WO1999003971A1 - Microorganism of the species lactococus lactis ssp. lactis cect 4478 usable for the fabrication of cheese - Google Patents

Microorganism of the species lactococus lactis ssp. lactis cect 4478 usable for the fabrication of cheese Download PDF

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Publication number
WO1999003971A1
WO1999003971A1 PCT/ES1998/000209 ES9800209W WO9903971A1 WO 1999003971 A1 WO1999003971 A1 WO 1999003971A1 ES 9800209 W ES9800209 W ES 9800209W WO 9903971 A1 WO9903971 A1 WO 9903971A1
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cect
cheese
ssp
microorganism
lactis
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PCT/ES1998/000209
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Spanish (es)
French (fr)
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Teresa Requena Rolania
Jesús RODRIGUEZ FERNANDEZ
Carmen Pelaez Martinez
Manuela Juarez Iglesias
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Consejo Superior De Investigaciones Cientificas
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Priority claimed from ES9701576A external-priority patent/ES2125205B1/en
Application filed by Consejo Superior De Investigaciones Cientificas filed Critical Consejo Superior De Investigaciones Cientificas
Publication of WO1999003971A1 publication Critical patent/WO1999003971A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/02—Making cheese curd
    • A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
    • A23C19/0323—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin using only lactic acid bacteria, e.g. Pediococcus and Leuconostoc species; Bifidobacteria; Microbial starters in general
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/21—Streptococcus, lactococcus
    • A23V2400/231—Lactis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Definitions

  • the technique is framed in the dairy sector, subsectors of cheeses and microbial cultures.
  • the starter cultures used for cheese making are generally made up of one or several strains belonging to one or several species of lactic bacteria, which are used to inoculate pasteurized milk in order to initiate fermentation.
  • the microorganisms that are part of the starter cultures in the Dairy Industry are bacteria of the genera Lactococcus, Streptococcus, Lactobacillus and Leuconostoc
  • modified lactic bacteria In addition to the use of high ripening temperatures, or the incorporation of enzymes, the addition of modified lactic bacteria has been suggested as a method with better future prospects to accelerate cheese maturation, by modifying to a lesser extent the natural enzymatic balance of the cheese and have greater specificity.
  • the inoculation of the milk in the curdling bowl of a concentration much higher than normal of viable lactic bacteria can produce an excess of lactic acid.
  • the research has been aimed at modifying the functions of the starter culture, reducing its ability to ferment lactose without detriment to other metabolic activities that play an important role in cheese maturation. This can be achieved by chemical, thermal treatments or genetic manipulation.
  • Lactis CECT 4478 together with the original microorganism, in addition to two strains of Lactobacillus and one of Leuconostoc, in low proportion, as a starter culture of interest to accelerate the ripening of semi-hard cheese obtained from mixed milk of cow, sheep and goat species.
  • the microorganism Lactococcus lactis ssp. lactis CECT 4478 was selected based on its acidifying and proteolytic activity.
  • the Lac Prot " variant, deposited in the crop collection with the registration number CECT 4595, can be added in significant quantities to the curdle, without causing an increase in acidity or bitterness due to high proteolysis.
  • By maintaining the microorganism its peptidic activity is an additional source of enzymes capable of increasing the production of aroma compounds.
  • the biochemical characteristics of the modified microorganism are the same as those of Lactococcus lactis ssp. lactis CECT 4478, listed in the patent already cited except for the loss of acidifying and proteinaceous activity.
  • the starter culture used in the process indicated below is that proposed in the aforementioned patent, formed by the microorganisms Lactococcus lactis ssp. lactis CECT 4478 in a proportion of 82%, together with Lactobacillus casei ssp. casei CECT 4598, Lactobacillus plantarum CECT 4599 and Leuconostoc mesenteroides ssp. dextranicum CECT 4597, in a proportion each of 6%, to which the Lac Prt variant was incorporated " , in a proportion equal to that of the original microorganism.
  • the starter culture was prepared by mixing the different lyophilized strains in the proportions indicated above:
  • the raw material used to make the cheese was: * 55% skimmed cow's milk * 15% sheep's milk * 30% goat's milk
  • the milk was cooled to 50 ° C to begin the microfiltration concentration (MF) process.
  • the concentration process was carried out at a temperature of 50 ° C ⁇ 2 ° C until a concentration factor (FC) of 3 was reached; then the diafiltration was carried out, adding water at 50 ° C in approximately the same proportion of concentrate.
  • the MF process was completed when a concentration factor of 6 was obtained.
  • the starter culture was resuspended in permeate and added to the concentrate in sufficient proportion to reach a number of viable of the order of 10 8 cfu (colony forming units) / g.
  • the ripening of the cheeses was carried out for 60 days by sampling every two weeks. Physicochemical characteristics. At the end of maturation, the pH value was 5.3, starting with an initial value of 6.6 in milk.
  • the levels found for these indices despite the delay in maturation that generally involves the use of concentrated milk by membrane processes, were of the same order as those obtained in cheeses made by traditional procedures.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Dairy Products (AREA)

Abstract

Microorganism of which the enzymatic equipment is modified in order to obtain the microorganism Lactococcus lactis ssp. Lactis CECT (Spanish Collection of Culture Type) n°4595. The use of the initiator culture is intended to accelerate the maturation of a low fat cheese, obtained from a mixture of milks from cows, goats and ewes, which have been concentrated by ultrafiltration.

Description

TITULOTITLE
MICROORGANISMO DE LA ESPECIE LACTOCOCCUS LACTIS SSP. LACTIS CECT 4478 UTILIZABLE PARA LA FABRICACIÓN DE QUESO.MICROORGANISM OF THE LACTOCOCCUS LACTIS SSP. LACTIS CECT 4478 USED FOR CHEESE MANUFACTURING.
SECTOR DE LA TÉCNICASECTOR OF THE TECHNIQUE
La técnica se encuadra en el sector lácteo, subsectores de quesos y de cultivos microbianos.The technique is framed in the dairy sector, subsectors of cheeses and microbial cultures.
ESTADO DE LA TÉCNICA La leche de oveja y cabra tiene gran importancia regional fundamentalmente en base a la aceptación de los productos derivados, sobre todo queso.STATE OF THE TECHNIQUE Sheep and goat milk is of great regional importance mainly based on the acceptance of derived products, especially cheese.
La industrialización de quesos tradicionalmente elaborados de forma artesanal proporciona productos con un mínimo de variación en sus características, capaces de competir en el mercado europeo. Para ello, es necesario restaurar la flora microbiana beneficiosa destruida durante el tratamiento térmico de la leche, adicionando cultivos iniciadores específicos que den lugar a productos similares a los elaborados artesanalmente.The industrialization of traditionally crafted cheeses provides products with a minimum of variation in their characteristics, capable of competing in the European market. For this, it is necessary to restore the beneficial microbial flora destroyed during the heat treatment of milk, adding specific starter cultures that give rise to products similar to those made by hand.
Los cultivos iniciadores empleados para la elaboración de quesos están constituidos en general por una o varias cepas pertenecientes a una o varias especies de bacterias lácticas, que se utilizan para inocular la leche pasterizada con objeto de iniciar la fermentación. Los microorganismos integrantes de los cultivos iniciadores en la Industria Láctea son bacterias de los géneros Lactococcus, Streptococcus, Lactobacillus y LeuconostocThe starter cultures used for cheese making are generally made up of one or several strains belonging to one or several species of lactic bacteria, which are used to inoculate pasteurized milk in order to initiate fermentation. The microorganisms that are part of the starter cultures in the Dairy Industry are bacteria of the genera Lactococcus, Streptococcus, Lactobacillus and Leuconostoc
En España se fabrican todavía a nivel artesanal una larga serie de quesos muy apreciados de leche de oveja, cabra y en mezcla con leche de vaca, cuyas características difieren bastante de los que ya se elaboran a partir de leche pasterizada adicionada de cultivos iniciadores comerciales inespecíficos [MARCOS, A.(1987) Spanish and Portuguese cheese varieties. En: Cheese. Chemistry, Physic and Microbiology. Vol. 2: Major cheese group, pp.185-219 (Ed. P.F. Fox). Elsevier Applied Science Publishers, London]. Las diferencias se basan en la inespecificidad de estos microorganismos en las elaboraciones industriales, que no proporcionan productos de calidad sensorial similar a la de los productos artesanales. Estudios sobre la evolución de la flora microbiana de algunas variedades de quesos artesanales españoles han conducido al aislamiento y selección de determinadas cepas de las principales especies de microorganismos que intervienen en la maduración de estos quesos. El estudio de las características fisiológicas y de aptitud tecnológica de estas cepas se ha dirigido, en algunos casos, al desarrollo de un cultivo iniciador específico para la variedad de queso estudiada. [NUÑEZ M. (1978) Microflora of Cabrales cheese.changes during maturation. Journal of Dairy Research 45, 501-508; BARNETO, R. y Ordóñez J.A. (1979) Método de elaboración de cultivos iniciadores para la fabricación de queso Manchego. Patente de Invención n° 476.810; ORDOÑEZ, J.A.; Masso, J.A., Marmol, M.P. y Ramos, M.(1980) Contribution á l'étude du fromage "Roncal". Le Lait 60, 283- 294; RAMOS, M., Barneto, R., Suárez J.A. y Iñigo, B. (1982) Contribution to study of Mahón cheese. I. Microbiological and biochemical aspects. Chemie, Mikrobiologie, Technologie der Lebensmittel 7, 167-172; POLLET, B., Huertas, M., Roa, I., Cáceres, P. y Larriba, G. (1990) Efectos de la inoculación de cultivos lácticos en la evolución de la flora microbiana durante la maduración de los quesos del Casar. Vil Reunión Científica de Microbiología de los Alimentos. Barcelona, 23]. No obstante, la amplia gama de quesos existente en nuestro país y la aplicación de nuevas tecnologías hace que los estudios no sean suficientes. En nuestro laboratorio se han abordado estudios bioquímicos y microbiológicos de quesos semiduros de cabra artesanales e industriales de nuestro país a lo largo de la maduración, destacándose las bacterias lácticas como la flora característica de estos quesos [GÓMEZ, R., Peláez, C y De La Torre E. (1989) Microbiological study of semi-hard goat's milk cheese (Majorero) International Journal of Food Science and Technology 24, 147-151; FONTECHA, J., Peláez, O, Juárez, M., Requena, T., Gómez, R. y Ramos, M. (1990) Biochemical and microbiological characteristics of artisanal hard goat's cheese. Journal of Dairy Scien∞ 73, 1150-1157; REQUENA, T., Peláez, O y Desmazeaud, M.J. (1991) Characterization of lactococci and lactobacilli isolated from semi-hard goat's cheese. Journal of Dairy Research 58, 137-145]. El estudio y caracterización de esta flora láctica ha proporcionado la información básica fundamental para comprender su mecanismo de actuación en la elaboración del queso, facilitando así su selección como cultivos iniciadores para la elaboración de queso semiduro, principalmente de leche de cabra [REQUENA T., Peláez, P. y Juárez, M. (1993) Microorganismo de la especie Lactococcus lactis ssp. lactis CECT 4478 utilizable para la fabricación de queso. Patente de Invención n° 9302713]. Por otra parte, durante los últimos años se han abierto diferentes líneas de investigación que tienen como fin nuevas formas de utilización de bacterias lácticas como cultivos iniciadores en la industria quesera, como son la adición de lactobacilos para mejorar el aroma, maduración acelerada de quesos y la mejora genética de cultivos iniciadores. Además de la utilización de temperaturas elevadas de maduración, o la incorporación de enzimas, se ha sugerido la adición de bacterias lácticas modificadas como un método con mejores perspectivas de futuro para acelerar la maduración de quesos, al modificar en menor medida el equilibrio enzimático natural del queso y poseer mayor especificidad. La inoculación de la leche en la cuba de cuajado de una concentración muy superior a la normal de bacterias lácticas viables puede producir un exceso de ácido láctico. Como consecuencia de ello, la investigación se ha encaminado a la modificación de las funciones del cultivo iniciador, reduciendo su capacidad para fermentar la lactosa sin detrimento de otras actividades metabólicas que juegan un papel importante en la maduración del queso. Esto puede conseguirse por tratamientos químicos, térmicos o manipulación genética.In Spain, a long series of highly prized cheeses of sheep, goat's milk and mixed with cow's milk are still manufactured at an artisanal level, the characteristics of which differ considerably from those already made from pasteurized milk added from non-specific commercial starter cultures [MARCOS, A. (1987) Spanish and Portuguese cheese varieties. In: Cheese. Chemistry, Physic and Microbiology. Vol. 2: Major cheese group, pp. 185-219 (Ed. PF Fox). Elsevier Applied Science Publishers, London]. The differences are based on the non-specificity of these microorganisms in industrial elaborations, which do not provide products of sensory quality similar to that of artisanal products. Studies on the evolution of the microbial flora of some varieties of Spanish artisan cheeses have led to the isolation and selection of certain strains of the main species of microorganisms involved in the maturation of these cheeses. The study of the physiological characteristics and technological aptitude of these strains has been directed, in some cases, to the development of a specific starter culture for the variety of cheese studied. [NUÑEZ M. (1978) Microflora of Cabrales cheese.changes during maturation. Journal of Dairy Research 45, 501-508; BARNETO, R. and Ordóñez JA (1979) Method of preparation of starter cultures for the manufacture of Manchego cheese. Invention Patent No. 476,810; ORDOÑEZ, JA; Masso, JA, Marmol, MP and Ramos, M. (1980) Contribution to l'étude du fromage "Roncal". Le Lait 60, 283-294; RAMOS, M., Barneto, R., Suárez JA and Iñigo, B. (1982) Contribution to study of Mahón cheese. I. Microbiological and biochemical aspects. Chemie, Mikrobiologie, Technologie der Lebensmittel 7, 167-172; POLLET, B., Huertas, M., Roa, I., Cáceres, P. and Larriba, G. (1990) Effects of inoculation of lactic cultures on the evolution of microbial flora during the maturation of Casar cheeses. Vil Scientific Meeting of Food Microbiology. Barcelona, 23]. However, the wide range of cheeses in our country and the application of new technologies means that studies are not enough. In our laboratory, biochemical and microbiological studies of artisanal and industrial goat semi-hard cheeses of our country have been approached throughout the ripening process, highlighting lactic bacteria as the characteristic flora of these cheeses [GÓMEZ, R., Peláez, C y De La Torre E. (1989) Microbiological study of semi-hard goat's milk cheese (Majorero) International Journal of Food Science and Technology 24, 147-151; FONTECHA, J., Peláez, O, Juárez, M., Requena, T., Gómez, R. and Ramos, M. (1990) Biochemical and microbiological characteristics of artisanal hard goat's cheese. Journal of Dairy Scien∞ 73, 1150-1157; REQUENA, T., Peláez, O and Desmazeaud, MJ (1991) Characterization of lactococci and lactobacilli isolated from semi-hard goat's cheese. Journal of Dairy Research 58, 137-145]. The study and characterization of this lactic flora has provided the basic basic information to understand its mechanism of action in cheese making, thus facilitating its selection as starter cultures for the production of semi-hard cheese, mainly from goat's milk [REQUENA T., Peláez, P. and Juárez, M. (1993) Microorganism of the species Lactococcus lactis ssp. lactis CECT 4478 usable for cheese making. Invention Patent No. 9302713]. On the other hand, during the last years different lines of research have been opened with the purpose of new ways of using lactic bacteria as starter cultures in the cheese industry, such as the addition of lactobacilli to improve the aroma, accelerated ripening of cheeses and the genetic improvement of starter cultures. In addition to the use of high ripening temperatures, or the incorporation of enzymes, the addition of modified lactic bacteria has been suggested as a method with better future prospects to accelerate cheese maturation, by modifying to a lesser extent the natural enzymatic balance of the cheese and have greater specificity. The inoculation of the milk in the curdling bowl of a concentration much higher than normal of viable lactic bacteria can produce an excess of lactic acid. As a result, the research has been aimed at modifying the functions of the starter culture, reducing its ability to ferment lactose without detriment to other metabolic activities that play an important role in cheese maturation. This can be achieved by chemical, thermal treatments or genetic manipulation.
Estudios realizados sobre el microorganismo Lactococcus lactis ssp. lactis CECT 4478, origen de la Patente de Invención 9302713 (1993), han permitido la localización genética de su actividad acidificante y proteinásica, y la obtención de una variante desprovista de esas actividades enzimáticas (Lac Prot"), que manteniendo intacta la actividad peptidásica, es de potencial utilización para acelerar la maduración de quesos.Studies conducted on the microorganism Lactococcus lactis ssp. lactis CECT 4478, origin of the Invention Patent 9302713 (1993), have allowed the genetic localization of its acidifying and proteinaceous activity, and obtaining a variant devoid of these enzymatic activities (Lac Prot " ), which keeping the peptidic activity intact , is of potential use to accelerate cheese ripening.
DESCRIPCIÓN DE LA INVENCIÓN Se trata de un microorganismo, modificado en su equipamiento enzimático respecto al original (Lactococcus lactis ssp. lactis CECT 4478, aislado de leche y quesos autóctonos). La modificación consiste en la pérdida de sus actividades proteinásica y acidificante conservando la actividad peptidásica. La utilización del microorganismo original y su variante (en una proporción del 82%), junto con dos cepas de Lactobacillus y una de Leuconostoc (en proporción del 18%), da lugar a quesos con aroma y sabor característico y más marcado. La presente invención se refiere a la utilización de la variante Lac Prt" del microorganismo Lactococcus lactis ssp. lactis CECT 4478, junto con el microorganismo original, además de dos cepas de Lactobacillus y una de Leuconostoc, en baja proporción, como cultivo iniciador de interés para acelerar la maduración de queso semiduro obtenido a partir de leche de mezcla de las especies vaca, oveja y cabra.DESCRIPTION OF THE INVENTION It is a microorganism, modified in its enzymatic equipment with respect to the original (Lactococcus lactis ssp. Lactis CECT 4478, isolated from milk and native cheeses). The modification consists in the loss of your activities Proteinic and acidifying while preserving peptidic activity. The use of the original microorganism and its variant (in a proportion of 82%), together with two strains of Lactobacillus and one of Leuconostoc (in proportion of 18%), gives rise to cheeses with characteristic and more marked aroma and flavor. The present invention relates to the use of the Lac Prt " variant of the microorganism Lactococcus lactis ssp. Lactis CECT 4478, together with the original microorganism, in addition to two strains of Lactobacillus and one of Leuconostoc, in low proportion, as a starter culture of interest to accelerate the ripening of semi-hard cheese obtained from mixed milk of cow, sheep and goat species.
Tal como figura en la Patente de Invención 9302713 (1993), ya citada, el microorganismo Lactococcus lactis ssp. lactis CECT 4478, se seleccionó en base a su actividad acidificante y proteolítica. La variante Lac Prot", depositada en la colección de cultivos con el número de registro CECT 4595, puede adicionarse en cantidades importantes a la cuba de cuajar, sin provocar un aumento de acidez, ni defectos de amargor debidos a una elevada proteolisis. Sin embargo, al mantener el microorganismo su actividad peptidásica es una fuente adicional de enzimas capaz de incrementar la producción de compuestos del aroma.As stated in the Invention Patent 9302713 (1993), already mentioned, the microorganism Lactococcus lactis ssp. lactis CECT 4478, was selected based on its acidifying and proteolytic activity. The Lac Prot " variant, deposited in the crop collection with the registration number CECT 4595, can be added in significant quantities to the curdle, without causing an increase in acidity or bitterness due to high proteolysis. However By maintaining the microorganism its peptidic activity is an additional source of enzymes capable of increasing the production of aroma compounds.
Características bioquímicas del Lactococcus lactis ssp. lactis CECT 4595, Lac PrfBiochemical characteristics of Lactococcus lactis ssp. lactis CECT 4595, Lac Prf
Las características bioquímicas del microorganismo modificado son las mismas que las de Lactococcus lactis ssp. lactis CECT 4478, que figuran en la patente ya citada a excepción de la pérdida de actividad acidificante y proteinásica.The biochemical characteristics of the modified microorganism are the same as those of Lactococcus lactis ssp. lactis CECT 4478, listed in the patent already cited except for the loss of acidifying and proteinaceous activity.
Estas actividades están asociadas a un plásmido de 40 MDa, habiéndose localizado mediante ensayos de curación con incubaciones a temperaturas subletales en presencia de novobiocina. Con este procedimiento se obtuvo la variante Lac" Prot-, Lactococcus lactis ssp. lactis CECT 4595.These activities are associated with a plasmid of 40 MDa, having been located by cure tests with incubations at sublethal temperatures in the presence of novobiocin. With this procedure the Lac " Prot-, Lactococcus lactis ssp. Lactis CECT 4595 variant was obtained.
EJEMPLO DE REALIZACIÓN DE LA INVENCIÓN El cultivo iniciador utilizado en el proceso que se indica a continuación es el propuesto en la patente ya citada, formado por los microorganismos Lactococcus lactis ssp. lactis CECT 4478 en una proporción del 82%, junto con Lactobacillus casei ssp. casei CECT 4598, Lactobacillus plantarum CECT 4599 y Leuconostoc mesenteroides ssp. dextranicum CECT 4597, en una proporción cada uno del 6%, al que se le incorporó la variante Lac Prt", en una proporción igual a la del microorganismo original. Las cepas conservadas en congelación se propagaron en medios específicos inoculados al 1% e incubados a 30 °C durante 16 h. Los microorganismos recuperados mediante centrifugación a 8.000 x g durante 10 min, se resuspendieron al 1% en leche en polvo desnatada reconstituida al 10% y posteriormente se liofilizaron. El cultivo iniciador se preparó mezclando las diferentes cepas liofilizadas en las proporciones antes indicadas. La materia prima utilizada para la fabricación del queso fue: *55% de leche de vaca desnatada *15% de leche de oveja *30% de leche de cabraEXAMPLE OF EMBODIMENT OF THE INVENTION The starter culture used in the process indicated below is that proposed in the aforementioned patent, formed by the microorganisms Lactococcus lactis ssp. lactis CECT 4478 in a proportion of 82%, together with Lactobacillus casei ssp. casei CECT 4598, Lactobacillus plantarum CECT 4599 and Leuconostoc mesenteroides ssp. dextranicum CECT 4597, in a proportion each of 6%, to which the Lac Prt variant was incorporated " , in a proportion equal to that of the original microorganism. The strains preserved in freezing propagated in specific media inoculated at 1% and incubated at 30 ° C for 16 h The microorganisms recovered by centrifugation at 8,000 xg for 10 min, were resuspended at 1% in 10% reconstituted skimmed milk powder and subsequently lyophilized.The starter culture was prepared by mixing the different lyophilized strains in the proportions indicated above: The raw material used to make the cheese was: * 55% skimmed cow's milk * 15% sheep's milk * 30% goat's milk
Inmediatamente después de la pasterización (72 °C, 4 s), la leche fue enfriada a 50 °C para comenzar el proceso de concentración por microfiltración (MF). El proceso de concentración se llevó a cabo a una temperatura de 50 °C ± 2 °C hasta alcanzar un factor de concentración (FC) de 3; a continuación se procedió a la diafiltración del mismo, añadiendo agua a 50°C aproximadamente en la misma proporción de concentrado. El proceso de MF se finalizó cuando se obtuvo un factor de concentración de 6.Immediately after pasteurization (72 ° C, 4 s), the milk was cooled to 50 ° C to begin the microfiltration concentration (MF) process. The concentration process was carried out at a temperature of 50 ° C ± 2 ° C until a concentration factor (FC) of 3 was reached; then the diafiltration was carried out, adding water at 50 ° C in approximately the same proportion of concentrate. The MF process was completed when a concentration factor of 6 was obtained.
El cultivo iniciador se resuspendió en permeado y se añadió al concentrado en proporción suficiente para alcanzar un número de viables del orden de 108 u.f.c. (unidades formadoras de colonias)/g.The starter culture was resuspended in permeate and added to the concentrate in sufficient proportion to reach a number of viable of the order of 10 8 cfu (colony forming units) / g.
Posteriormente se añadió al prequeso, CINa y cuajo. El queso obtenido se colocó en moldes y se mantuvieron durante una noche a 30 °C para posteriormente pasarlos a las cámaras de maduración.Later it was added to the prequese, CINa and rennet. The cheese obtained was placed in molds and kept overnight at 30 ° C and then passed to the ripening chambers.
La maduración de los quesos se llevó a cabo durante 60 días realizando toma de muestra cada dos semanas. Características físico-químicas. Al final de la maduración el valor de pH fue de 5,3 habiéndose partido de un valor inicial de 6,6 en la leche. Los valores obtenidos en las fracciones nitrogenadas: nitrógeno no caseínico (NNC) y nitrógeno no proteico (NNP), aumentaron principalmente durante el primer mes de maduración, para continuar incrementando hasta el final de la misma, aunque de forma menos marcada, sobre todo en el caso del NNC. Los niveles encontrados para estos índices, a pesar del retraso en la maduración que en general conlleva la utilización de leche concentrada por procesos de membrana, fueron del mismo orden que los obtenidos en quesos elaborados por procedimientos tradicionales. Al comparar estos valores con los obtenidos para el mismo tipo de queso, pero elaborado con el cultivo iniciador que no llevaba incorporado Lactococcus lactis ssp. lactis CECT 4595, se puede concluir que este microorganismo da lugar a una apreciable aceleración de la maduración.The ripening of the cheeses was carried out for 60 days by sampling every two weeks. Physicochemical characteristics. At the end of maturation, the pH value was 5.3, starting with an initial value of 6.6 in milk. The values obtained in the nitrogen fractions: non-casein nitrogen (NNC) and non-protein nitrogen (NNP), increased mainly during the first month of maturation, to continue increasing until the end of the same, although less markedly, especially in the case of the NNC. The levels found for these indices, despite the delay in maturation that generally involves the use of concentrated milk by membrane processes, were of the same order as those obtained in cheeses made by traditional procedures. When comparing these values with those obtained for the same type of cheese, but made with the starter culture that did not incorporate Lactococcus lactis ssp. lactis CECT 4595, it can be concluded that this microorganism results in an appreciable acceleration of maturation.
Características sensoriales. El queso obtenido en las condiciones desαitas presentó un olor y sabor típico a queso y más marcado que el mostrado en quesos elaborados con cultivos iniciadores comerciales no específicos. Sensory characteristics. The cheese obtained under the conditions described showed a typical cheese smell and taste and more marked than that shown in cheeses made with non-specific commercial starter cultures.

Claims

REIVINDICACIONES
1. Microorganismo de la especie Lactococcus lactis ssp. lactis CECT 4478 utilizable para la fabricación de queso, el cual, se modifica en su equipamiento enzimático, y da lugar al microorganismo Lactococcus lactis ssp. lactis CECT (Colección Española de Cultivos Tipo) n° 4595.1. Microorganism of the species Lactococcus lactis ssp. lactis CECT 4478 usable for the manufacture of cheese, which is modified in its enzymatic equipment, and gives rise to the microorganism Lactococcus lactis ssp. CECT lactis (Spanish Type Culture Collection) No. 4595.
2. Utilización del microorganismo CECT 4478, mezclado con su variante CECT 4595, según reivindicación 1 , en una proporción del 82%, junto con dos cepas de Lactobacillus: Lactobacillus casei ssp. casei CECT 4598, Lactobacillus plantarum CECT 4599 y una de Leuconostoc: Leuconostoc mesenteroides ssp. dextranicum CECT 4597, en proporción del 18%, como cultivo iniciador para la fabricación de quesos.2. Use of the CECT 4478 microorganism, mixed with its CECT 4595 variant, according to claim 1, in a proportion of 82%, together with two strains of Lactobacillus: Lactobacillus casei ssp. casei CECT 4598, Lactobacillus plantarum CECT 4599 and one of Leuconostoc: Leuconostoc mesenteroides ssp. dextranicum CECT 4597, in a proportion of 18%, as a starter for cheese production.
3. Utilización del cultivo iniciador, según reivindicación 2, para acelerar la maduración de un queso semiduro obtenido a partir de leche de mezcla de las especies vaca, oveja y cabra. 3. Use of the starter culture, according to claim 2, to accelerate the ripening of a semi-hard cheese obtained from mixed milk of the cow, sheep and goat species.
REIVINDICACIONES MODIFICADASMODIFIED CLAIMS
[recibidas por la oficina Internacional el 30 de deciembre de 1998 (30.12.98); reivindicaciones 1-3 reemplazadas por las reivindicaciones 1-2 modificadas (1 página)][received by the International Bureau on December 30, 1998 (30.12.98); claims 1-3 replaced by modified claims 1-2 (1 page)]
1.- Microorganismo Lactococcus lactis ssp lactis CECT (Colección Española de1.- Lactococcus lactis ssp lactis CECT Microorganism (Spanish Collection of
Cultivos Tipo) n° 4595 obtenido a partir del microorganismo Lactococcus lactis ssp lactis CECT (Colección Española de Cultivos Tipo) n° 4478, modificándolo en su equipamiento enzimático que conduce a la pérdida de actividad proteinásica y de capacidad de metabolizar la lactosa.Type cultures) No. 4595 obtained from the microorganism Lactococcus lactis ssp lactis CECT (Spanish Type Culture Collection) No. 4478, modifying it in its enzymatic equipment that leads to the loss of proteinaceous activity and the ability to metabolize lactose.
2.- Procedimiento de elaboración de quesos caracterizado porque se procede de la siguiente manera:2.- Cheese production procedure characterized in that it proceeds as follows:
- Se forma un cultivo iniciador compuesto por los microorganismos Lactococcus lactis ssp. lactis CECT 4478 en una proporción del 82%, junto con Lactobacillus casei ssp. casei CECT 4598, Lactobacillus plantarum CECT 4599 y Leuconostoc mesenteroides ssp. dextranicum CECT 4597, en una proporción cada uno del 6%, al que se le incorpora la variante modificada según reivindicación 1 , en una proporción igual a la del microorganismo original. - Las cepas conservadas en congelación se propagan en medios específicos y se concentran por liofilización. El cultivo iniciador se prepara mezclando las diferentes cepas liofilizadas en las proporciones antes indicadas.- A starter culture consisting of the microorganisms Lactococcus lactis ssp is formed. lactis CECT 4478 in a proportion of 82%, together with Lactobacillus casei ssp. casei CECT 4598, Lactobacillus plantarum CECT 4599 and Leuconostoc mesenteroides ssp. dextranicum CECT 4597, in a proportion each of 6%, to which the modified variant according to claim 1 is incorporated, in a proportion equal to that of the original microorganism. - Frozen strains are propagated in specific media and concentrated by lyophilization. The starter culture is prepared by mixing the different lyophilized strains in the proportions indicated above.
- Se utiliza como materia prima para la fabricación del queso, 55% de leche de vaca desnatada, 15% de leche de oveja y 30% de leche de cabra. Inmediatamente después de la pasterización (72 °C, 4 s), la leche se enfria a 50 °C para comenzar el proceso de concentración por microfiltración (MF). El proceso de concentración se lleva a cabo a una temperatura de 50 °C ± 2 °C hasta alcanzar un factor de concentración (FC) de 3; a continuación se procede a la diafiltración del mismo, añadiendo agua a 50°C aproximadamente en la misma proporción de concentrado. El proceso de MF se finaliza cuando se obtiene un factor de concentración de 6.- It is used as raw material for the manufacture of cheese, 55% skimmed cow's milk, 15% sheep's milk and 30% goat's milk. Immediately after pasteurization (72 ° C, 4 s), the milk is cooled to 50 ° C to begin the microfiltration concentration (MF) process. The concentration process is carried out at a temperature of 50 ° C ± 2 ° C until a concentration factor (FC) of 3 is reached; Thereafter, the diafiltration is carried out, adding water at approximately 50 ° C in the same proportion of concentrate. The MF process is terminated when a concentration factor of 6 is obtained.
- El cultivo iniciador se resuspende en permeado y se añade al concentrado en proporción suficiente para alcanzar un número de viables del orden de 108 u.f.c. (unidades formadoras de colonias)/g. Posteriormente se añade al prequeso, CINa y cuajo. El queso obtenido se coloca en moldes y se mantiene durante una noche a 30 °C para posteriormente pasarlos a las cámaras de maduración. - The starter culture is resuspended in permeate and added to the concentrate in sufficient proportion to reach a number of viable in the order of 10 8 cfu (colony forming units) / g. Later it is added to the prequese, CINa and rennet. The cheese obtained is placed in molds and kept overnight at 30 ° C and then passed to the ripening chambers.
PCT/ES1998/000209 1997-07-15 1998-07-14 Microorganism of the species lactococus lactis ssp. lactis cect 4478 usable for the fabrication of cheese WO1999003971A1 (en)

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ES9701576A ES2125205B1 (en) 1993-12-29 1997-07-15 IMPROVEMENTS IN THE PATENT OF INVENTION N 9302713 MICROORGANISM OF THE SPECIES LACTOCOCCUS LACTIS SSP. LACTIS CECT 4478 USABLE FOR THE MANUFACTURE OF CHEESE.

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2072827A1 (en) * 1993-12-29 1995-07-16 Consejo Superior Investigacion Microorganism of the species Lactococcus lactis ssp. lactis CECT 4478, which can be used for the manufacture of cheese.

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2072827A1 (en) * 1993-12-29 1995-07-16 Consejo Superior Investigacion Microorganism of the species Lactococcus lactis ssp. lactis CECT 4478, which can be used for the manufacture of cheese.

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
REQUENA ET AL.: "Plasmid profiles and relationship to lactose utilization proteinase activiti in a lactococcal strain isolated from semi-hard natural cheese", MILCHWISSENSCHAFT,, vol. 48, no. 5, 1993, pages 264 - 268 *
RODRIGUEZ J. ET AL.: "Accelerated ripening of reduced fat semi-hard cheese from a mixture of cow's goat's and ewe's ultrafiltraled milk by using a Lac Prt strain of lactococci", vol. 76, no. 6, 1996, pages 513 - 522 *

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