Posters: Metabolism, physiology & biochemistry - Centro de ...

IV International Symposium on Lactic Acid Bacteria Food, Health and Applications

Posters: Metabolism, physiology & biochemistry

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 A1. N. Romano, L. Pedroni, E. Gerbino, P. Mobili, A. Gómez-‐Zavaglia A2. M.F. Vega, S.N. Diéguez, B. Riccio, S. Aranguren, A. Giordano, A. L. Soraci, M.O. Tapia, S.N. González A3. A.A. Hugo, P. Alves, E. Tymczyszyn, Felipe Szymanowski, P. Perez, J.F.J. Coelho, P.N. Simões, A. Gomez-‐Zavaglia A4. A.N. Ramos, S. Lindon, M.E. Cruz, M.E. Sesto Cabral, C.A. Cabrera, J.C. Valdez A5. C.A. Cabrera, A.N. Ramos, M.E. Sesto Cabral, S. Lindón, M.E. Cruz, J.C. Valdez A6. B.M. Bravo-‐Ferrada, A. Hollmann, L. Delfederico, D. Valdés La Hens, A. Caballero, L. Semorile A7. M.V. GangoiS, M.F. Hamet, M. Medrano, A.I. Puertas, J, Piermaria, M.T. Dueñas, A.G Abraham A8. F. Guzmán, V.G. Herrera, S.N González, R. Oliszewski A9. S. Layús Paz, E.M. Hébert, G. Font de Valdez and M.I. Torino A10. M. Pescuma, E. M. Hébert, F. Mozzi, G. Font de Valdez A11. L.G. Ruiz Rodríguez, J. Bleckwedel, R. R. Raya, E. M. Hébert, F. Mozzi A12. C.E. Ale, S.E. Pasteris, M.C. Otero, V.I. Morata A13. J.M. Villegas, E.M. Hebert, G. Savoy de Giori A14. M. Fernández de Ullivarri, L.M. Mendoza, R.R. Raya A15. M. Novicov FancioY and M.C. Audisio A16. L. Pinuer, J. Ferrer, R. Bórquez, A. García A17. J.M. Jiménez Medina, E. Salas Osorio, G. Medina Ramírez A18. M. E. Lucca, F. Siñeriz A19. M.E. OrSz, R.R. Raya, F. Mozzi A20. G. Marcial, J. Messing, G. Faller, G. Font de Valdez, A. Hensel A21. G. Marcial, A. Hensel, J. Messing, G. Faller, G. Font de Valdez A22. N. Taboada, M. Molina, S. Lopez Alzogaray, R. Medina A23. C.E. Ale, S.E. Pasteris, E. Bru, V.I. Morata, A.M. Strasser de Saad

San Miguel de Tucumán, Tucumán, ARGENTINA. October 16-18, 2013

IV International Symposium on Lactic Acid Bacteria: Food, Health and Applications

A1

EFFECT OF ETHANOL PRETREATMENT AND FRUCTO-‐ OLIGOSACCHARIDES ON THE GROWTH OF BACTERIAL STRAINS ISOLATED FROM AGAVE MUST IN A SYNTHETIC MEZCAL MEDIUM N. Romano, L. Pedroni, E. Gerbino, P. Mobili and A. Gómez-‐Zavaglia

Centro de InvesSgación y Desarrollo en Criotecnología de Alimentos (CIDCA, CCT-‐La Plata). Calle 47 y 116. La Plata, ArgenSna. E-‐mail: [email protected]

Mezcal, a tradiSonal beverage from Mexico, is obtained by disSllaSon of the fermented must obtained from the "cores" (leaf bases and stems) of Agave salmiana and Agave americana. Agave cores are rich in inulin, and afer thermal hydrolysis and pressing they release a juice rich in fructose that is then fermented by many different micro-‐organisms present in the environment. Unlike tequila, the disSllate of Agave tequilana fermented must, mezcal is mainly a homemade product. Therefore, the fermentaSon condiSons and the use of starter microorganisms, among other aspects of the process, are mostly not controlled variables. Thus, the idenSficaSon and characterizaSon of the microorganisms involved in the fermentaSon and their growth properSes are relevant to opSmize the producSon process. Along the fermentaSon process, microorganisms have to overcome stress condiSons such as low pH and increasing ethanol concentraSons. Fructo-‐ oligosaccharides (FOS) are widely known prebioScs and, due to their hydroxylated nature, may act as membrane protectants under such stress condiSons. The CIDCA collecSon has more than 50 isolates of bacteria from mezcal must, and several of them are Gram (+), catalase (-‐) bacilli. In the present work we evaluated the ability of 9 of these isolates to grow in an arSficial medium with a composiSon resembling that of the late fermented must (named SMM from “syntheSc mezcal medium”), and the effect of acclimaSzaSon with ethanol and the FOS supplementaSon. To determine the ethanol tolerance the isolates were pretreated as follows: 1) shock: 30 min incubaSon at high ethanol concentraSons (12 or 25 % v/v in saline soluSon), or 2) acclimaSzaSon: 48 h incubaSon in modified MRS with sub lethal ethanol concentraSons (6 or 8 % v/v). In both cases, afer treatment the cells were inoculated into SMM (ethanol 12 % v/v and pH 3.5). To analyze the protecSve effect of FOS, the syntheSc mezcal medium was supplemented with increasing concentraSons (0 to 5 % w/v) of FOS (Orafi p95). Growth kineScs at 30 °C were followed by recording the opScal density values (OD) at 590 nm. The lag phase extension and the final OD of the cultures were considered as the response parameters. Afer a shock with 12 % v/v ethanol, the lag Sme and final OD did not change with respect to the control. No growth in SMM was detected for any of the isolates afer the exposure to 25 % v/v ethanol for 30 min. The acclimaSzaSon with 6 or 8 % v/v ethanol increased the tolerance of some strains to the stress condiSons in SMM, as shown by the shorter lag phases and higher final OD. The addiSon of FOS to the SMM posiSvely affected bacterial growth, showing shorter lag phase and higher final OD than those observed in pretreated or untreated cells. This growth promoSng effect was increased at higher FOS concentraSons and might act in a synergisSc way with the acclimaSzaSon.



A2

FEED ADDITIVE WITH ANTI-‐ZEARALENONE EFFECT TO IMPROVE REPRODUCTIVE HEALTH OF PIGS M.F. Vega1, S.N. Diéguez2, B. Riccio2, S. Aranguren2, A. Giordano2, A.L. Soraci2, M.O. Tapia2, R. Ross3, A. Apás3 and S.N. González3

1Departamento de Tecnología y Calidad de los Alimentos. Facultad de Ciencias Veterinarias. Universidad Nacional del

Centro de la Provincia de Buenos Aires. Paraje Arroyo Seco S/N, 7.000 Tandil, Buenos Aires, ArgenSna. 2Laboratorio de Toxicología (SNITV). Facultad de Ciencias Veterinarias. Universidad Nacional del Centro de la Provincia de Buenos Aires. 3Departamento de Salud Pública. Facultad de Bioquímica Química y Farmacia. Universidad Nacional de Tucumán. E-‐ mail: [email protected]

Zearalenone (ZEA) is a mycotoxin with estrogenic effects in humans and animals. This mycotoxin affects mostly pigs, causing severe reproducSve disorders and economic losses for producers. To develop techniques for detoxificaSon, the ability to adsorb ZEA was evaluated for bacterial precipitates obtained by reacSvaSon and centrifugaSon of lactobacilli strains isolated from rectal swabs of pigs from Tandil, Buenos Aires, ArgenSna. The bacterial cultures were incubated, centrifuged, washed and resuspended in contact soluSon with ZEA. To evaluate the degree of adsorpSon (%ADS), the remaining mycotoxin was measured in soluSon by HPLC-‐UV. The volume and type of iniSal culture medium, and the contact soluSon used in the adsorpSon process were opSmized. The strength of the ZEA-‐bacteria complex was analyzed by successive rinsing. In every case the bacterial adsorpSon capacity was higher than 40%, and the final adsorpSon percentage of the selected strain (Lactobacillus plantarum) was 68.20%. Afer three rinses the amount of retained ZEA in complex was 15.82% of that originally added. During the lyophilizaSon process, the Lactobacilli were suspended in lactose, skim milk, and ascorbic acid, and were then freeze-‐dried. This process resulted in 109 CFU/g in the final powder, and an adsorpSon percentage of 87.9%. In order to conduct a live test, ZEA concentrate was obtained by incubaSng Fusarium graminearum strain NRRL 28063 in corn producing only zearalenone. This concentrate was obtained afer two extracSons, first alkaline and then with chloroform, and contained 6.5 g total of the mycotoxin. Prepuberal gilts were fed a diet containing 1 ppm ZEA for 15 days, resulSng in oestrogenic prevenSon during the first four, which jusSfies further studies of immunotoxicity.



A3

EVALUATION OF LIPID OF LACTIC ACID BACTERIA FOR THE STABILIZATION OF POLYMER-‐LIPOSOME COMPLEXES WITH POTENTIAL APPLICATIONS IN DRUG DELIVERY A.A. Hugo1, P. Alves2, E. Tymczyszyn1, Felipe Szymanowski1, P. Perez1, J.F.J. Coelho2, P.N. Simões2 and A. Gomez-‐Zavaglia1

1Centro

de InvesSgación y desarrollo en Criotecnología de Alimento. Calle 47 y 116 – La Plata – Buenos Aires – ArgenSna (1900). 2Department of Chemical Engineering, University of Coimbra, P-‐3030-‐790 Coimbra, Portugal

Liposomes are awracSve materials for drug delivery and the lipid composiSon is responsible for the stabilizaSon of liposome formulaSons. Bacterial lipids are mainly found in cell membranes and have a crucial role in stabilizing the membrane structure when cells are exposed to stress processes. The lipid composiSon of lacSc acid bacteria includes cardiolipin and phosphaSdylglycerol as main phospholipids and three different glycolipids. In addiSon, the unsaturated/saturated fawy acids raSo is also related with the stability to different kinds of stresses involving membrane damage (i.e.: freeze-‐thawing, lyophilizaSon). Thus, lipids from lacSc acid bacteria represent natural formulaSons that may be potenSally useful in drug delivery. The internalizaSon of liposomes in the Sssues and the acSon on drug entrapped in target cells can be favoured by the incorporaSon of pH-‐sensiSve polymer, such us Poly(2-‐(dimethylamino)ethyl methacrylate) (PDMAEMA). With this background, the aim of this work was to study the stability of liposomes formed by a lipid extract of Lactobacillus delbrueckii subsp. lac9s CIDCA 133 with different concentraSons of PDMAEMA. The leakage at different pHs was evaluated using calcein entrapped liposomes. The formulaSon containing 5 and 10% of PDMAEMA showed highest stability at pH 7 and 37°C. When pH decreased to 5, a rapid release of calcein was observed. Similar results were found in lecithin/PDMAEMA liposomes. Liposome formulaSons were preserved at 4°C, frozen at -‐20 and -‐80°C and freeze-‐dried. 250 mM Trehalose was used as cryoportectant agent in all cases. Afer freeze-‐drying, liposome formulaSons containing bacterial lipids showed higher stability than those prepared with lecithin. The presence of trehalose in the medium increased the stability of lipid formulaSon containing bacterial lipids and PDMAEMA 5% along Sme. In vitro studies on Raw 264.7 and Caco-‐2/TC7 cells demonstrated an efficient incorporaSon of liposomes into the cells. These results substanSate the efficiency of PDMAEMA incorporated onto bacterial lipids liposomes to assist for the release of the liposome content in mildly acidic environments, like those found in early endosomes where pH is slightly lower than the physiologic. In summary, the main achievement of this work was the uSlizaSon of bacterial lipids from Lactobacillus delbrueckii subsp. lac9s in the formulaSon of polymer-‐liposomes with noSceable advantages on the stabilizaSon respect to lecithin based formulaSons.



A4

PHARMACEUTICAL BIOTECHNOLOGY: METHODS TO OPTIMIZE THE PRODUCTION OF METABOLITES FROM Lactobacillus PHARMACOLOGICALLY ACTIVES A.N. Ramos, S. Lindon, M.E. Cruz, M.E. Sesto Cabral, C.A. Cabrera and J.C. Valdez

Cátedra de Inmunología. InsStuto de Microbiología. FBQyF-‐UNT. Ayacucho 471 CP 4000. San Miguel de Tucumán, ArgenSna. Email: [email protected]

Our medical team applied Lactobacillus plantarum (whole culture and supernatants) in chronic wounds (diabeSc foot ulcers, venous ulcers, burns, etc) with very encouraging results. In previous works we demonstrated that L. plantarum culture supernatants (LpS) have anSpathogenic acSviSes (on bacteria typically isolated from chronic wounds), pro-‐healing and anestheSc properSes. Also was determined the bioacSve metabolites responsible for the aforemenSoned properSes and its acSon mechanism was hypothesized. The aim of this work was to design methods and culture media that maximize the producSon of the above metabolites to increase the therapeuSc effecSveness of the supernatants. ModificaSons were made in MRS broth on its composiSon and concentraSon of carbohydrates, proteins, boron, caSons and surfactants sources. L. plantarum was culSvated in the different media for 12 h at 37 °C with disSnct physicochemical condiSons (CO2 concentraSon, agitaSon, etc.). Modified supernatants (LpSm) were obtained by centrifugaSon and filtraSon. AnSpathogenic acSvity of LpSm was quanSfied using LpS as control: 1) bacteriostaSc and bactericidal (growth curves in microplates), 2) inhibiSon of biofilm formaSon and disrupSon of pre-‐formed biofilm (crystal violet technique in microplates and polypropylene beads). The tested pathogenic strains represent 90% of the aerobic isolates in chronic wounds. The concentraSon of bioacSve metabolites in LpSm was quanSfied by gas chromatography in tandem with mass spectrometry (GCMS). In healthy volunteers, we evaluated the LpSm relaSve anestheSc potency and effect duraSon compared to lidocaine 2%. The LpSm obtained from media with high concentraSons of yeast extract possessed the greatest anestheSc power due to a greater producSon of barbiturates (GCMS). Those containing greater amount of meat extract, caSons and surfactants had the highest capacity of biofilm disrupSon. When the glucose and galactose concentraSon were increased in media, the LpSm had the greatest bacteriostaSc and bactericidal power as well as those grown with higher concentraSons of CO2. This was due to increased producSon of organic acids, mainly lacSc acid. Finally, media with higher concentraSons of boric acid had higher inhibitory capacity of biofilm formaSon by increased producSon of furanosyl borate diester (autoinducers type 2). The idenSficaSon of the precursor substrates of the bioacSve metabolites allows in the future an LpSm manufacture with greater therapeuSc power than LpS and even custom properSes for each type of wound.



A5

PHARMACEUTICAL BIOTECHNOLOGY INTERFACE IN THE DESIGN OF FORMULATIONS CONTAINING ACTIVE METABOLITES FROM LAB C.A. Cabrera, A.N. Ramos, M.E. Sesto Cabral, S. Lindón, M.E. Cruz and J.C. Valdez

Cátedra de Inmunología. InsStuto de Microbiología. FBQyF-‐UNT. Ayacucho 471 CP 4000. San Miguel de Tucumán, ArgenSna. Email: [email protected]

Chronic wounds are those that are arrested in the inflammatory stage of healing. Regardless of the background pathology, the main cause of chronicity is infecSon produced by biofilm forming bacteria. These can adhere to a living Sssue and grow embedded in a matrix of polymers producing by themselves. This confers them resistance to anSbioScs, anSsepScs and immune system. Therefore, current therapies for the treatment of these wounds require anSpathogenic and pro-‐ healing properSes. Some authors ensure that Pseudomonas aeruginosa causes chronicity and perhaps represent a predisposing factor for addiSonal bacteria colonizaSon. In previous works we demonstrate that Lactobacillus plantarum culture supernatants of (Lps) have anSpathogenic acSvity against P. aeruginosa inhibiSng in vitro adhesion, growth, quorum sensing system, biofilm and virulence factors. Also Lps showed a significant pro-‐healing capacity in human chronic wounds (diabeSc foot ulcers, burns, etc.). However, its liquid state makes difficult the applicaSon and therefore it reduced its therapeuSc effecSveness. The aim of this work was to contribute to the development of pharmaceuScal biotechnology, by designing a low-‐cost effecSve, stable and secure medicament using Lps as acSve pharmaceuScal ingredient (API) for chronic wounds topical treatment. Semisolid formulaSons were designed. Besides pharmacotechnical quality controls (established by Pharmacopoeia) and in vitro release studies of API were carried out. To confirm the conservaSon of previously reported acSviSes, convenSonal microbiological assays were adapted to evaluate the selected semisolid formulaSons: 1) growth inhibiSon (growth curves in microplates), 2) biofilm formaSon inhibiSon and preformed biofilm disrupSon (crystal violet technique in microplates). The selected formulaSons showed opSmum mechanical and rheological properSes and a pH within the accepted range. The pharmacotechnical and anSpathogenic properSes were maintained for at least six months. From in vitro release studies, it was found that the acSve pharmaceuScal ingredients were released from the formulaSons in opSmal quanSSes to maintain its acSviSes. These studies allowed also determine the repeSSon Sme of treatment and posology. This vehicle provides occlusive properSes to keep moisture in wounds and promote the development of granulaSon Sssue as required in ischemic wounds such as diabeSc foot ulcers. The larger-‐scale producSon will allow the applicaSon of an adjuvant treatment for infected chronic wounds paSents in hospitals of San Miguel de Tucumán, increasing the effecSveness of convenSonal treatments.



A6

PATAGONIAN RED WINES: SELECTION OF Lactobacillus plantarum ISOLATES AS POTENTIAL STARTER CULTURES FOR MALOLACTIC FERMENTATION B.M. Bravo-‐Ferrada1, A. Hollmann1, L. Delfederico1, D. Valdés La Hens, A. Caballero2 and L. Semorile1

1Laboratorio de Microbiología Molecular, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes,

Bernal, ArgenSna. 2Facultad de Ciencia y Tecnología de los Alimentos, Universidad Nacional del Comahue, 25 de Mayo y Reconquista, (8336) Villa Regina, Río Negro, ArgenSna e IDEPA CONICET-‐Universidad Nacional del Comahue, Buenos Aires 1400, (8300) Neuquén, ArgenSna. Email: [email protected]

SelecSon of LAB strains for wine inoculaSon should essenSally be based on a high malolacSc acSvity under harsh condiSons encountered. Further features to be taken into account correspond to interesSng oenological properSes such as the presence of enzymaSc acSviSes involved in the producSon of desirable wine aromas and the absence of biogenic amines producSon. North Patagonia is the southernmost wine-‐producing region of ArgenSna. Both, spontaneous and conducted grape juice fermentaSons (AF) are carried out and young dry red (85%) and white wines (12%) from neutral Vi9s vinifera varieSes are mostly produced. In these wines, malolacSc fermentaSon (MLF) takes place spontaneously, and it is ofen unpredictable. For this reason, it would be important to dispose of indigenous malolacSc starters to ensure that the process is carried out successfully, keeping the characterisScs of the terroir. Previous studies of LAB populaSon diversity in Patagonian red wines showed that both Oenococcus oeni and Lactobacillus plantarum are the major species present through spontaneous MLF. The aim of this study was to evaluate fify three Lb. plantarum isolates obtained from a Patagonian red wine, molecularly idenSfied and typified using RAPD analysis, in order to select starter cultures for MLF. The results obtained suggest a considerable geneSc diversity, taking into account that all Lb. plantarum isolates were obtained from one cellar and one vintage. Based on the capacity to tolerate a concentraSon of 14% ethanol in MRS broth, eight isolates were selected for the subsequent analysis. The incidence of various wine stress factors (ethanol, acid pH, lysozyme and sulfur dioxide) on isolates growth was studied. Besides, glucosidase and tannase acSviSes were evaluated, and the presence of genes involved in the synthesis of biogenic amines was examined by PCR. A previously characterized indigenous O. oeni strain was included with comparaSve purposes. Differences in technologically relevant characterisScs were observed among the eight selected isolates, revealing an isolate-‐dependent behavior. Detectable glucosidase and tannase acSviSes were found in all isolates. The presence of genes encoding hisSdine and tyrosine descarboxylases and putrescine carbamoyltransferase was not detected. The ability of Lb. plantarum isolates to grow and consume L-‐malic acid in simulated laboratory-‐scale vinificaSons revealed that two of them could be considered as possible MLF starter cultures for Patagonian red wines. These isolates will be subjected to further analysis, for a final winery technological characterizaSon.



A7

GENOTYPIC AND PHENOTYPIC CHARACTERIZATION OF EXOPOLYSACCHARIDE-‐PRODUCING Lactobacillus STRAINS ISOLATED FROM KEFIR GRAINS M.V. GangoiS1, M.F. Hamet2, M. Medrano2, A.I. Puertas3, J, Piermaria, M.T. Dueñas3, A.G Abraham1,2

1Area Bioquímica y Control de Alimentos – Facultad de ciencias Exactas, UNLP. 47 y 115 (1900) La Plata, Buenos Aires,

ArgenSna. 2Centro de InvesSgación y Desarrollo en Criotecnología de Alimentos (CIDCA). 47 y 116 (1900) La Plata, Buenos Aires, ArgenSna. 3Dpto. de Química Aplicada, Dpto. de Ciencia y Tecnología de Polímeros, Facultad de Ciencias Químicas. Universidad del País Vasco. Paseo Manuel de Lardizabal, 3 (20018), San SebasSán, País Vasco. E-‐mail: [email protected]

Some lacSc acid bacteria (LAB) isolated from kefir grains, exert the ability to develop ropy colonies when growing in MRS-‐Agar, that could be associated to exopolysaccharide (EPS) producing bacteria. EPS producing LAB have the ability to synthesize in situ these biopolymers, which could act as natural thickening agents, improving texture on fermented products. This ability, and the GRAS status that LAB possesses, confers these microorganisms an addiSonal value to be used on dairy industry. The aim of the present study was to conduct a genotypic and phenotypic characterizaSon of the EPS producSon of 3 Lactobacillus paracasei strains (CIDCA 8339, CIDCA 83123, CIDCA 83124) and 2 L. kefiranofaciens strains (CIDCA 83118, CIDCA 83119) isolated from kefir grains. For genotypic characterizaSon, the presence of the priming enzyme glycosyl transferase (pGT) -‐essenSal for heteropolysaccharides synthesis-‐ was determined by PCR. The obtained products were amplified and sequenced, finding that the pGT gen was present in all the studied strains. For phenotypic characterizaSon, all the microorganisms were grown in milk under their respecSve opSmal culture condiSons, and EPS producSon was characterized. EPS produced by these strains was between 140 and 301,9 mg/l. Molecular weight of EPSs was determined by high pressure liquid chromatography (HPLC) using a molecular exclusion column associated to a refracSon index detector. Different fracSons were found, with molecular weights between 104-‐106 Da for EPSs produced by L. paracasei and 102-‐105 Da for those produced by L. kefiranofaciens. To conduct all the determinaSons, two EPS producing strains were used as posiSve controls: L. delbrueckii subsp bulgaricus CIDCA 332 and L. kefiranofaciens subsp kefiranofaciens JCM 6985. The evaluaSon of the flow curves of the fermented products of all the studied strains showed a pseudoplasSc fluid behavior, and presented apparent viscosity values and hysteresis areas higher to acid milk gels obtained by chemical acidificaSon of milk with δ-‐gluconolactona. It can be concluded that Lactobacillus studied possess the gen encoding enzyme responsible for heteropolysaccharide synthesis. The phenotypic expression of this gen results in the producSon of EPSs that modify rheological behavior of fermented milks. Taking into account the present results these strains and the EPSs they produce are interesSng candidates for their applicaSon in food industry.



A8

MANUFACTURE OF GOAT CHEESE WITH ADDITION OF AUTOCHTHONOUS STARTER AND ADJUNCT ACID LACTIC CULTURES: IMPACT ON CHEESE FLAVOUR F. Guzmán1, V.G. Herrera2, S.N González3 and R. Oliszewski1

1LACALAC. Fac. Agronomía y Zootecnia UNT. Av. Roca 1900. 4000. Tucumán, ArgenSna. 2INTA EEA Catamarca. Campo

Anexo Santa Cruz. 3CERELA-‐CONICET. E-‐mail: [email protected]

The use of autochthonous acid lacSc bacteria cultures contribute to the typicality of the cheese and ensure food safety. The acid lacSc cultures govern during cheese ripening, quality parameters such as texture, flavor and aroma, of fundamental importance in the final product. The aim of this work was to manufacture a pilot scale semi-‐hard goat cheese with autochthonous lacSc acid bacteria starter and adjunct cultures to evaluate the performance of autochthonous adjunct strains on the sensory percepSon of cheeses. Two batches were made of semi-‐hard cheeses from 40 L of goat milk. Lb bulgaricus ETC2 was used as starter and E. faecium BM6 and BM18 as adjunct cultures. Control batch (C) was manufactures only with starter culture (1% w/v) while Experimental batch (E) with starter culture (1% w/v) and the addiSon of adjunct cultures (0.5% w/v). The milk was pasteurized, acid lacSc cultures were added at 42 °C, then calcium chloride (0.01% w/v) and bovine liquid rennet (0.05% v/v) were added, the mass was cut corn grain size, molded and pressed up to pH 5.2 and then salted in brine (20% w/v, 12 h/K cheese). Two cheeses of 600g per batch were obtained. There were two independent trials on consecuSve days, using the same milk to avoid variaSons in composiSon. Cheeses microorganisms were analyzed by plaSng: lactococci by LAPTg at 30° and 45°C, lactobacilli by MRS at 30° and 45°C and enterococci by KF at 45°C, all incubated 48 to 72 h, coliforms at 30°C using VRBA coliforms at 45°C by VRBGA, incubated 24 to 48 h, and fungi and yeasts at 30°C incubated 5 d. Sensory analysis was performed by triangle test. Experimental and Control batches were evaluated by 24 panelists. 3 porSons cheese randomly and unidenSfied (2 porSons for a batch and 1 porSon for another) were evaluated by each judge. The choice of the different porSon by each judge allows to evaluate staSsScally the number of posiSve hits of the panel, for which a minimum of 13 panelists from a total of 24 must find the different sample (significance level p