Novel microorganism and use thereof in ripening cheese

Abstract

The invention relates to Lactobacillus helveticus AGCl, a sample of which has been deposited on 29 September 1988 at The National Collections of Industrial And Marine Bacteria Limited under the accession number NCIB 40051, or a mutant or derivative thereof. This strain is useful as part of a starter culture addition to cheese milk, for accelerated ripening of cheddar style cheeses.

Description

This invention relates to a novel microorganism and its use in a method for the accelerated ripening of hard type cheeses of the Cheddar and related variant types, including low fat cheddar style cheese.

After initial manufacture, Cheddar cheese and Cheddar style varieties of cheese require a storage period of the order of four to six months at about 7.degree. C., prior to sale . This storage period is necessary to allow the body of the cheese to acquire the typical characteristics of Cheddar cheese in terms of texture, consistency, and flavour. This extended storage period has evident disadvantages with respect to the financing costs of the cheese stocks involved, and with respect to marketing and production planning.

The development of typical Cheddar cheese, body, texture and flavour is the end result of complex physical and biochemical processes. These processes are influenced by a wide range of factors such as the composition and bacterial flora of the raw milk, the hygienic and manufacturing conditions used, the type of and condition of the starter bacteria used and the type of adventitious organisms present in the finished cheese. The composition of the cheese produced, the length of ripening period, and the temperature of storage during the ripening period are also important with respect to the development of a typical Cheddar cheese texture and flavour.

It is recognized that the body of the cheese is mainly modified from the initial `curdy` texture and appearance of freshly produced cheddar cheese to that of a typical cheese as purchased by the consumer, by the proteolytic action of retained chymosin, by the proteolytic and peptidase enzymes, produced by the lactic acid starter bacteria and by adventitious organisms. These adventitious non-starter bacteria form the major flora of cheddar cheese after a relatively short period of ripening. The influence and interrelationships of these factors, in terms of cheese flavour and cheese texture, are as yet relatively undefined.

Manufacture of low fat cheeses presents problems in that flavour development is extremely slow and consequently such cheeses have a low consumer appeal.

A number of attempts to accelerate the flavour development of Cheddar and low fat Cheddar-style cheeses have been described.

EP-A-No. 0150743 describes a method for accelerating the aging process of Cheddar style cheese which is based on the use of preserved, partially disrupted preparation of the lactic acid bacterium Lactobacillus caesi, Lactobacillus lactis, Lactobacillus olantarum and a blend of pre-gastric lipases.

WO No. 82/03971 describes a method for the production of a low fat cheese product with enhanced flavour, using a culture of Lactobacillus bulgaricus and Streptococcus thermophilus and with a culture of Lactobacillus caesi, in addition to a normal cheese starter.

A number of methods to achieve accelerated ripening of Cheddar style cheeses, mainly based on enzyme additions are now commercially available or publicized (refs: 1,4,5,6).

However, one of the disadvantages of some of the systems is that the enzyme preparation has to be added to the cheese curd during the salt addition process and can give rise to enzyme distribution problems in the finished cheese, potentially resulting in flavour variations and physical defects such as cheese mottling. In the United Kingdom these systems have an additional disadvantage in that enzyme additions, other than coagulating enzymes, are not permitted under current cheese regulations, in traditional English type cheeses manufactured for direct sale to the consumer.

EP-A-No. 0150743 suffers from the disadvantage that cells of the lactic acid bacteria must be partially disrupted and then preserved before use. Such a process is technically difficult to operate and adds to the cost of cheese manufacture. WO No. 82/03971 suffers from the disadvantage that three cultures, in addition to a normal cheese starter, must be used.

The present invention relates to the surprising discovery that addition of a specific culture of Lactobacillus helveticus AGC1, characterized by its carbohydrate utilization pattern, and by its content of the nucleotides, guanine and cytosine (38.5%), to a typical manufacturing process for Cheddar and low fat Cheddar-style cheeses results in accelerated ripening.

The invention thus provides Lactobacillus helveticus AGC1, a sample of which has been deposited on 29 Sept. 1988 at The National Collections of Industrial and Marine Bacteria Ltd (NCIMB), P.O. Box 31, 135 Abbey Road, Aberdeen AB9 8DG, UK, under accession number NC1B 40051, or a mutant or derivative thereof.

L. helveticus AGC1 can be distinguished from other strains of L. helveticus by its characteristic carbohydrate utilization pattern (see Table 1). Thus, the invention provides a novel strain of L. helveticus which can be used to accelerate ripening of Cheddar style cheeses.

In practice, the additional culture is added at the normal point of starter culture addition to cheese milk, during the cheese making process. The rate of culture addition can be varied as required and is normally in the range of 0.01-1% and preferably 0.05% to 0.3%.

The addition of L. helveticus AGC1 in this manner may be regarded as the deliberate addition of a non-traditional (in this application) lactic acid starter bacterium, as a source of additional peptidases and proteolytic enzymes. This method produces the correct amount and type of breakdown in the body of the cheese, as determined by an expert cheese grader, and confirmed rheologically by means of measurements using an Instron universal tester instrument.

Novel aspects of our invention include the following:

1. A method for producing Cheddar cheese or Cheddar-like cheeses of composition in the range of 20-38% fat content and 30-45% moisture content made in a usual Cheddar cheese manufacturing type operation, with the specific addition of the identified strain of Lactobacillus helveticus AGC1 in addition to the usual cheese starter culture organisms. Such a method results in a cheese characterized by an accelerated ripening profile and in which unacceptable changes in texture do not occur.

2. A method for the production of a low fat Cheddar style cheese with a fat content in the range 8-20% and a moisture content in the range of 37-54% made with the specified strain of Lactobacillus helveticus AGC1, in addition to the usual cheese starter culture organisms. Such a method results in a cheese characterized by an accelerated ripening profile and in which unacceptable changes in texture do not occur.

L. helveticus AGC1 is a thermophilic strain and has an optimum temperature for growth that is higher than that of normal starter cultures. For that reason it is important that the scald temperature is maintained in the range of 95.degree.-108.degree. F. (35.degree.-42.2.degree. C.). Within this temperature range growth of all the starter cultures is maintained in balance. At higher temperatures L. helveticus AGC1 will overrun traditional cultures resulting in a poor quality cheese.

The application of the invention is exemplified in the following Examples.

EXAMPLE 1

Typical Cheddar Cheese Manufacturing Schedule, with the addition of Lactobacillus helveticus AGC1

Raw Materials

A. Milk which should be clean fresh and free from off flavour, antibiotics or any other inhibitory substance and be of good bacteriological quality. B. Starters--lactic cultures (i) Normal S. lactis/S. cremoris type. and (ii) L. helveticus AGC1 C. Rennet: Standard rennet, as necessary. D. Anatto: Coloured variety only. E. Salt: Standard cheese salt.

PROCESSING CONDITIONS

A Blend of normal starter cultures and the L. helveticus AGC1 culture, depending on the activity of the cultures and the type of acid profile required, is added at the rate of 0.5-2%. The L. helveticus AGC1 culture is added at the rate of 0.05-1%. Both cultures are added simultaneously at the beginning of the usual milk `ripening` period, immediately prior to renneting. The scald temperature should be sufficiently high to promote growth of the L. helveticus AGC1 culture (i.e. in the range of 95.degree.-108.degree. F. (35.degree.-42.2.degree. C.). Temperatures outside this range may result in an excessive outgrowth of the L. helveticus ACG1 culture. No significant difference from control cheese should be noted in the rennet to mill time.

Salt addition must be targeted to give a salt level in the finished cheese of 1.6-1.8%. The uniformity of salt distribution is of prime importance. The culture will be inhibited by a high salt concentration, thereby nullifying the effect of accelerated ripening. The cheese is thereafter pressed and transferred to storage as per routine procedures.

Typical results are shown as compared to the control cheese, in Table 2, demonstrating the initial rapid textural change in cheese produced by the method of this application, as compared to the control sample. This process also results in the production of the appropriate level and type of proteinases and peptidases which, with the retained chysmosin, are responsible for the breakdown of the initial cheese body and the production of a typical body texture and flavour profile, or the precursor chemicals to produce characteristic Cheddar cheese flavour, body and texture.

One of the traditional flavour defects in Cheddar cheese is a "bitter" flavour, which is thought to be due to the production of hydrophobic peptides due to insufficient hydrolysis of the peptide chains. Our process appears to result in the rapid breakdown of peptides, in excess of that encountered in a control sample, with the production of greater amounts of single amino-acids, without the production of bitter `off` flavours. An amino acid analysis of trichloroacetic acid (TCA) extracts of Control and Accelerated cheeses at various time intervals is given in Table 3.

We believe that the mode of action of the L. helveticus ACG1 culture is effectively the production of proteolytic and peptidase enzymes of the right quantity, type and character resulting in rapid breakdown of the protein structure to give the required texture, accompanied by extensive peptide hydrolysis, resulting in the production of a correct blend of amino acids, peptides, lipases, fatty acids, etc. which act as either the main flavour ingredient in cheese or the precursor for such flavour development. Our process produces a cheese which is ready for pre-packing or sale to consumer or customers in approximately 8 to 10 weeks, as shown in Table 2, as compared to sixteen to twenty weeks for cheese produced using standard techniques.

A typical comparison of the aging process for an accelerated Cheddar cheese, as compared to a control Cheddar cheese, is given in Table 2. In addition, surprisingly, we have noted that whereas flavour development continues at an accelerated rate, the texture changes in cheese produced by our method effectively reach a plateau for an indefinite period. In other words, once the initial texture changes are completed, evident, continuing unacceptable changes in texture do not occur, thus avoiding the production of an unacceptable product at any point in the life of the cheese.

EXAMPLE 2

Production of Low Fat Hard Type Cheese

To produce low fat cheese using L. helveticus ACG1 , particular attention needs to be given to certain key areas:

Standardization of the cheese milk and its heat treatment. The blending and addition of the lactic cultures. Setting and cutting of the curd. Scalding, stirring and pitching the curd. Curd treatment or cheddaring. Salting. Pressing. Fresh raw milk needs to be standardized, by the addition of skim milk or by partial separation to a given fat percentage, or fat to protein ratio.

The fat levels will depend upon:

1. Fat required in the end product. 2. Fat lost in the whey, or conversely, fat retained in the cheese. 3. Protein retained in the cheese.

For most factory conditions, the fat in the cheese will probably be less than 17% fat, therefore a fat to protein ratio of the order of 1:2, is required.

The fat reduced milk is pasteurized at 161.degree.-162.degree. F. (71.7.degree.-72.2.degree. C.) and cooled to 88.degree. F. (31.1.degree. C.) for incubation.

A blend of cultures is added to the vat milk and this consists of the normal cultures of the day and L. helveticus ACG1. The amount of culture and the ratio between cultures is governed by:

1. Acid development required to give the correct Rennet to Mill time (normally 3 hours 20 minutes to 3 hours 40 minutes). 2. Rate of maturation required.

At ripening temperature of 88.degree. F. (31.1.degree. C.) significant acidity development from the L. helveticus AGC1 cultures is not expected, or encountered. Too high ripening temperatures will result in the L. helveticus AGC1 culture outgrowing the normal culture, and over maturation of the resulting cheese.

Starter addition mix needs to be in the order of 1-2% and the L. helveticus AGC1 being added at 5-15% of the total inoculum. The length of ripening influences the moisture retaining properties of the curd and at least 35-45 minutes is required. Acidity at the end of the period is normally in the range 0.16-0.19% lactic acid.

At the end of ripening, standard rennet is added at the rate of 40 oz per 1000 gallons (0.249 g/1) of cheese milk.

The cheese milk is allowed to settle for approximately 45 minutes, or until the curd is firm. The exact cutting operation will vary from plant to plant, but the aim is to cut the curd fairly large, so as to retain as much moisture as possible. The stirring of the curd should be as gentle as possible, but obviously sufficient to prevent the curd matting at the bottom of the vat.

Scald temperatures ranging from 94.degree.-96.degree. F. (34.4.degree.-35.6.degree. C.) are used. The curd is milled with a Cheddar chip mill at an acidity of 0.55% lactic acid.

Salt addition is targeted to give approximately 1.4-1.5% salt in the finished cheese. Under salting may result in rapid proteolysis with bitter off flavour, whereas over salting will give cheese of poor texture and sweeter flavours. The cheese is pressed following standard cheese pressing procedures.

Typical recipes for production of conventional low fat Cheddar cheeses and for production of low fat Cheddar cheeses using L. helveticus AGC1 are given in Table 4. A typical comparison of the aging process for an accelerated low fat Cheddar cheese, as compared to a conventional low fat Cheddar cheese, is given in Table 5. Cheese produced by our method develops a mature flavour by 7 weeks whereas conventional low fat cheese does not reach this level of flavour development unless stored for unrealistic periods (i.e. greater than 18 weeks). In addition, surprisingly, low fat cheeses produced by our method do not undergo any unacceptable changes in texture.

REFERENCES

1. ARBIGE M.V. et al, Novel Lipases for Cheddar Cheese Flavour Development Food Technology 91-98, April 1986 2. DAVIS F. L. AND LAW B. L. (Editors), Advances in the Microbiology and Biochemistry of Cheese and Fermented Milks. Elsevier Applied Science Publishers. Pages 209-227 3. GRAPPIN R. et al, Primary Proteolysis of Cheese Proteins during Ripening. A Review. Journal of Dairy Science 68, 531-540, 1985. 4. LAW B. A., Accelerated Ripening of Cheddar Cheese with Microbial Proteinases. Netherlands Milk and Dairy Journal 35 (3), 313-327, 1981. 5. LAVERY AND SON PROPRIETARY LTD. WO No. 82/03971 6. MILES LABORATORIES, INC. EP-A-No. 0150 743 7. THOMAS, T. D., Oxidate Activity of Bacteria from Cheddar Cheese. New Zealand Journal of Dairy Science and Technology 21, 37-47, 1986. 8. RANK, T. C. et al, Secondary Proteolysis of Cheese During Ripening. Journal of Dairy Science 68, 801-805, 1985. __________________________________________________________________________ Fermentation Activity ** (3 = high, 2 = medium, 1 = low, 0 = negative) NCDO NCDO NCDO NCDO NCDO NCDO ATCC ATCC ATCC*Carbon Source AGC1 H3 H6 H13 H17 28 30 39538 39539 39542__________________________________________________________________________Glucose 3 3 3 3 3 3 3 3 3 --mannose 3 3 3 3 3 3 3 3 3 --lactose 3 3 3 3 3 1 3 1 3 --galactose 3 0 1 3 3 1 3 0 3 --fructose 3 2 2 1 1 1 2 3 3 --n-acetyl 1 0 0 3 0 1 3 3 3 --glucosamineribose 0 0 0 0 0 0 0 0 3 --trehalose 0 0 0 3 0 0 0 3 3 --maltose 0 0 0 0 0 0 0 1 0 --sucrose 0 0 0 0 0 0 0 3 3 --adonitol 0 0 0 0 0 0 0 0 1 --mannitol 0 0 0 0 0 0 0 0 3 --sorbitol 0 0 0 0 0 0 0 0 2 --inulin 0 0 0 0 0 0 0 0 3 --turanose 0 0 0 0 0 0 0 0 3 --lyxose 0 0 0 0 0 0 0 0 3 --sorbose ALL TESTS NEGATIVErhamnosemethyl-glucosideamygdalinarbutinesculincellobiosemelibiosemelezitoseraffinosestarchxylitolgentiobiosetagatosefucosearabitolgluconateketo-gluconate__________________________________________________________________________ *Morphologically dissimilar from LBI Not tested **Based on readings 24h after incubation at 37.degree. cC. NCDO type cultures were all L. helveticus strains ATCC 39538 was L. lactis strain ATCC 39539 was L. casei strain ATCC 39542 was L. plantarum strain TABLE 2__________________________________________________________________________TYPICAL AGEING PROFILE FOR ACCELERATED AS COMPAREDTO CONVENTIONAL CHEDDAR CHEESE__________________________________________________________________________ GRADING/COMMENTSAGE OF CHEESE VAT 9 (CONTROL) VAT 10 (ACCELERATED)__________________________________________________________________________ 3 weeks Typical young curd Good body and texture Insipid similar to 8-9 week cheese 7 weeks Good body - texture Good body and texture, Mild breaking down very nicely similar to 4-5 month Cheddar 9 weeks Good body - texture Good body and texture, on Mild par with 5-6 month Cheddar10 weeks Good body - texture Similar to a good 5-6 Mild month Cheddar12 weeks Typical good 3 month good body and texture old Cheddar equal to 6 month old16 weeks Good cheese 3-4 Good body and texture month age close and smooth, equal to 6 months old18 weeks Good 4-5 month Equal to 6 month old Cheddar20 weeks Good body and texture Good body and texture Reasonable flavour, Close and smooth waxy, equal to 5 month 7-8 month old Cheddar22 weeks Good body and texture Good body and texture Reasonable flavour Close and smooth waxy Equal to 5 month 7-8 month old. Cheddar__________________________________________________________________________ GRADING AT 11 WEEKS OLD (NACPE POINTS) VAT 9 VAT 10__________________________________________________________________________Flavour 40 40Body Texture 33 33Colour 9 10Finish 5 5 87 points 88 points__________________________________________________________________________ TABLE 3__________________________________________________________________________COMPARISON OF TCA SOLUBLE AMINO ACIDS IN CONTROLAND ACCELERATED CHEESES WITH TIME Nano Moles/per ml of sample 20 days 20 days 84 days 89 days 172 days 172 daysTIME Control Accel. Control Accel. Control Accel.__________________________________________________________________________Taurine --* -- -- -- -- --Urea -- -- -- -- -- --Asp 40 45 44 190 79 300Thre 9.1 17 14 69 29 140Ser 12 21 11 98 18 120Asn -- -- -- -- -- --Glu 87 140 130 610 240 980Gln -- -- -- -- -- --Sarcosine -- -- -- -- -- --.alpha.-amino adipic -- -- -- -- -- --Pro -- -- -- -- -- --Gly 11 22 14 105 31 170Ala 24 70 26 134 49 190Citrulline -- -- -- -- 3.9 37.beta.-amino butyric -- -- -- -- -- --Val 20 41 39 220 80 360Cystine -- -- -- -- -- --Met 2.4 2.0 11 46 23 85Cystathionine -- -- -- -- -- --Ileu 4.2 15 8.9 120 20 250Leu 55 78 94 370 190 530Tyr 7.7 13 9.2 70 11 110Phe 30 41 52 170 100 250.beta.-amino butyric -- -- -- -- -- --acid -- -- -- -- -- --.beta.-alanine -- -- -- -- -- --.gamma.-amino butyric -- -- -- -- -- --acid -- -- -- -- -- --Ornithine 3.5 0.5 25 3.1 58 63Lys 41 94 55 370 110 610Tryp -- -- -- -- -- --His 7.0 15 8.4 83 14 1701-Me-His -- -- -- -- -- --3-Me-His -- -- -- -- -- --Arg 23 46 18 170 22 120__________________________________________________________________________ *not determined TABLE 4__________________________________________________________________________LOW FAT CHEDDAR CHEESE RECIPES CONVENTIONAL ACCELERATED__________________________________________________________________________MILK 1100 gallons 1100 gallonsFat 1.62% these will 1.62% these willProtein 3.20% vary 3.20% varySTARTER/S Starter of the Starter of the day but reduced day reduced by 15% by 10% of normal of normal rate. rate. Acidity: 1.58% LA Acidity: 1.58% LA 2 gallons of L. helveticus AGC1 culture. Acidity: 1.50% LARIPENING TEMP. 90.degree. F. 90.degree. F.RIPENING TIME 30 mins 30 minsRENNET 43 oz 43 ozSETTING TIME 45 mins 45 minsCUTTING SPEED Speed 8 for 8 mins Speed 8 for 8 mins (large cut) (large cut)SCALD TEMP. 92.degree. F. 96.degree. F. (Acidity: 0.12% LA) (Acidity 0.12% LA) Scald up in 20 mins Scald up in 20 mins (Acidity: 0.125% LA) (Acidity: 0.125% LA)PITCHED 10 minutes after 10 minutes after scald up scald upWHEY OFF 15 minutes 15 minutes (Acidity: 0.15% LA) (Acidity: 0.16% LA)ACID DEVELOPMENT 2 hrs 0.18% LA 2 hrs 10 mins 0.21% LA(Time from Renneting) 2.5 hrs 0.27% LA 2 hrs 40 mins 0.27% LA 3 hrs 0.35% LA 3 hrs 20 mins 0.45% LA 3.5 hrs 0.49% LA 3 hrs 50 mins 0.55% LA 3 hrs 50 mins 0.57% LAMILLED @ 0.57% LA @ 0.55% LA__________________________________________________________________________ TABLE 5______________________________________TYPICAL AGEING PROFILE FOR ACCELERATEDAS COMPARED TO CONVENTIONAL LOW FATCHEDDAR CHEESEGRADING/COMMENTS ACCCELERATEDAGE OF CONVENTIONAL (NORMAL STARTER +CHEESE (NORMAL STARTER) L. HELVETICUS AGC1)______________________________________3 weeks Good body and texture Good body and texture Mild flavour Clean, good flavour, more flavour than conventional4 weeks Good body and texture Good body and texture Clean, mild flavour Good, clean flavour6 weeks Good body and texture Good body and texture Clean, mild flavour Good, clean, well developed flavour7 weeks Good body and texture Good body and texture Clean, mild flavour Good, clean, mature flavour11 weeks Good body and texture Good body and texture Clean flavour Good, clean mature flavour18 weeks Good body and texture Good body and texture Clean and slightly Good mature flavour sweet______________________________________

Claims

1. A method of making cheddar-style cheeses which comprises:

a. adding to milk a mixture of starter cultures which includes Lactobacillus helveticus AGC1 (NCIB 40051);

b. permitting the milk to ripen;

c. adding rennet to the ripened milk;

d. permitting the resulting milk to set and form a curd;

e. cutting the curd, stirring and heating to scald temperature;

f. separating the curd from the whey;

g. cheddaring the curd; and

h. recovering the cheeses.

2. A method according to claim 1 wherein the curd from step g is milled, salted, pressed and allowed to mature.

3. A method according to claim 1, in which the cheese has a fat content of 20 to 38% and a moisture content of 30 to 45%.

4. A method according to claim 1, in which the cheese has a fat content of 8 to 20% and a moisture content of 37 to 54%.

5. A method according to claim 1, in which Lactobacillus helveticus AGC1 is added to a proportion of 0.05 to 0.3%, based on the total weight of the starting milk.

6. A method according to claim 1, in which the scald temperature is in the range of 94.degree. to 108.degree. F.

7. A method according to claim 1 wherein the starter cultures are added to the milk in a proportion of 0.0l to 1%.