Oct 16, 2013 ... 2Insëtuto de Lactología Industrial (INLAIN,. UNL-‐CONICET). Sanëago del
Estero 2829. 3000. Santa Fe. 3Facultad de Ingeniería, Universidad ...
IV International Symposium on Lactic Acid Bacteria Food, Health and Applications
Posters: Applied biotechnology in food industry
Centro de Referencia para Lactobacilos (CERELA-CONICET) San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013
IV International Symposium on Lactic Acid Bacteria: Food, Health and Applications
D1. S. R. Chañi, M.O. Lobo, N.C. Samman, L.Rodriguez da Silva and E. Velazquez D2. T. Nediani, L. García, S. López Alzogaray and S. Fadda
D3. B.M. Bravo-‐Ferrada, A. Gómez-‐Zavaglia, L. Semorile and E.E. Tymczyszyn D4. V. Terán, M.F. Zacarías, P. Luna Pizarro, G. Vinderola, R. Medina and C. Van Nieuwenhove D5. E. Fabersani, S. Gonzalez and P. Gauffin Cano
D6. R.I. Limón, M.I. Torino, C. MarPnez-‐Villaluenga and J. Frías
D7. J.E. Laiño, M. Juárez del Valle, G. Savoy de Giori and J.G. LeBlanc
D8. S.L. Carrizo, M. Juarez, J.E. Laiño, J.G. Leblanc, G. Vignolo and G. Rollán D9. N. Palavecino Prpicha, M. Castroa, F. Rivasa; M. Cayré, O. Garroa and G. Vignolob D10. M. Juarez del Valle, J. Laiño, G. Savoy de Giori, JG. LeBlanc
D11. M. L. Cruz Pintos, V. Molina, M.P. Taranto and G. Font de Valdez D12. G.H. Peralta, M.C. CandioV, C.V. Bergamini, E.R. Hynes D13. R. Santos, E. Ramos, G. Vignolo and D. Zúñiga
D14. F. Cuffia, I.V. Wolf, E.R. Hynes, C.V. Bergamini and M.C. Pero[ D15. F.F.P. da Silva, J.G. LeBlanc and B.D.G.M. Franco D16. A. Rodríguez de Olmos and M.S. Garro
San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013
IV International Symposium on Lactic Acid Bacteria: Food, Health and Applications
D1
LACTIC ACID BACTERIA ISOLATED FROM ANDEAN FOOD. IDENTIFICATION AND APLICATION AT REGIONAL FOODS S. R. Chañi1,3, M.O. Lobo1, N.C. Samman1, E. Velazquez2 and L. Rodriguez da Silva3
Engineering Faculty. University NaVonal of Jujuy. MarVarena esq. Italia. 4600. Jujuy. ArgenVna. E-‐mail: nsamman@fi.unju.edu.ar. 2 Departament of Microbiology and GeneVcs. University of Salamanca. 37007 Salamanca. Spain. 3 Requimte/Laboratory of Pharmacognosy. Department of Chemistry Sciences. Faculty of Pharmacy. University of Porto, Portugal. 1
LacVc acid bacteria (LAB) may be used as bioprotecVve agent. Some strains of LAB may increase the safety and quality of fermented products due to the producVon of different anVmicrobial compounds, which can prevent the growth of pathogenic and spoilage bacteria. In this work, indigenous LAB strains were isolated from fermented foods without the addiVon of starter cultures (goat cheese, sausage and “Chicha” beverage) and non-‐fermented foods (llama meat, goat milk and several vegetables), all arVsans producing in Quebrada de Humahuaca, ArgenVna. All strains were propagated in MRS broth and cell-‐free supernatants were separated for parVal characterizaVon. Those supernatants were tested concerning their anVbacterial acVvity by a well-‐ diffusion assay against bacterial pathogens associated with food. Proteic nature of inhibitory substances was analysed by the acVon of digesVve enzymes and the stability at different temperatures and pH. Strains revealing anVbacterial acVvity were idenVfied by 16S rRNA gene sequencing analysis. One hundred seventy six (176) colonies Gram (+) and catalase (-‐) were isolated; 60 showed anVbacterial acVvity against Listeria monocytogenes, Listeria innocua and/or Enterococcus faecalis. The strains with anVbacterial acVvity could group in four genders: Pediococcus, Enterococcus, Lactobacillus and Leuconostoc. AnVmicrobial substances produced by Pediococcus acidilac7ci showed greater stability to sterilizing temperatures and wide pH range (2 to 10). When bacteriocin was incubated with proteinase K and chymotrypsin, the extract lost inhibitory acVvity. Llama meat and pre-‐cooked andean potatoes were co-‐inoculated with 107 cfu/g and 104 cfu/g of Pediococcus acidilac7ci and L. innocua, respecVvely and store to 12 °C, 7 days, a reducVon in total mesophylic and L. innocua (1 logarithmic cycle) were observed. This results show that the use of P. acidilac7ci as biopreservante in food could be possible.
San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013
IV International Symposium on Lactic Acid Bacteria: Food, Health and Applications
D2
DETERMINATION OF SAFETY PROPERTIES OF LACTOBACILLI STRAINS ISOLATED FROM GOATMEAT SAUSAGES T. Nediani1; L. García1; S. López Alzogaray1; S. Fadda2
1Facultad de Agronomía y Agroindustrias. UNSE. Av. Belgrano 1912. 4200. SanVago del Estero. ArgenVna. 2CERELA-‐
CONICET. Chacabuco 145. 4000. Tucumán. ArgenVna.
[email protected]
LacVc acid bacteria play an essenVal role in fermented sausage processing. The objecVve of this work was to study the hygienic properVes (acidifying capacity, synthesis of biogenic amines and anVmicrobial substance producVon) of 170 lactobacilli strains isolated from spontaneously fermented sausages elaborated with different proporVons of goat meat. The strains were isolated and idenVfied by phenotypic tests as L. curvatus (28%), L. sakei (24%), L. alimentarius (13.33%), L. casei subsp. tolerans (9.33%), L. plantarum (8.66%), L. brevis (6%), L. casei subsp. rhamnosus (5.33%) and L. farciminis (5.33%). Even if none of the lactobacilli strains were found to produce bacteriocin when tested against Listeria inocua, a high acidifying capacity in sarcoplasmic juice (24 h, 30°C) was observed in L. plantarum (pH 3,86± 0.03) and L. curvatus (pH 3,93±0,02) strains while only the 19% of the L. sakei (pH 3,89±0,05) strains showed a good acidogenic potenVal. These three species exhibited the greatest viability in the sarcoplasmic juice (around 8 Log CFU/ml at 24 h). The absence of lysine-‐, tyrosine-‐ and hisVdine-‐descarboxylase acVvity was detected indicaVng no biogenic amine producVon. The suitable combinaVon of the most efficient strains in the formulaVon of a starter culture could contribute to assure the homogeneity and the hygienic and sensorial quality of meat fermented foods.
San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013
IV International Symposium on Lactic Acid Bacteria: Food, Health and Applications
D3
EFFECT OF PRE-ACCLIMATION MEDIUM ON CELL VIABILITY, MEMBRANE INTEGRITY AND MALOLACTIC FERMENTATION OF Lactobacillus plantarum INOCULATED IN SYNTHETIC WINE B.M. Bravo-‐Ferrada1, A. Gómez-‐Zavaglia2, L. Semorile1 and E.E. Tymczyszyn2
1Laboratorio de Microbiología Molecular, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes,
Bernal, ArgenVna.
[email protected] 2Centro de InvesVgación y Desarrollo en Criotecnología de Alimentos (CIDCA) (CONICET La Plata, UNLP), La Plata, ArgenVna.
Lactobacillus plantarum has been described as one of the LAB species responsible for malolacVc fermentaVon in winemaking. Due the stress factors present in wine (low pH, high ethanol concentraVon, etc.), starter cultures shall be previously acclimated. The aim of this work was to evaluate the effect of acclimaVon on the viability, membrane integrity and technological properVes of three oenological strains of Lb. plantarum exposed to wine condiVons. Cultures in the exponenVal phase were inoculated in an acclimaVon medium containing 0, 6 and 10% v/v ethanol and incubated 48 h at 28 °C. Ayer incubaVon, cells were harvested by centrifugaVon and inoculated in a syntheVc wine, containing 14% v/v ethanol and pH 3.5. The membrane integrity and viability were measured by flow cytometry using two fluorescent dyes: propidium iodide (PI) and carboxyfluorescein diacetate (cFDA). Strains previously acclimated were inoculated in syntheVc wine and incubated at 28 ºC without shaking. CulVvability and L-‐malic acid consumpVon were monitored during 15 days. In non-‐acclimated strains, the damage of bacterial membranes produced a rapid increase of PI uptake. At the same Vme, a noVceable decrease of microbial viability was observed. AcclimaVon in both 6% and 10% v/v ethanol, noVceable increased the microbial viability of all strains in syntheVc wine, and contributed to maintain the membrane integrity. The evoluVon of viability and malolacVc fermentaVon of acclimated strains, grown 15 days in syntheVc wine, showed the high effecVveness of pre-‐acclimaVon at 10% v/v ethanol. AcclimaVon of oenological strains in media containing ethanol prior to inoculaVon of wines significantly improves the viability and decreases the membrane damage in the harsh wine condiVons. These results represent a contribuVon for the development of LAB starter cultures for winery.
San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013
IV International Symposium on Lactic Acid Bacteria: Food, Health and Applications
D4
CONJUGATED BIOACTIVE LIPID PRODUCTION BY BIFIDOBACTERIA STRAINS ISOLATED FROM HUMAN BREAST MILK
V. Terán1; M.F. Zacarías2; P. Luna Pizarro3, G. Vinderola2, R. Medina1,4; C. Van Nieuwenhove 1,4 1CERELA-‐CONICET. Chacabuco 145. 4000. S.M. de Tucumán-‐ ArgenVna. 3Facultad
UNL-‐CONICET). SanVago del Estero 2829. 3000. Santa Fe. 4Universidad Nacional de Tucumán. E-‐mail:
[email protected]
2InsVtuto de Lactología Industrial (INLAIN, de Ingeniería, Universidad Nacional de Jujuy.
Among bioacVve compounds, there are different conjugated fa|y acids with beneficial health properVes for humans. Conjugated linoleic (CLA) and linolenic acid (CLNA) are the most important ones in this group, being posiVonal and geometric isomers of linoleic (LA) and linolenic acid (LNA), respecVvely. Many biological effects have been a|ributed to them, including anVcarcinogenic acVvity. CLA has also properVes as anV-‐obesity and immunomodulatory compound, but CLNA isomers are most effecVve against cancer. Milk and meat of ruminants are the main source of these biolipids for humans. Many bacteria are able to form different conjugated fa|y acids, included lacVc acid bacteria, propionibacteria and bifidobacteria. This bioprocess allows the use of microorganisms as adjunct culture to increase the level of CLA and CLNA in fermented products or as probioVc strains for bioproducVon at intesVnal level. The aim of this work was to determine CLA and CLNA producVon in four Bifidobacterium strains isolated from human breast milk (at the INLAIN). Strains were cultured in MRS-‐cys, supplemented with LA or LNA for 48 h at 37ºC under anaerobic condiVons. Total conjugated fa|y acid levels were first determined by UV method (233 nm), ayer which possiVve strains were evaluated by gaseous chromatography. Results showed that strains were able to form CLA or CLNA in MRS-‐cys broth, having percentages of conversion from 1 % to 14% for CLA, and 1% to 65% for CLNA. The highest CLNA producVon was determined in Bifidobacterium animalis subsp. lac7s INL2, selected for further studies. In this strain, LNA was mainly converted to the c9,t,11,c12 isomer. Increasing substrate concentraVons were added to the broth (200 to 1000 µg/mL) to evaluate tolerance. No signifficant inhibitory effect of LA or LNA on bacterial growth was observed during 48 h compared to control (without fa|y acid addiVon). According to results, 500 µg/mL of substrate was selected for further assays. Regarding Vme of conjugated fa|y acids producVon, CLA/CLNA begins ayer 8-‐12 h of incubaVon. This study demonstrates that bifidobacteria are good producers of conjugated bioacVve lipid in vitro. Bacteria conjugated LNA more efficiently than LA. As far as we are concerned, this study informed for the first Vme CLNA producVon by strains in our country. Since CLNA has higher effect on cancer prevenVon than CLA, the use of these bacteria able to produce it could be an advance to develop funcVonal products enriched in CLNA. For this purpose, bacteria must be carefully selected taking into account the opVmal condiVons for the biolipid producVon.
San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013
IV International Symposium on Lactic Acid Bacteria: Food, Health and Applications
D5
EFFECTS OF LACTIC ACID BACTERIA ON LEPTIN SECRETION IN MURINE ADIPOCYTES E. Fabersani1, S. Gonzalez1,2, P. Gauffin Cano1,3
1 Centro
de Referencia para Lactobacilos (CERELA)-‐CONICET-‐Chacabuco 145-‐ (4000) Tucumán-‐ArgenVna-‐ pgauffi
[email protected]. 2Universidad Nacional de Tucumán (UNT). 3Universidad del Norte Santo Tomás de Aquino (UNSTA).
LepVn, a pleiotropic hormone mainly produced by the adipose Vssue, regulates mulVple homeostaVc funcVons such as food intake, reproducVve and immune funcVons, besides basal metabolism. The objecVve of this study was to evaluate the influence of different lacVc acid bacteria (LAB) on lepVn and cytokine secreVon by murine adipocyte cells. These parameters and the Obr-‐ lepVn receptor expression were also evaluated in co-‐cultures of adipocytes and macrophage cells in order to select a microorganism with low pro-‐inflammatory potenVal, able to posiVvely and negaVvely modulate lepVn secreVon. Bacterial cell suspensions of 14 LAB strains were used (Lactobacillus casei CRL431, Lactobacillus acidophilus CRL258, Lactobacillus acidophilus CRL1063, Lactobacillus casei CRL66, Lactobacillus casei CRL72, Lactobacillus casei CRL117, Lactobacillus fermentum CRL1446, Lactococcus lac7s CRL1434, Lactobacillus plantarum CRL350, Lactobacillus plantarum CRL352, Lactobacillus plantarum CRL353, Lactobacillus plantarum CRL355, Lactobacillus paracasei CRL575 y Lactobacillus casei ssp rhamnosus CRL576). The influence of different LAB on immunological properVes (cytokine) and Obr receptor expression in RAW 264.7 macrophage cell line was evaluated. These macrophages were sVmulated with bacterial cell suspensions (1x107 UFC/mL) at 37°C under 5% CO2 for 24 h. Adipocytes isolated from Balb/c mice were sVmulated with LAB suspensions (1x107 UFC/mL) under the same condiVons, and adipokines levels were determined. For co-‐culture experiments, adipocytes were cultured with Raw 264.7 macrophage cells (5x104 cels/mL, 1:1). LepVn, TNF-‐α, IL-‐6, IL-‐10 and MCP-‐1 levels were determined in the culture supernatants by ELISA test. The principal component analysis (PCA) allow the classificaVon into three disVncVve groups of LAB according to their ability to modulate both lepVn and lepVn receptor expression, and inflammatory profile. We selected one strain from each group in order to perform further studies: 1) Lactobacillus casei CRL431 (low lepVn levels, medium inflammatory profile and ObR expression), Lactobacillus fermentum CRL1446 (medium lepVn levels, low inflammatory profile and ObR expression) and Lactococcus lac7s CRL1434 (high lepVn levels, medium-‐high inflammatory profile and high ObR expression). Our results suggested that different strains may have different funcVonal roles and applicaVons in diverse pathologies. Therefore, modulaVon of circulaVng lepVn levels may be considered as a promising novel strategy to intervene on different nutriVonal condiVons (obesity and undernutriVon).
San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013
IV International Symposium on Lactic Acid Bacteria: Food, Health and Applications
D6
BIOACTIVE COMPOUNDS AND MICROBIOLOGICAL ANALYSIS IN FERMENTED KIDNEY BEANS R.I. Limón1, M.I. Torino2, C. MarPnez-‐Villaluenga1 and J. Frías1
1InsVtuto de Ciencia y Tecnología de Alimentos y Nutrición (ICTAN)-‐CSIC. Juan de la Cierva 3, 28006 Madrid, España.
2CONICET-‐CCT Tucumán, Centro de Referencia para Lactobacilos (CERELA). Chacabuco 145, 4000 Tucumán, ArgenVna.
E-‐mail:
[email protected]
Phaseolus vulgaris is a valuable source of phenolic compounds and precursor proteins of bioacVve pepVdes exhibiVng several bioacVviVes. FermentaVon has been used as a cost-‐effecVve process for biopepVdes release, γ-‐aminobutyric acid (GABA) producVon and bioconversion of phenolic compounds which ulVmately lead to enhancements of their bioacVvity. Therefore, there is an increased interest in the applicaVon of fermentaVon for producVon of bioacVve compounds. The objecVve was to produce bioacVve compounds with anVoxidant and potenVal anVhypertensive acVviVes by liquid (LSF) and solid state fermentaVon (SSF) of kidney beans (P. vulgaris var. pinto). LSF of bean flour was performed either naturally (NF) or induced by Lactobacillus plantarum (LP). SSF of cracked seeds was carried out by Bacillus sub7lis (BS). Soluble fracVons were used to analyse free-‐amino groups (by TNBS method), GABA (by HPLC), anVoxidant acVvity (by ORAC-‐FL), total phenolic compounds (TPC, by the Folin Ciocalteu method) and angiotensin I-‐converVng enzyme inhibiVon (by fluorescence). In addiVon, selecVve and differenVal agarized media were used for specific microbiological counts (Log CFU/ml). LSF of kidney bean for 48 h (both NF and LP) led to an extended protein hydrolysis and higher (P93% inhibiVon). In contrast, no significant differences for TPC were found. SSF for 48h and 96h gave rise to water-‐soluble fracVons with higher TPC that was accompanied with a noVceable increase in anVoxidant acVvity. Unlike LSF, protein hydrolysis was observed ayer 48 h of SSF. In addiVon, kidney bean fermented by B. sub7lis for 48 and 96 h brought about lower (P 260 ng / ml), whereas L. pentosus produced the highest concentraVon of intracellular B2 (31.8 ± 0.1 ng / ml), and L. rhamnosus the highest concentraVon of extracellular B2 (364 ± 0.1 ng/ml). All isolated strains (35) grew in syntheVc medium without B9. The total concentraVon of this vitamin was determined in a range between 16 and 76 ng / ml. L. pentosus produced the highest concentraVon of extracellular (49.84 ± 0.1 ng / ml) and L rhamnosus the highest concentraVon of intracellular B9 (30.08 ± 0.1 ng / ml). The results obtained put in evidence that naVve LAB isolated from quinoa and amaranth from NOA, produce significant levels of vitamin B2 and B9, indicaVng their potenVal to be included as starter cultures in pseudocereals containing food preparaVon with higher nutriVonal values.
San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013
IV International Symposium on Lactic Acid Bacteria: Food, Health and Applications
D9
MIXED STARTER CULTURES FROM AUTOCHTHONOUS STRAINS IN FERMENTED MEAT MODEL SYSTEMS N. Palavecino Prpich1, M. Castro1, F. Rivas1, M. Cayré, O. Garro1 and G. Vignolo2
1CONICET.
Universidad Nacional del Chaco Austral, Cte. Fernández 755. Sáenz Peña, Chaco. 2CERELA-‐CONICET. Chacabuco 145. San Miguel de Tucumán. E-‐mail:
[email protected]
The introducVon of starter cultures designed using autochthonous microflora can enhance safety of fermented meat products while preserving their sensory qualiVes. The selecVon of potenVal starter strains is based on their technological and safety characterisVcs. Each of the strains of a mixed culture meant to be added to a meat product should be compaVble. A first approach to this situaVon can be simulated in model systems where assessments compiled in vitro can be checked. Hence, the aim of this study was to evaluate the behavior of pre-‐selected autochthonous strains, as mixed cultures, in meat model systems. From a compaVbility trial, carried out by means of the agar well diffusion method, comprising seven strains of Lactobacillus sakei and three strains of coagulase negaVve cocci (CNC) (Staphylococcus vitulus and two S. xylosus strains), two pairs of strains were selected. Culture A: L. sakei 487 and S. vitulinus; culture B: L.sakei 442 and S xylosus C8. The meat model contained: minced beef and pork meat (70:30), NaCl, milk powder, sugar, spices and KNO3. The ingredients were thoroughly mixed and placed in a beaker. The meat matrix was separated into three batches, which were then subdivided into six batches to create the corresponding duplicates. Batches A and B corresponded to the aforemenVoned mixed cultures and the third one corresponded to the control system, with the addiVon of sodium azide and no added cultures. The batches were incubated at 20°C and samples were withdrawn at the beginning of the trial, one and seven days ayer inoculaVon. Microbial counts were assessed on MRS agar and mannitol salt agar (AMS). Total protein content on sarcoplasmic fracVon was determined by means of the bicinchoninic acid method and free aminoacid concentraVon was determined by the OPA method. pH measurements were also registered. Both mixed cultures adapted well to the meat environment; LAB final counts reached values of 8.6 and 8.8 log colony forming units per gram (cfu/g) and CNC final counts were 7.4 and 8.3 log (cfu/g) in batch A and B, respecVvely. Regarding pH values, they diminished from 5.54 (iniVal Vme) to 4.66 (A) and 4.68 (B) at the end of the trial, while the control system did not show pH variaVons. Final pH values from both batches are similar to those found in local dry sausages at the end of the ripening stage. Total protein content reducVon was 16% (control), 77.9% (A) and 78.4% (B) indicaVng a strong metabolic acVvity of both mixed cultures. Besides, the release of soluble aminoacids started being 0.98 mMl, showed a peak of 3.11 mM (A) and 2.43 mM (B) at 24 h, followed by a reducVon (1.99 mM) in both batches. Protein fracVon released at 24 h may have contributed to aminoacid supply for bacterial growth. The acidogenic capacity and the hydrolysis of meat proteins observed in batches A and B highlight the potenVal contribuVon of both mixed cultures to meat fermentaVon.
San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013
IV International Symposium on Lactic Acid Bacteria: Food, Health and Applications
D10
PRODUCTION OF RIBOFLAVIN BY L. PLANTARUM CRL725 IN SOYMILK. INFLUENCE OF ENVIRONMENTAL PH CONDITIONS M. Juarez del Valle1, J. Laiño 1, G. Savoy de Giori1-‐2, and JG. LeBlanc1-‐3
1Centro de Referencia para Lactobacilos (CERELA-‐CONICET), Chacabuco 145. 4000, Tucumán, ArgenVna, 2Cátedra de
Microbiología Superior, Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, Tucumán, ArgenVna. 3Cátedra de Metodología de la InvesVgación CienPfica, Facultad de Medicina, Universidad Nacional de Tucumán, Tucumán, ArgenVna. E-‐mail:
[email protected]
Soymilk, the water extract of soybean, is a rich source of high quality proteins, amino acids, unsaturated fa|y acids and isoflavones. Soymilk contains sucrose as the main carbohydrate source, which makes it a good alternaVve from milk for lactose-‐intolerant people. However, soymilk contains low concentraVons of B-‐group vitamins such as riboflavin. Riboflavin (vitamin B2) is important water-‐soluble vitamin available only through food intake, because humans lack the ability to synthesize it. Vitamin B2 is the precursor of two co-‐enzymes: flavin mononucleoVde (FAD) and flavin adenin dinucleoVde (FMN) that regulate the acVvity of many different metabolic enzymes of the redox pathways associated with energy producVon. Although riboflavin is found in a wide variety of foods, subclinical deficiency of riboflavin is common in many parts of the world, even in highly industrialized countries. Some lacVc acid bacteria (LAB) have ability to produce riboflavin but it producVon depends on the strain used and the culture condiVons. The ability of riboflavin producVon by 180 strains of LAB, belonging to CERELA culture collecVon, was evaluated using a commercial riboflavin-‐free growth medium. Only 43 strains were able to grow in this broth without the exogenous addiVon of riboflavin, and the vitamin producVon was evaluated by a microbiological method using Lactobacillus (L.) rhamnosus ATCC7469 as the indicator strain. L. plantarum CRL 725 was able to grow and increase 2.3 Vmes the riboflavin concentraVon in soymilk (300ng/ml). In this study, the influence of controlled and uncontrolled pH condiVons on riboflavin producVon by L. plantarum CRL 725 during soymilk fermentaVon was evaluated. The highest riboflavin producVon (900ngB2/mL) was achieved ayer 8 h of incubaVon under controlled pH (pH 6.0) whereas lower values were obtained during fermentaVon both at controlled pH of 5.0 or uncontrolled pH. These results showed that the ability of L. plantarum CRL 725 to produce riboflavin in soymilk is influenced by the environmental pH condiVons. This strain could be used to obtain soy foods enriched in riboflavin, taking into account the growth condiVons of this substrate.
San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013
IV International Symposium on Lactic Acid Bacteria: Food, Health and Applications
D11
STORAGE OF LYOPHILIZED PROBIOTIC STRAINS WITHOUT USING COLD CHAIN M.L. Cruz Pintos, V. Molina, M.P. Taranto and G. Font de Valdez
CONICET-‐CCT-‐Tucumán, Centro de Referencia para Lactobacilos (CERELA). Chacabuco 145. 4000 Tucumán, ArgenVna. E-‐mail:
[email protected]
The lacVc acid bacteria (LB) are subject to constant scienVfic studies in the field of biotechnology. The use of LB as probioVc cultures has grown significantly in last years. The incorporaVon of the probioVc cultures more adapted to the new technological processes is a requirement for their use in the modern food industry. While the fermented foods containing probioVcs LB are generally associated with milk based products that require cold chain for storage, the need to expand the use of probioVc strains in foods not requiring refrigeraVon for storage is a valuable contribuVon for the industry. The objecVve of this work was to develop new strategies of conservaVon at room temperature of probioVc strains previously selected (Lactobacillus (L.) casei CRL 731 and L. reuteri CRL 1098). In this context, the formulaVon of an economic culture media for the development of probioVc strains; the evaluaVon of different lyophilizaVon substrates and the study of storage condiVons without using cold chain considering Vme/temperature and cell viability variables were carried out. Biomass producVon was carried out with two defined experimental media (M1 and M2 for L. reuteri CRL 1098 and L. casei CRL 731, respecVvely) under controlled condiVons (temperature and pH constants). LyophilizaVon process was standardized evaluaVng different substrates: powdered skim milk (10 and 20%) and two type of soy flour-‐based beverage (A and B) to 10%; in all cases, the monosodium glutamate 5% (w/v) was used as lyoprotector. For conservaVon studies, lyophilized microorganisms were packed in bi-‐laminated polyester metallic containers with polyethylene, vacuum sealed and stored at 22 ± 2ºC during 5 months. Cell viability was determined by cell plate count using specific culture medium for each strain. The best results in terms of low cost biomass recovering were obtained with the medium containing glucose as a carbon source, manganese sulfate as essenVal salt and yeast extract, trypVcase and meat peptone as nitrogen source. With respect to the studies of different lyophilizaVon supports, 20% and 10% skim milk powder and soy beverage B (flavored soy meal) showed the best results. L. reuteri CRL 1098 retained viability during more than 2 months, yielding 107 CFU/g ayer 42 days of storage when milk at 20% was used as lyophilizaVon support and 106 CFU/g during 56 days when the support used was soy beverage B. L. casei CRL 731 remained viable at room temperature for only 14 days at the assayed storage condiVons. The minimum cell concentraVon required of probioVc microorganism to exert a beneficial effect in the host ranges from 106 to107 CFU/g. In consequence the results obtained in this study are promising as they would allow expanding the use of probioVc strains in products that not require cold chain for markeVng consVtuVng an interesVng technological applicaVon for the food industry.
San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013
IV International Symposium on Lactic Acid Bacteria: Food, Health and Applications
D12
PRODUCTION OF FLAVOUR COMPOUNDS BY INDIVIDUAL OR MIXED CULTURES OF LACTIC ACID BACTERIA G.H. Peralta, M.C. CandioV, C.V. Bergamini and E.R. Hynes
InsVtuto de Lactología Industrial, UNL/CONICET. Facultad de Ingeniería Química, SanVago del Estero 2829. 3000 Santa Fe, ArgenVna. gperalta@fiq.unl.edu.ar
The cooperaVon of lacVc acid bacteria with different enzymaVc abiliVes involved in the catabolism of amino acids (AA) has been proposed as a possible approach to increase flavour formaVon in cheese. In this work, we studied the influence on fermentaVon and volaVle compounds producVon of two strains of lactobacilli, L. paracasei 90 (Lp90) and L. casei 72 (Lc72), and their potenVal cooperaVon with a strain of S. thermophilus (St). The thermophilic culture was assayed both as viable cells (Stv) or a|enuated culture, in which case ethanol (Sta) or ultrasound waves were applied (Stu). Experiments were carried out in a sterile extract which consisted of the aqueous phase obtained from soy cheeses of 15 days of ripening, standardized to pH 5.2 and NaCl 1.5%. Experimental extracts were inoculated with Lp90 or Lc72, which possess aminotransferase (AT) acVvity towards different AA, and Stv, Sta or Stu, all derived from a strain with high glutamate dehydrogenase (GDH) acVvity. Control extracts without inoculaVon were also obtained. The pH of all the experimental extracts decreased significantly during incubaVon, due to fermentaVon of lactose and galactose, with the consequent producVon of lacVc acid. As for amino acids metabolism, significant differences were observed in α-‐ketoglutarate concentraVon. This key intermediate compound is consumed as amino group acceptor in the first step of the catabolism of AA and it is replenished from glutamic acid by GDH acVvity. ConcentraVon of α-‐ketoglutarate was lower than the controls in the extracts containing Lp90+Stv and Lp90+Sta, but higher than in the controls in extracts with Lc72, Lc72+Sta and Lc72+Stu. Diacetyl and acetoin were significantly higher in all extracts inoculated with Lp90 in comparison with the control; evidence of some cooperaVon with a|enuated cells of S. thermophilus was found, as both volaVle compounds were highest when Lp90 was combined with Sta o Stu. In extracts containing Lc72, diacetyl was higher than in the controls; the highest producVon was found when Lc72+Stv were added. Extracts with Lp90 single or mixed with Stv, Sta and Stu always contained more diacetyl and acetoin than those with Lc72. This result correlates with the aminotransferase profile of Lp90, which was highest towards Asp, as diacetyl and acetoin may derive from Asp transaminaVon in cheese. On the other hand, 3-‐methyl butanal and 3-‐methyl butanol, derived from the catabolism of Leu, were higher in all extracts with Lc72 than in the controls. This finding also matches with aminotransferase specificity of Lc72 towards branched chain AA. In extracts with Lp90, these compounds were only slightly higher than in the controls. In the present work, we found that fermentaVon of lactose and galactose and formaVon of volaVle compounds were led by the strain of Lactobacillus tested. Only minimal changes were observed which could be a|ributed to the combinaVon of these strains with St in different physiological states, and they always reinforced the influence detected for the Lactobacillus strains when used alone.
San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013
IV International Symposium on Lactic Acid Bacteria: Food, Health and Applications
D13
MOLECULAR CHARACTERIZATION OF LACTIC ACID BACTERIA ISOLATED FROM « TUNTA » PRODUCTION CHAIN R. Santos1, E. Ramos1, G. Vignolo2 and D. Zúñiga1
1 Laboratorio de Ecología Microbiana y Biotecnología Marino Tabusso. Departamento de Biología. Universidad Nacional
Agraria La Molina. Av. La Molina s/n, Lima 12, Perú. E-‐mail:
[email protected] Centro de Referencia para Lactobacilos. Chacabuco 145-‐T4000ILC, San Miguel de Tucumán, ArgenVna.
The tunta, is a fermented food which is made from naturally lyophilized naVve potatoes. Its resistance to weather and high caloric content, higher than fresh potatoes, make it a strategic product in the fragile economy of the AlVplano. Puno region (south-‐East Peru) accounts for about 70% tunta producVon in the country and actually, an esVmated 6000 tonnes of this product is traded annually. Because there is not enough informaVon of microbial populaVons in naVve products or natural fermented foods, this study aims to molecularly characterize isolates of lacVc acid bacteria (LAB) and determine their phylogeneVc affiliaVons. The study area was located in Ilave river basin, province of El Collao, southern Puno, with temperatures ranging from 1 -‐ 15 °C and average annual rainfall of around 620 mm. Two sampling were carried out in different steps within the producVon chain of tunta. Samples of raw materials, naVve potatoes immersed in the river for processing into tunta and final product were taken. QuanVficaVon and isolaVon of LAB was performed under aerobic and anaerobic condiVons, in Man, Rogosa & Sharpe (MRS) media supplemented with different sugars and yeast glucose lactose peptone agar (YGLP). The confirmaVon of presumpVve colonies was given by Gram staining and catalase tests. IsolaVon of 126 strains was achieved, they have been stored in 25% glycerol at -‐80 °C.. Ayer performing the molecular characterizaVon of 52 LAB strains by BOX-‐PCR amplificaVon (Versalovic et al. 1991) and 16S ribosomal gene sequencing, it was determined that isolates belong to the genera Lactobacillus and Leuconostoc. Eighteen strains showed 99.71% similarity to Lactobacillus curvatus LMG 9198T [AJ621550]. Five were classified into the cluster including L. curvatus LMG 9198T [AJ621550] and L. graminis DSM 20719T [AM113778]. Four strains were idenVfied as Lactobacillus sakei subsp. sakei DSM 20017T [AY204893] (100% similarity) and other as Lactobacillus brevis ATCC 14687T [EF120367] (100% similarity). Likewise, two of the strains were idenVfied characterized (100% similarity) and Leuconostoc mesenteroides subsp. dextranicum NRIC 1539T [AB023246] and 1 was associated with a 99.93% similarity to Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293T [NR 074 957]. Furthermore, two of characterized strains were idenVfied (100% similarity) as Leuconostoc mesenteroides subsp. dextranicum NRIC 1539T [AB023246] and one was related to Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293T [NR 074 957] with 99.93% similarity. Assays evaluaVng bacteriocin-‐producing capacity of the strains reflect that 41% of isolates inhibited the growth of Listeria innocua and 1% inhibited the growth of Lactobacillus plantarum. Strains were found in this study have been previously reported as bacteriocin producers and anVmicrobial compounds, others could be also used as bio-‐thickeners and even have probioVc properVes. This research aims to revaluate the biotechnological importance of LAB from naVve Andean products, taking advantage of their qualiVes in nutriVonal and hygienic improvements and quality food, for industrial and pharmaceuVcal industries. In a second stage of this study, the funcVonal and probioVc properVes of isolates and their possible importance for industry will be idenVfied
San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013
IV International Symposium on Lactic Acid Bacteria: Food, Health and Applications
D14
BIOGENERATION OF FLAVOUR COMPOUNDS BY L paracasei I90 AND L. helveticus B02 IN A HARD CHEESE MODEL F. Cuffia, I.V. Wolf, E.R. Hynes, C.V. Bergamini and M.C. Pero[.
InsVtuto de Lactología Industrial UNL-‐CONICET. SanVago del Estero 2829. 3000 Santa Fe, ArgenVna. E-‐mail: fcuffi
[email protected]
Amino acid (AA) catabolism by lacVc acid bacteria is one of the most important biochemical events that lead to bioformaVon of cheese flavour. The selecVon of adjunct strains of mesophilic lactobacilli possessing key enzymaVc acVviVes to AA catabolism and its combinaVon with appropriate primary cultures represents an interesVng strategy to accelerate or diversify the flavor development in cheeses. The aim of the present work was to evaluate the ability to produce volaVle compounds derived from AA in a hard cheese model by a strain of mesophilic lactobacilli, Lb. paracasei I90, and a strain of thermophilic starter, L. helve7cus B02, which were added individually and mixed. Hard cheese model consisted in a sterile extract standardized in salt content and pH, obtained from the aqueous phase of a Reggianito cheese manufactured with L. helve7cus B02 and ripened for 3 months. From this model system were prepared: extracts inoculated with the same strain of L. helve7cus (re-‐inoculaVon) used in cheese-‐making, extracts inoculated with L. paracasei I90, extracts inoculated with both strains, and control extracts with not added lactobacilli. They were incubated at 37ºC for 14 days. The assays were performed in duplicate, by using two independent cultures of each strain for the inoculaVon. The producVon of volaVle compounds was monitored by solid-‐phase microextracVon (SPME) coupled to GC-‐FID and GC-‐MS. A total of 38 volaVle compounds were idenVfied in the cromatographic profiles of extracts. However, only 13 were related to AA catabolism: acetaldehyde, 2-‐ and 3-‐methyl butanal, ethanol, diacetyl, acetoin, 3-‐methyl 1-‐butanol, benzaldehyde, acetophenone, aceVc acid, 3-‐methyl butanoic acid, phenylmethanol and phenol. The levels of acetaldehyde were staVsVcally higher in the extracts inoculated with both strains in comparison to the rest of the extracts. Diacetyl, 3-‐methyl 1-‐ butanol and aceVc acid had higher area values in all inoculated extracts in comparison to the controls. In parVcular, aceVc acid was produced in higher levels in the extracts containing the mixed strains than in those with individual strains. A preferenVal producVon of isovaleric acid was observed in the extracts with L. paracasei I90 both alone as combined with L. helve7cus B02. The cooperaVon effect between both strains was only detected for acetaldehyde and aceVc acid. The benzaldehyde and phenylmethanol levels were always higher in the control extracts. Both strains did not significantly increase the amount of the remaining volaVle compounds derived from AA. In the present study, L. helve7cus B02 and L. paracasei I90 affected the producVon of some volaVle compounds that play a key role in the aroma, therefore, these strains could be used in cheese-‐ making in order to improve flavour development.
San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013
IV International Symposium on Lactic Acid Bacteria: Food, Health and Applications
D15
PRODUCTION OF NATURAL FOLATE AND RIBOFLAVIN BY POTENTIAL LACTIC ACID BACTERIA ISOLATED FROM GOAT’S MILK AND CHEESE PRODUCED IN SÃO PAULO, BRAZIL F.F.P. da Silva1, J.G. LeBlanc2 and B.D.G. M. Franco1
1Faculdade de Ciências FarmacêuVcas, Universidade de São Paulo Avenida Professor Lineu Prestes 580, São Paulo, SP,
Brasil. E-‐mail:
[email protected]. 2Centro de Referencia para Lactobacilos (CERELA-‐CONICET). Chacabuco 145, Tucumán, ArgenVna.
Goat milk is considered an important product in many countries due to its economic and nutriVonal values. The important nutriVonal properVes and organolepVc characterisVcs have made its dairy products, such as cheeses, recognized as high value-‐added products. The majority of fermented products are produced by lacVc acid bacteria (LAB) which have a lot of beneficial properVes. Some LAB possess the ability to produce vitamins which are micronutrients that are essenVal for the metabolism of living organisms. B-‐group vitamins, such as folate and riboflavin (B2), play important roles in metabolic processes such as energy producVon and nucleoVdes and enzymes synthesis. The aim of this study was to determinate the producVon of natural folate and riboflavin by potenVal LAB isolated from goat’s milk and cheeses produced in São Paulo, Brazil. A total of 127 potenVal LAB (Gram posiVve and catalase negaVve) were isolated from goat’s milk and cheeses samples. A group of 34 isolates (10 isolates from cheeses and 24 isolates from goat’s milk) were able to grow in the absence of folate ayer 7 passages in folate-‐free medium. These 34 isolates were used to determinate the extracellular (EC) and intracellular (IC) producVon of natural folate and riboflavin. Folate determinaVon was performed using a modified microbiological assay using Lactobacillus casei subsp. rhamnosus NCIMB 10463 as indicator strain. Riboflavin determinaVon was performed using a similar microbiological assay using Lactobacillus rhamnosus ATCC 7469 as indicator strain. The average of total natural folate concentraVon produced was 91.2 ng/ml and the values ranged from 2.4 to 340.6 ng/ml. A total of 18 isolates (52.9%, 5 isolates from cheeses samples and 13 isolates from goat’s milk samples) were able to produce very high levels of natural folates (value over 91.2 ng/ml). The mean value of total folate produced by isolates from goat’s milk samples were 98.4 ng/ml and by isolates from cheeses samples were 73.8 ng/ml. The average of extracellular concentraVons was 25.8 ng/ml and the values ranged from 1.1 to 227.7 ng/ml with 9 isolates producing more than average. The mean value of intracellular concentraVons was 65.3 ng/ml, ranging from 1.3 to 150.1 ng/ml and half of the isolates (17) produced more than mean value. One isolate from a goat’s milk sample was the best folate producer, yielding 340.6 ng/ ml total folate (EC = 227.7 ng/ml and IC = 112.9 ng/ml). Even though some isolates were able to grow without riboflavin, none were able to produce this vitamin. The potenVal lacVc acid bacteria isolated in this study showed good results for folate producVon and probably they will have good potenVal to be used in the producVon of a variety of bioenriched foods and are currently being idenVfied.
San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013
IV International Symposium on Lactic Acid Bacteria: Food, Health and Applications
D16
INOCULUM VARIATION STUDY OF Lactobacillus paracasei AND Bifidobacterium longum ON SOY SOLID STATE FERMENTATION A. Rodríguez de Olmos, M.S. Garro
Centro de Referencia para Lactobacilos (CERELA-‐CONICET CCT Tucumán). Chacabuco 145, (T4000ILC) Tucumán, ArgenVna.
[email protected].
Solid state fermentaVon (SSF) is a very interesVng technological alternaVve to obtain new products due to it uses waste and cheap raw material like substrate. In this sense soy is a great substrate to use for its high nutriVonal value and low cost. In previous studies the behavior of Bifidobacterium (B.) longum CRL 849 and Lactobacillus (L.) paracasei subsp paracasei CRL 207 on soy SSF with different moisture content (50, 55, 65, 75, 80 %) and several temperatures (31, 33, 37, 41, 43 ºC) were analyzed. These assays were performed using 4% inoculum. These results demonstrated that both strains were able to develop on SSF proposed and some parameters of fermentaVve process were opVmized. The condiVons selected were: moisture 65% and 37ºC. The aim of this study was to analyze different inoculum concentraVons on the behavior of both strains under previously opVmized condiVons of soy SSF. Pastes were made from commercial soy flour and disVllated water to obtain 65% moisture; they were sterilized and inoculated with 1, 2 and 4% cultures of each strain. These were incubated at 37ºC for 24 h in adequate condiVons for each strain and samples at different Vmes (0, 4, 8, 12, 16, 24 h) were taken. The growth, through the measurement of pH and plate counts, proteolyVc acVvity and β-‐glucosidase acVvity were analyzed. At the end of fermentaVon, CRL 849 populaVon reached around 9 log CFU g-‐1 for the three assayed inoculum and pH values decreased c.a. 1,55 Vmes respect to the control, nevertheless the acidificaVon rate was lower for 1% inoculum. The behavior was similar for the three condiVons assayed respect to amino acids intake, soluble protein concentraVon decrease and β-‐glucosidase acVvity enhancement compared with non-‐inoculated control 4). On the other hand, populaVon of CRL 207 reached around 9.6 log CFU g-‐1 for the three inoculum, however pH values decreased slightly in all cases. Increase of amino acids amount and evidence of β-‐glucosidase acVvity was observed without significant differences between inoculum concentraVons. Taken together, obtained results show a different behavior for each strain. In addiVon, we can conclude that it is not necessary to work with 4% inoculum because with lower iniVal bacterial amounts is possible to obtain results with significant technological impact.
San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013