CA2072159A1 - Accelerated maturation of cheddar cheese by the addition of live and heat-shocked lactobacilliand neutrase - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Live and heat-shocked Lactobacillus casei subsp case; (L2A) cultures, supplemented with the proteolytic enzyme Neutrase, have been developed by means of biotechnology, which accelerate Cheddar cheese ripening by up to 60% in flavor intensity, as compared to control cheese. The present invention studied Cheddar cheese evolution in detail over a 9-month ripening period by evaluating microbiological, physico-chemical, sensory and rheological parameters.

This novel integrated process comprises the addition of (1) live Lactobacillus casei subsp casei L2A to control the undesirable microflora, (2) heat-shocked cells of the same culture at a level of 1% (v/v), and (3) Neutrase at a level of 1.0 x 10-5 Au/g of cheese.

Description

LACTOBACILLI CELLS AND EXTRACTS
FOR ACCELE5:lATED CHEESE RIPENING

The production of matured Cheddar cheese involves considerable costs for the cheese industry, mainly due to a siow microbial process which incurs high costs for refrigeration and warehousing.

Furthermore, impending legislation for mandatory pasteurization to destroy pathogens (e.g.
Listeria) will lower the content of useful lactic acid bacteria (i~B), increasing the time necessary to produce matured cheese.

Cheese flavor development is a dynamic process which represents a finely orchestrated series of successive and concomitant biochemical events over a period of time, leading to products with highly desirable aromas and flavors. None are characterized sufficiently to permit duplication of their complete flavor by mixtures of pure compounds thus using indirect methods to speed up cheese ripening ~1).

Elevated temperatures during cheese ripening, added enzymes, mociified starter and slurry method have been commonly used, but the most widely employed method for accelerated cheese ripening is the addition of extraneous enzymes to the cheese (2).

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Although some cheap commercial food grade enzymes are available for the above purpose, almost all have their limitations, especially regarding the control of their action on milk components, resulting in a rheologically poor final product, and often with bitter flavor.
Lactococcus lactis enzymes were introduced to the market as the product "Accelerase" by Imperial Biotechnology Ltd (London), but most star~er lactococci are unable to multiply in cheese and contain less active peptidases (3), ancl esterases (4) than Lactobacillus strains (5).

The present novel process involves the addition of live and heat-shocked cells of Lac~obacillus casei subsp-casei L2A, combined with the enzyme Neutrase and elevated ripening ternperature.

The Lactobacillus strain selected was typical of the microflora of good quality Cheddar cheese, and shows good growth and ensured control of undesirable microflora (6, 7). This strain is known for its rich peptidases (43, which are responsible for advanced hydrolysis of milk casein, and in order to develop desirable flavors without quality defects.

DESC:RIPTION OF THE INVENTIC)N

Fresh raw milk was pasteurized p3C, 16 s) immediately before cheesemaking, and cheeses were made in pilot cheese vats containing 225 L of milk. The starter culture, a mixture of Lactococcuscremorisand Leuconostoccremoris(Agropur, Granby, Quebec, Canada) was added to the milk (1.~% v/v) after three transfers for 18 hr at 20C in 12% (w/v) reconstituted skim milk at a level of 2% (v/v) inoculum for the acidification of all vats. Lactobacillus casei-subsp-casei L2A
grown in 12% (wlv) reconstituted skim milk (30C, 48 hr) was added to the milk at 0.01% (vlv) inoculum.

When the milk acidity had increased by 4 Dornic (0.04% Lactic Acid), rennet (50% bovine rennet + 50% porcine pepsin; Chr. Hansens Lab, Inc., Miiwaukee, Wl) was added at a level of 0.02%
(vlv). The miik was set at 30C for 30 min, the curd was cut, cooked at 38C, and ~he whey drained off. After chedclaring at 38C, milling, and salting (2%, w/w), following ovemight pressing of the curd ~approx. 10 kg), all cheeses were vacuum packed in plastic bags (4 mil, Winpak Co., Winnipeg, Canada), and ripened at 4C for one week, followed by 2 months at 13C, and 7 months at 7C.

2~721~9 Heat-shocked lactobacilli were prepared by adjusting the pH of the culture to 6.5 using 1 M
NaOH, and by pumping through a stainless stell coil immersed in a water bath (67r:;) to achieve a 22-s contact time. Mortality rate was 94.5% but proteolytic activity, measured by Hide Powder Azure (HPA) method (8) remained unchanged. Duplica~e cheeses were made on ssparate days in four identical vats and the effects of the various concentrations of bacterial additives compared.
After milling, the curd from each vat was divided in two and different concentrations of Neutrase added.

Control cheese, which constituted the first of three groups, is a standard cheese without the additives. Live and heat-shocked cultures were added to the second group of cheeses, while various concentrations of Neutrase besides bacterial additives were added to the third group. The experimental design is shown in Table 1.

Cheèse samples taken after 0, 1, 2, 3, 4 weeks and 2, 4, 6, 9 months were analyzed for lactic acid bacteria (LAB) and lactobacilli counts. LAB were counted on Lactobacilli MRS agar (Difco), following incubation at 30C for 48 h under anaerobic conditions (BBL Gaspak system).
Lacto~acilli numbers were determined with Rogosa agar (Difco). Each cheese sample (25 9) was sliced and added to 225 ml of 2.0% citrated water and homogenized for 5 min in a Stomacher (Lab Blender, Model 400, A.~. Seward Lab., London, UK). Dilutions were plated on Lactobacilli MRS and Rogosa Agars.

Samples of cheeses were taken for duplicate determination of fat, protein, moisture, salt and pH
by APHA (9) methods. Physico-ch0mical ratios were calculated as:
% M/NFS = (Moisture/Non-fat solids (100-fat)) X 100, % F/S = (FaVSolids (100-rnoisture)) X 100, % S/M = (SalVMoisture~ X 100.

Rheological parameters were determined by double compression tests using the Instron Universal Testing Mashine (Modei 1101, Instron Corp., Canton, U.S.A.). The 500-kg cell was fixed to the crosshead, adjusted to 5 cm/min, and chart speed was 5 cm/min.

2~72159 The compression unit and a stainless steel cylinder (Diameter: 3.5 cm) caused 80% deformation of cyiindrical cheese samples (diameter: 1.25 cm; height: 1.0 cm) held at room temperature 1 hr before testing. Three parameters, fracturability (kPa), firmness (kPa), and cohesiveness (%), were studied with ten replicates of each cheese. Cheeses coded with three-digit numbers were presented to a panel of three expert graders from Agriculture Canada at 1, 2, 4, ~ and 9 months of ripening. Two blocks of cheese were graded for flavor and texture. Flavor intensity was graded on a scale of 1 to 10, while flavor and texture defects were evaluated on a scale of 1 to 100. The minimum points of grade 1, 2, 3 Canada Cheddar were 2 92, 91 to 87, and < 86, respectively.

Analysis of variance (F test) was performed on microbial counts, proximate analysis (moisture, fat, protein, salt), physico-chemical (pH, fracturability, firmness, cohesiveness), and flavor intensity (%) by sensory evaluation.

The variance hornogeneity was verified by the 8artlett test, and Duncan's multiple range test used to determine significant differences (p < 0.05) among treatments (10).

The results of the experimental design for cheesemaking showed that the proximate analysis (moisture, fat, protein, salt) of the one month-old cheeses was comparable for all cheeses, within the normal range for Cheddar cheese, except for protein content (Table 2).

The protein content of the experimental cheeses was significantly higher than the control cheese throughout the ripening period (Fig. 1). This increase was likely due to the presence of the bacterial and enzyme additives. The pH values of experimentai cheeses were lower than the control cheese (Fig. 2), mainly due to the production of lactic acid by live lactobacilli. ~/Vhere ~/
added, Neutrase liberated neutral peptides that partly neutralized the lactic acid.

Significant difference in LAB and lactobacilli counts were observed between the control and experimental cheeses up to 2 months, except for the beginning of maturation (Figs. 3, 4). - ' Cheeses produced with added cultures of lactobacilli had LAB counts of 7.0 to 8.5 log within 1 week, while that of the control was 5.7 log/g of cheese. Lactobacilli counts showed a similar growth pattern to the LAB, but the corresponding control counts were 4.0 log, while the 2~2~ $~

experimental cheeses had an average count of 6.0-6.7 log/g. Cheeses supplemented with either 1.0 to 2.0% of heat-shocked cells showed comparable counts because of the rapid growth of residual live heat-shocked cells. Although certain strains of Lactobacillus casei contributed to increased acidity to the point where the cheeses were graded as acidic and bitter-tasting (11), the addition of lactobacilli cultures, heat-shocked cells and Neutrase did not affect the cheese quality. All cheeses were classifled as first class and many of them would qualify as premium class (Table 3). The pH values were between 5.01 and 5.13. pH values above 5.2 or below 4.85 are considered undesirable (12). Low pl~ is known to influence cheese texture by decreasing the fracturation force (13, 14), but the effect of pH was not clearly demonstrated (Fig. 5), because of the more pronounced effect of Neutrase on the weakening of the casein network. Fracturability, firmness and cohesiveness values in Figs 5, 6 and 7 showed a decreasing pattern over the whole i maturation period, with relative stabilization during the last 3 months. These changes are due to the degradation of aS1-casein, and agree with the results of others (8, 13, 15). Cheese texture continued to soften during the last 3 months, but was counterbalanced by the rise in pH, thus leading to a final stabilization of rheological parameters.

Cheeses supplemented with Neutrase at a concentration of 4 X 105 Au/g of cheese were more fracturable and less cohesive than those with Neutrase at 2.0 or 1.0 X 105 Au/g, thereby confirrning the results of others (8, 15). The effect of heat-shocked cells on rheological parameters compared with Neutrase was not significant, as its activity was mainly directed towards the degradation of small peptides as well as casein. We confirmed this previously by a good correlation between the nitrogen soluble in phosphotungstic acid (PTA-sol N) and the amount of heat-shocked cells added (16). The interpretation of rheological parameters is sometimes rendered difficult, as evidenced in firmness (Fig. 6). This large variability associated with rheologica~ measurements is often caused by the non-homogeneity of cheese (17, 18, 19).
Statistical analysis of rheological parameters showed that the addition of Neutrase appears to be the most eflective treatment, unless this causes sensory deterioration.

Flavor intensity scores given by expert graders permitted calculation of % increase in flavor during maturation. A good correlation was found among % increase in (1) flavor intensity, (2) heat-shocked cells (HS) and (3) Neutrase concentration.

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The order of efficiency in accelerating ripening at 6 months was: HS (2.0%~ ~ Neutrase B ~ HS
(2.0%~ ~ Neutrase C > HS (1%) ~ Neutrase A, B, C. Although % increases varied from 100% to 50%, HS (2.0%) plus Neutrase C showed a significantly greater increase in flavor intensity as compared to the other treatments throughout maturation, except at 6 months. These results cornpared favorably with the reduction of 30 to 50/O in ripening time claimed by many authors (9, 21, 22, 23, 24).

The quality of control cheese and cheeses supplemented with live (LL) and heat-shocked (HS) ceils remained e)~cellent (class 1) throughout maturation. However, total cheese quality (flavor &
texture) was strongly influenced by bittemess development in cheeses with added Neutrase.
Cheeses which received live + HS cells and Neutrase showe~ a good quality (class ll) after 6 and 9 months. However, biKemess developed significantly with increased Neutrase concentration.
Texture defects were not directly associated with intensity of treatment but partly with pH of the cheese.

Based on total quality score and % increase in flavor, this novel process recommends the addition of 1.0% HS cells, along with Neutrase (1.0 X 105 Au/g of cheese). To control the undesirable microflora, addltion of live Lactobacillus casei-subsp-casei (l2A) cells is also suggested at a concentration not highsr than 4.0 (Log10 CFU/ml of milk) to maintain the desired pH.

This novel process does not appear to be expensive, given the low concentrations of additives necessary, and the simple ap,oaratus required for the heat-shock treatment. When we combine the effect of elevated storage temperature, as shown in the present study, this process represents an important technoiogy for accelerating Cheddar cheese ripening.

Claims (6)

1. A process for preparing lactococci starter and lactobacilli adjunct cultures in pilot plant vats wherein 225 L of fresh raw milk was pasteurized (73°C, 16 s), inoculated with a mixted culture (1:1 ratio) of Lactococcus cremoris and Leuconostoc cremoris at a level of 1.5%
(v/v) after three transfers grown for 18 hr at 20°C in 12% (w/v) reconstituted skim milk, and that Lactobacillus casei-subsp-casei L2A grown in 12% (w/v) reconstituted skim milk (30°C, 48 hr) was added to the milk at a level of 0.01% (v/v), before renneting.

2. A process for making Cheddar cheese in pilot plant scale according to Claim 1 in which rennet was added at a level of 0.02% (v/v) when milk acidity had increased by 4° Domic (0.04% Lactic Acid), the curd was cut, cooked, and cheddared at 38°C, followed by milling, salting (2%, w/w), packing and ripening at 4°C for one week, and then 2 months at 13°C
and 7 months at 7°C.

3. A process for preparing heat-schocked Lactobacillus casei-subsp-casei L2A cells on a laboratory scale, wherein the culture was adjusted to pH 6.5 using 1 M NaOH and passed through a stainless steel coil immersed in water bath (67°C) for 22 s to achieve mortality rate of 94.5% without destroying proteolytic enzyme activity, and the heat-shocked cells were added to cheeses prepared according to claim 2 at 1.0% or 2.0% (v/v) levels.

4. A process for making Cheddar cheese on a pilot plant scale according to claim 3, wherein different concentrations of the proteolytic enzyme Neutrase, at (1) 1.0 X 10-5 (Arson Unit), (2) 2.0 X 10-5 Au, and (3) 4.0 X 10-5 Au/g of cheese were added at salting stage.

5. A combined process for making Cheddar cheese by adding (1) 0.01% (v/v) of live Lactobacillus casei-subsp-casei L2A cells of claim 1, (2) 1.0% or 2.0% (v/v) levels of heat-shocked cells of claim 3, (3) Neutrase at 1.0 x 10-5 Au and/or 2.0 X 10-5 Au/g of cheese as in claim 4, and by maturing those cheeses at 4°C for one week, followed by 2 months at 13°C, and 7 months at 7°C, as in claim 2.

6. A process for reducing the Cheddar cheese ripening period by up to 60% on a laboratory scale, pilot scale or commercial scale according to any of claims 1 to 6 inclusive.