METHOD FOR THE PREPARATION OF ATTENUATED STARTER CULTURES

Abstract: A process for the preparation of an attenuated starter culture which can be used in the preparation of cheese, cheese analogues and cheese-derived products comprises treatment of the cells with microwave radiation.

Description: METHOD FOR THE PREPARATION OF ATTENUATED STARTER CULTURES Field of the invention The present invention relates to a process for the preparation of attenuated starter cultures and the use of these cultures in, for example, the production of cheese, cheese analogues or cheese-derived products Background of the invention Cheese ripening is a time-consuming process involving complex and well-balanced reactions between glycolysis, proteolysis and lipolysis of milk components Whatever cheese is considered, bacterial enzymes play a major role in this process The proteases, peptidases, esterases, and the enzymes of ammo acid catabo sm in the lactic acid bacteria involved in these processes have been extensively studied (Law, J. and Haandnkman, A , 1997, International Dairy Journal 7 1-1 1 ) It is well known that changing the bacterial enzyme content in cheese directly affects the rate of cheese ripening and its final flavour One of the most efficient ways to increase the bacterial enzyme pool in cheese curd, without altering the primary starter and the type of cheese , is to add whole lactic acid bacteria, which are unable to grow and to produce a significant quantity of lactic acid, but which can still deliver active ripening enzymes during cheese ageing These starter cultures are normally weakened and are called "attenuated" The preparation of attenuated starter cultures can be achieved by various treatments like heating, freezing, spray drying, freeze-drying, fragilisation using lysozyme or solvents, or by the selection of lactose negative mutants The first preparation and use of attenuated starter cultures as a cheese additive was reported in 1975 by Pettersson and Sjostrom (Pettersson, H and Sjostrom, G , 1975, Journal of Dairy Research 42, 313-326) At that time, their main purpose was to accelerate proteolysis and shorten ripening time, which was of great economic interest Today, in most developed countries, milk is of such high hygienic quality that the problem is not only to shorten the ripening time but also to improve the flavour The most widely studied method for the preparation of attenuated starter cultures is heat-treatment This attenuation method is based on sub-lethal heat- treatment of a bacterial cell suspension The attenuated cells are then added to cheese milk along with the primary starter culture It was shown that heat-treatment destroyed or delayed the acidification ability of the cells for several hours Therefore the attenuated starter cultures do not significantly modify the acidification course of the curd, and subsequently the fat/moisture, and pH of the cheese Most often, the loss of lactic acid production closely follows the loss of viability (Pettersson and Sjostrom, 1975, ibid ) In CA 2072 159 a method was disclosed to accelerate the maturation of cheddar cheese by using live and heat-attenuated L casei Although there is still little agreement on which heat-treatment procedure is best, in most cases proteolysis and lipolysis were increased, leading to a reduced ripening time and improved flavour (bitterness reduction) However, a critical point for heat attenuation remains in that the correct temperature/time combination must be defined so that acidification is eliminated or delayed without excessively denaturing potential ripening enzymes (e g proteases, peptidases, esterases, or others) Furthermore, the very narrow difference between adequate and excessive heat treatments remains a major practical problem (Castaneda, R , Vassal, L , Gripon, J and Rousseau, M , 1990, Netherland Milk and Dairy Journal 44, 49-52) Alternative methods for attenuation have also been described, for example attenuation by freezing-thawing (Johnson, J A C , Etzel, M R , Chen, C M and Johnson, M E , 1995, Journal of Dairy Science 78, 769-776), by lysozyme treatment (EP 02 46 163), by the use of solvents or by spray- and freeze-drying (Johnson, J A C and Etzel, M R, 1995, Journal of Dairy Science 78, 761-768) Despite promising results in cheese, the industrial use of attenuated starters is not widespread The main reason for this is the empirical character of the attenuation methods which have been proposed so far The present invention seeks to solve some of the problems encountered in prior art processes, by providing a novel process for the preparation of attenuated starter cultures which is reproducible and well-balanced Furthermore, the process of the invention is rapid and simple and it can be carried out easily and at low cost on an industrial scale Summary of the invention The present invention discloses a process for the preparation of an attenuated starter culture, which process comprises exposing cells of at least one strain in a starter culture to microwave radiation Attenuated starter cultures of the invention can be used in, for example, production of cheese, cheese analogues or cheese- derived products Attenuated starter cultures of the invention contain cells that have been treated with microwave radiation The invention also provides An attenuated starter culture obtainable by a process of the invention for the preparation of an attenuated starter culture, An attenuated starter culture which has a viability of less than 30% and an enzyme activity of more than 50% that of a non-attenuated starter culture, A process for the preparation of a cheese, a cheese analogue as a cheese- derived product which process comprises contacting a cheese, a cheese analogue or a cheese-derived product or a precursor thereof with an attenuated starter culture of the invention, and Use of an attenuated starter culture of the invention in the preparation of a cheese, a cheese analogue or a cheese-derived product Optionally, the starter culture can be contacted with untreated cells following exposure to microwave radiation In a particular embodiment, the starter culture can be exposed to from 1000 to 30000 Joule of microwave radiation Preferably cells can be treated for from 5 to 30 seconds at a power of from 200 to 1000 Watts (Joule/s), or most preferably for 15 to 20 seconds at from 300 to 650 Watts per 10ml of an aqueous composition for example a suspension containing ~10 9 cells / ml Preferably, attenuated starter cultures comprise cells that have been treated with microwave radiation in such a way that the viability of the attenuated starter culture is less than 20%, more preferably from between 1 and 10%, as compared to untreated starter cultures when measured in CFU/ml Furthermore, preferably the killed cells are not lysed Typically the same time, the treated cells retain their enzyme activity In general, more than 50%, more preferably more than 90% of the enzyme activity of the attenuated starter culture is maintained The activity of one enzyme, in particular preferably peptidase, present in the attenuated starter culture is more than 90% as compared to the activity in untreated cells, as can be determined via the method indicated in Example 2 This peptidase activity is used as indication for the whole enzyme activity present in treated cells Ripening enzymes can be chosen from the group containing proteases, peptidases, decarboxylases, amine oxidases, alcohol dehydrogenases, ammo acid dehydrogenases, ammo acid oxidases, ammo transferases or transammases Attenuated starter cultures can contain cells belonging to one or more of the strains selected from the group of species consisting of Lactobacillus, Lactococcus, Streptococcus, Corynebacteπum, Bifidobacteπum, Micrococci anά yeasts An attenuated starter culture of the invention may be in a concentrated form and optimally may also be in a frozen form Such attenuated starter cultures may be prepared by carrying out the process of the invention for the preparation of an attenuated starter culture and subsequently concentrating the resulting attenuated starter culture, optionally followed by freezing Detailed description of the invention In present invention a process for the preparation of an attenuated starter culture using microwave radiation is described The use of microwaves is well known in the art for killing cells in food preparations From these studies it has been concluded that the exposure of cells to microwave radiation results in inactivation of microorganisms While an inhibitory effect of microwave radiation on the viability of microorganisms is established, the question how microwave radiation brings about that effect has been a matter of discussion It is most likely that the effect is of a thermal character However, experiments performed by Rosenberg and co-workers revealed that microwave treatment resulted in a loss of viability at a rate which was higher than that obtained with conventional heating alone (Rosenberg, U and Smell, H J , 1989, Zentralbl Hyg Umweltmed 188 271-283) This suggests the co-existence of a so-called "athermal effect" during microwave treatment as well This hypothesis is strengthened by the fact that recent studies have shown that microwave treatment at reduced temperatures (below 40°C) also results in decreased viability of microorganisms (Kozempel, M F , et al J food Prot , 1998, 61 582-585) This is likely to be due to the fact that bacterial DNA, RNA and proteins absorb microwaves of specific frequencies which results in genetic and protein disorders (Webb, S J , Nature, 1969, 222 1199-1200) Taking the thermal and athermal effect together it can be concluded that microwave treatment can not be seen simply as an equivalent to heat treatment On the contrary, its destructive action on cells does not favour the use of microwaves as a method to generate attenuated starter cultures with high intact enzyme activity Surprisingly, the present invention discloses that a treatment of cells whereby cells are exposed to microwave radiation (also indicated as the "microwave treatment") shows to be very favourable for the attenuation of starter cultures which can be used in, for example, cheese making The reason for this is that the microwave treatment of present invention appears to have a wider gap between adequate and excessive treatments, as compared to attenuation by conventional heat treatment Furthermore, this simple and rapid method results in the appearance of cells with low viability and high enzyme activity, is highly reproducible and can be applied to concentrated cell suspensions, making it highly suitable for low cost use and continuous use on an industrial scale By the term starter culture we mean a particular strain of bacterium or yeast, or a combination of one or more species or strains from one or more genera, which by growing and metabolising, assist in the manufacture of the product, for example, by formation of taste, flavour and/or texture For example, dairy starter cultures can contain lactic acid bacteria that grow and metabolise in milk and curd and assist in the production of mature cheese Also starter cultures can be used in the production of meat and derivatives thereof By the term cheese we mean a dairy product belonging to a soft cheese variety such as Camembert, Munster or Livarot, to a semi-hard variety such as Tome de Savoie, Gouda, Edam or Mimolette or to a hard cheese variety such as Cheddar, Gruyere or the Swiss cheeses or Parmesan By the term cheese analogue we mean a product composed of dairy proteins such as casein, casemate, whey protein concentrate coagulated by acidification or by heat-treatment, or a combination of both, to which cheese-flavouring agents are added By the term cheese-derived product we mean either a processed cheese, i e a preheated mixture of cheeses, butter or other fatty materials, melting salts or any type of cheese curds to which enzymes are added in order to give desired texture- and flavouring properties By the term precursor we mean a substance that is present in the process of the invention which will ultimately become a cheese, a cheese analogue or a cheese-derived product Thus a precursor could be milk, butter or cheese curds for example In present invention, attenuated starter cultures can comprise lactic acid bacteria, which are highly affected in their ability to grow (i e their viability) and to produce significant quantities of lactic acid but which are still capable of delivering active ripening enzymes during cheese ageing Attenuated starter cultures can comprise lactic acid bacteria or any other type of cells that have been treated in such a way that the viability of the attenuated starter culture is less than 20%, more preferably less than 10%, most preferably less than 1%, as compared to an untreated starter culture as measured in CFU/ml Preferably more than 50%, more preferably more than 90% of the activity of the ripening enzymes in an attenuated starter culture is retained as compared to an untreated starter culture Ammopeptidase activity can be used to determine how much activity is retained (see example 2) Attenuation can be performed on bacterial or fungal starter cultures Preferably, Lactobacillus, Lactococcus, Streptococcus, Corynebacteπum, Bifidobacteπum, Micrococci or yeast species can be used Most preferably, L helveticus, L lactis, L casei, L plantarum, L acidophilus, L delbrueckn, L bulgaπcus, S thermophilus, L lactis ssp cremoπs, K lactis or B linens can be used Attenuated starter cultures of present invention contain one or several strains from which at least one might be attenuated by treatment with microwave radiation Thus, a starter culture may be attenuated by exposure to microwaves and be mixed with manufactured cells and/or starter cultures Ripening enzymes are enzymes involved in the formation of taste, texture and/or flavour of cheese, cheese analogues, cheese-derived products and/or processed cheese. Formation of taste, flavour and texture involves complex and well-balanced reactions between glycolysis, proteolysis and lipolysis of the milk components and further degradation of ammo acids into amines, keto-acids aldehydes, ketones, alcohols, esters, acid and sulfur compounds by a cascade of enzymatic reactions Preferred ripening enzymes can be proteases (for example serme-, cysteine- aspartic-, and/or metallo-endopeptidases), peptidases (for example ammo-, carboxy-, dι-, dipeptidyl-, tπpeptidyl-, serme-type carboxy-, metallocarboxy-, cysteme-type carboxy-, and/or omega peptidases), decarboxylases (for example lysme-, arginme- and/or histidme decarboxylase), amine oxidases, aldehyde dehydrogenases, alcohol dehydrogenases, ammo acid dehydrogenases, ammo acid oxidases, ammo transferases or transaminases Preferably the microwave treatment is carried out using a microwave or the like Preferably, a conventional household mιcrowave(oven) is used For attenuation at an irradiation frequency of 2450 MHz, the microwave power can range from 10- 1800W More preferably, at a power of between from 300W to 650W, the duration of the treatment can be from 5 to 30 sec This treatment is especially suitable in case the starter cultures are present in an aqueous environment e g are suspended in water In case the starter cultures are in dried form (e g freeze dried) or in a concentrated state (10 to 40 w% dry matter) the skilled will appreciate the microwave treatment conditions will have to be adjusted to obtain the desired state of killed but not lysed Most preferably, conditions for the microwave-treatment are chosen which result in at least 90%, more preferably 99% of the cells in the starters culture being killed but not lysed, while in general more than 50%, more preferably 90% of the activity of ripening enzymes is retained Viability can be measured in, for example, CFU/ml (CFU = Colony Forming Unit) by plating a dilution of the attenuated cells on suitable MRS or YEG agar plates. Lysis can be determined by, for example, conventional methods using microscopy Activity of ripening enzymes can be determined, for example, as described for peptidase in Example 2 Of course, all attenuation protocols in which a microwave-treatment is used in combination with an alternative attenuation treatment forms part of this invention We have found that in the "killed but not lysed" state of the starter culture alcohol formation or acidification were prevented and the flavour related enzymes were still active So the disadvantages of the starters were reduced because the starter cultures were catabohtically inactive whereas the enzymes present were available for the cheese making process Another aspect of present invention provides an attenuated starter culture which consists of an attenuated starter culture comprising cells prepared via the method of the invention which are subsequently concentrated by, for example, centnfugation and optionally deep-freezing or lyophilismg (freeze-drying) Also, a method for preparing such attenuated starter cultures, the use of said attenuated starter cultures in cheese, cheese analogue or cheese-derives products making, and a method for making cheese, cheese analogues and/or cheese-derived products with the said attenuated starter cultures are parts of present invention The following examples are only to be considered as illustration of the present invention, and are not to be construed as limiting Example 1 Killing of starter cultures using microwave radiation Killing activity of a conventional household microwave of 2450 MHz frequency on Lactobacillus helveticus strain CNRZ-32 (available from INRA-collection, Institut National de la Recherche Agronomique, Gouy-en-Josas, France) and Kluyveromyces lactis strain 2166 (ATCC No. 8585) was investigated by exposing the cells to 300 W and 650 W microwaves for different lengths of time. Thus, cells were cultured in 1 litre of appropriate (MRS for L helveticus and YEG for K. lactis) medium and in the required (37°C, unstirred for L. helveticus ; 30°C, stirred for K. lactis) conditions until an OD650 of 1 and 2 for L. helveticus and K. lactis respectively (about 5x10 8 to 10 9 cells/ml) was reduced. Cells were harvested in the early exponential stage under sterile conditions and washed twice with distilled water. Pellets were resuspended in 100 ml of sterile distilled water and divided into portions of 10 ml in test tubes. Test tubes were placed in a water bath at 20°C until treatment. Microwave treatment was carried out by placing the one glass test tube containing 10 ml of cell suspension in an almost horizontal position onto two beakers of different size. At a power of 650 Watts the following timepoints were measured: 5, 10, 15, 20 and 25 sec. At a power of 300 Watts, cells were radiated for 15, 20, or 30 sec. After treatment test tubes were placed in a water bath again at 20° C for 10 min before determination of the killing rate. Viability was measured by plating the cells onto MRS (a medium for cultivating lactobacilli, the reference is "de Man JC, Rogosa M., and Sharpe E., 1960. A medium for cultivation of lactobacilli. J. appl. Bacteriol., 23, 130-135) or YEG (YEG = yeast extract 10g/l; bacto pepton 20g/l; dextrose 20g/l; pH6.5) plates. Also lysis of cells was monitored by microscopy. Table 1 shows the effect of the different microwave treatments on the viability of the two strains in log CFU/ml. A decrease in viability with a 2 log factor could be obtained for both cell types within 20 sec at a power of 650 W, and within 30 sec at 300 W. Table 1 : shows the viability of Lactobacillus helveticus and Kluyveromyces lactis after microwave radiation applied for different lengths of time (t=0 to t=30 seconds) at different power levels (300W and 650W). The viability is given in CFU/ml. Example 2 Determination of intracellular peptidase activity after attenuation of starter cultures by microwave treatment The impact of attenuation by microwave treatment on proteolytic activity was systematically followed by measuring the increase in free amino groups during proteolytic degradation of β-casein by an attenuated cell-free extract. Since the proteolytic activity measurements as described by Doi and co-workers (Doi et al 1981 , Anal. Biochem. 118, 173-184) could not properly be carried out in whole cells, cell-free extracts were prepared, which were subsequently exposed to microwave radiation. It is reasonably expected that the enzyme activity as measured in radiated cell free extracts is an underestimation of the enzyme activity that would be present after radiating whole cells. Cell free extracts were prepared by disruption of the cells at 1000 Bars in a refrigerated French press. Undisrupted cells and cell debris were removed by cent fugation and the collected supernatant was filter-sterilised. The protein content of the cell free extracts was determined according to Lowry. (Lowry O.H., Rosebrouch N.J., Farr A.L., and Randall R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275) β-Casein was purified from milk by methods known in the art [PCT/FR 91/00506] and subsequently partially hydrolysed using endoproteases trypsiπ (EC.3.4.21.4) and chymotrypsin (EC.3.4.21.1 ) until a hydrolysis degree of 16% was obtained as measured by free amino -group determination as described by Doi and co-workers (Doi et al .1981 , Anal. Biochem. 1 18, 173-184). The peptide mixture obtained in this way contained 34 different peptides with molecular weights ranging from 373 to 3864 D as determined by mass spectrometry. Proteolytic degradation of the β-casem peptide mixture was performed by incubating 1 ml of 0 7 mg/ml beta-casein together with 125 μg of cytoplasmic protein of L helveticus or K lactis at 24°C in a buffer of 50 mM sodium phosphate at pH 5 7 Samples were taken after 10 or 24 hours, after which the increase in free ammo groups was measured as described by Doi [Doi et al 1981 , ibid] It was thus shown that the microwave treatment had no significant effects on the peptidase activity of K lactis , whereas the peptidase activity of L helveticus had dropped with 16% Furthermore, the release of intracellular constituents during the microwave treatment was determined (not shown) and found to be non-significant Table 2 Release of free ammo groups (in mM) during the kinetic degradation (t=0, t=10 and t=24 hours) of a β-casem hydrolysate by microwave-treated cell free extracts from Lactobacillus helveticus and Kluyveromyces lactis

Claims: CLAIMS 1 A process for the preparation of an attenuated starter culture, the process comprising exposing cells of at least one strain in a starter culture to microwave radiation. 2. A process according to claim 1 , wherein the exposure to microwave radiation is such that the cells of at least one strain in the starter culture are killed bur not lysed 3 A process according to claim 2, wherein the viability of at least one strain in the starter culture is less than 20%, as measured in CFU/ml 4 A process according to claim 3 wherein the viability of at least one strain in the starter culture is less than 10%, as measured in CFU/ml 5 A process according to any one of the preceding claims, wherein the exposure to microwave radiation is from 1000 to 30000 Joule per 10 ml of an aqueous composition which comprises the cells of at least one strain A process according to claim 5, wherein the exposure to microwave radiation is for from 5 to 30 seconds at a power of from 200 to 1000 Joule/s, preferably the exposure to microwave radiation is for from 15 to 20 seconds at a power of from 300 to 650 Joule/s A process according to any one of the preceding claims, wherein there are cells in the attenuated starter culture capable of expressing a ripening enzyme 8 A process according to claim 7, wherein the ripening enzyme is a protease, a peptidase, a decarboxylase, an amine oxidase, an aldehyde dehydrogenase, an alcohol dehydrogenase, an ammo acid dehydrogenase, an ammo acid oxidase, an ammo transferase or a transammase A process according to claim 8, wherein the peptidase activity of the attenuated starter culture is more than 50% that of a non-attenuated starter culture, preferably is more than 90% that of a non-attenuated starter culture A process according to any one of the preceding claims, wherein the cells present in the attenuated starter culture are Lactobacillus, Lactococcus Streptococcus, Micrococci, Corynebacteπum, Bifidobactenum or yeast cells or any combination thereof A process according to any one of the preceding claims which process further comprises concentrating the starter culture before or after it has been exposed to microwave radiation A process according to claim 1 1 which further comprises freezing the starter culture after concentrating and exposure to microwave radiation An attenuated starter culture obtainable by a process according to any one of the preceding claims An attenuated starter culture which has a viability of less than 20% and/or an enzyme activity of more than 50% that of a non-attenuated starter culture An attenuated starter culture according to claim 14 which has a viability of less than 90% that of a non-attenuated starter culture An attenuated starter culture according to claim 14 or 15 which has an enzyme activity of more than 90% that of a non-attenuated starter culture A composition comprising an attenuated starter culture according to any one of claims 13 to 16 A process for the preparation of a cheese, a cheese analogue or a cheese derived product which process comprises contacting a cheese, a cheese analogue or a cheese-derived product or a precursor thereof with an attenuated starter culture according to any one of claims 13 to 16 or a composition according to claim 17. 19. Use of an attenuated starter culture according to any one of claims 13 to 16 or a composition according to claim 17 in the preparation of a cheese, a cheese analogue or a cheese-derived product. 20. A cheese, a cheese analogue or a cheese-derived product comprising an attenuated starter culture according to any one of claims 13 to 16 or a composition according to claim 17.

Inventors: KLEIN NATHALIE, LORTAL SYLVIE, KLEIN, NATHALIE, LORTAL, SYLVIE
Applicants: DSM NV, KLEIN NATHALIE, LORTAL SYLVIE, DSM N.V., KLEIN, NATHALIE, LORTAL, SYLVIE
Application year: 2000
Main classification: A23C19
IPC codes: A23C19