc/ml 1,1x10 6 ufc/ml 1,1x10 3 ufc/ml 1,2x10 5 ufc/ml Pseudocereals (quinoa/amaranth grains) - Isolation from grains (Argentina)

8. 296 bacterial colonies were picked from different seeds. Gram and Catalase Test Number of bacterial colonies picked in each sample Samples Seed 1 (broth) Seed 1 (slurry) Seed 2 (broth) Seed2 (slurry) Seed 3 (broth) Seed 3 (slurry) Seed 4 (broth) Seed 4 (slurry) Colony numbers 5036174940353435 Pseudocereals (quinoa/amaranth grains) - Isolation from grains (Argentina)

9. Determination of pH Sample Seed 1 (broth) Seed 1 (slurry) Seed 2 (broth) Seed2 (slurry) Seed 3 (broth) Seed 3 (slurry) Seed 4 (broth) Seed 4 (slurry) Day 1 6,036,276,036,026,096,56,086,55 Day 3 6,046,096,14,864,445,545,065,13 Day 7 5,196,115,675,044,34,464,544,94 Pseudocereals (quinoa/amaranth grains) - Isolation from grains (Argentina)

10. Number Gram (+) catalase (-) of isolated bacteria Samples Seed 1 (broth) Seed 1 (slurry) Seed 2 (broth) Seed2 (slurry) Seed 3 (broth) Seed 3 (slurry) Seed 4 (broth) Seed 4 (slurry) Total counts 5036174940353435 Gram (+) Catalase (-) 1758932352226 Identification, Characterization and biodiversity analysis…………..in progress - Isolation from grains (Argentina) Pseudocereals (quinoa/amaranth grains)

11. Activities - Sampling (Argentina) - Isolation of LAB (Argentina) - Identification by RAPD-PCR profiles (M13 and XD9) (Argentina) - Sequencing of 16S rRNA (Argentina) In progress…. Quinoa / amaranth sourdough Involved people Jenni Korhonen Luciana Ruiz Rodriguez Lorena Carrizo

12. Quinoa / amaranth sourdough Analyzed flours Quinoa Amaranth Ying Yang® (commercial)Sturla® (commercial) Real Hornillos (natural) Pseudocereals flour used

13. Samples were withdrawn daily at refreshment steps to evaluate: -pH -Cell counts (CFU/g) on BHI and MRS-5 -Isolation was done on day 0, 1, 3, 5, 8 and 10 on MRS-5 agar. dough yield [(doughmass/flourmass)×100] daily back-slopping (10%) during 10 days Incubation 30°C - Dough preparation and sampling (Argentina) Quinoa / amaranth sourdough

14. Changes in pH during a 10-day sourdough fermentation with daily back- slopping. Quinoa Real ( ), Quinoa Yin Yang (  ) and Amaranth (  ) The pH decreased from 6.1 ± 0.3 to 4.2 ± 0.2 after 2 days of incubation and remained approximately constant until the end of the fermentation period. Quinoa / amaranth sourdough - pH determination

15. The cell count of LAB in the sourdough samples ranged from 2.0 ± 0.5 to 8.3 ± 0.5 log CFU/g during the 10 days-fermentation period incubation. At the beginning of the fermentations, low LAB colony counts (2.0 ± 0.5 log CFU/g) were observed. However, LAB counts increased approximately 4 log after the first day of fermentation in all sourdoughs; reaching up to 10 8 CFU/g at day 6. Population dynamics during 10-day sourdough fermentation with daily back- slopping. Counts were performed in triplicate, and mean values ± standard deviations are represented. Quinoa / amaranth sourdough - LAB counts in MRS-5 (Argentina)

16. At least one isolate representative of each RAPD-pattern was sequenced and analyzed using the Ribosomal Database Project and GeneBank Database. Quinoa / amaranth sourdough - LAB identification by RAPD-PCR (M13 and XD9) and 16S rDNA (Argentina) A total of 247 Gram-positive and catalase negative isolates from MRS-5 were analyzed.

17. Bacterial population dynamics during Quinoa Real, Quinoa Yin Yang and Amaranth sourdough fermentations Days of fermentation Quinoa / amaranth sourdough - LAB dynamics during fermentation (Argentina)

18. -LAB analysis from grains and sourdough from other quinoa/amaranth varieties (Argentina) Quinoa / amaranth grains andsourdough Analyzed varieties Quinoa Amaranth (Chenopodium quinoa) (Amaranthus caudatus) Sturla® (commercial)Cauqueva Cica (natural) In progress…………..