Note: Descriptions are shown in the official language in which they were submitted.
- - ~,AZ~621~ 2
This novel integrated process comprises the addition of (t) live ~acfo~ac~llus casei subsp casel
L2A to control the undesirable micro!lora, ~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 ci~eese.
LA~::TOBAC~LLI CELLS AND EXTRACTS
FOR ~CCELERATED CHE~SE RIPENING
The production of matured Chedd~r cheese involves considerable co~s for the cheese industry,
mainly due to a slow microbial process which incurs higll costs for refri~eration and warehousing.
Furlhermore, impending legisiation for mandatory pasteurization to destroy pathogens (e.g.
List~ria) will lower the content of useful lactic acid bacteria (LAB), increasin~ the time necessary
to produce maturecl 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 ch~racterized sufficiently to permit dup~ication of
their complele flavor by mixtures of pure compounds lhus using indirect methods to speed up
-cheese ripening (1~.
Elevated temperatures during cheese ripening, added enzymes, modilied starter and slurry
method have been commonly used, but ~he most widely employed method for accelerated cheese
ripening is the addition of extraneous enzyrmes to the cheese (2).
~. CA2162181
Although some cheap commercial food Qrade 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 Jactis enzymes were introduced to the market as the product "Accelerase" by
Imperial Biotechnology Ltd (London), but most starter lactococci are unable to multiply in cheese
and contain less active peptidases ~3), and esterases (4~ than Lactobacillus strains (5).
The present novel process involves the addition of live and heat-shocked cells of Lactobacillus
casei subsp-casei L2A, combined with the enzyme Neutrase and elevated ripening temperature.
Ti~e Lactobacillus strain selected was typical of the microfiora 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 (4), which are responsible for adval1ced hydrolysis of milk casein, and in
order to develop desirable flavors without quality defects.
DESCRI-?TION OF THE INVENTION
Fresh raw milk was pasteurized (73~C, 16 s) immediately before cheesemaking, and cheeses
were made in pilot cheese vats containing 225 L of milk. The starter culture, a mixture of
Lactococc~ls cremorisand Leucor~ostoccremoris(Agropur, Granby, Quebec, Canada) was added
to the milk (1.5% viv) after three transfers for 18 'nr at 20~C in 12% (w/v) reconstituted skim milk
at a level of 2% (vlv) inoculum for the acidification of all vats. Lactobaci/Jus casei-subsp-casei i~A
grown in 12% (w/v) reconstituted skim milk (30"C, 48 hr) was added to the milk at 0.01% (v/v)
inoculum.
When the milk acidity had increased by 4~ Dornic (0.04% Lactic Acid), rennet (50% bovine rennet
+ 50% porcine pepsin; Chr. i-iansens Lab, Inc., Milwaukee, Wl) was added at a level of 0.02%
(v/v). The miik was set at 30~C for 30 min, the curd was cut, cooked at 38~C, and the whey
drained off. After cheddaring at 38~C, milling, and salting (2%, w/w), following overnight pressing
of the curd (approx. 10 kg), all cheeses were vacuum packed in plastic bags (4 mil, Winpak Co.,
Winnipeg, Canada), and ripened at 4~C for one week, foilowed by 2 months at 13~C, and 7
months at 7~C.
- CA21621 81
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 (67~C) 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. Duplicate cheeses were made on separate 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 cu!tyres 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.
Cheese samples taken after ~, 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 30~C for 48 h under anaerobic conditions (BBL Gaspak system).
Lactobacilli numbers were determined with Rogosa agar (Difco). Each cheese sample (25 g) was
sliced and added to 225 ml of 2.0% citrated water ahd homogenized for 5 min in a Stomacher
(Lab Blender, Model 400, A.J. 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-chemical ratios were calculated as:
% M/NFS = (Moisture/Non-fat solids (100-fat)) X 100,
% F/S = (FaVSolids (100-moisture)) X 100,
% SIM = (Salt/Moisture) X 100.
Rheological parameters \Ivere determined by double compression tests using the Instron Universal
Testing Machine (Model 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 cmimin.
~A21 621 81
The compression unit and a stainless steel cylinder (Diameter: 3.5 cm) caused 80% deformation
of cylindrical cheese samples (diarneter: 1.25 cm; height: 1.0 cm) held at room temperature l hr
before testing. Three parameters, fracturability (kPa), firn~ness (kPa), and cohesiveness (~,h), 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, 6 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 1 O, while flavor and texture defects were evaluated on a scale of 1 to 100. The
rninimum 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-chernical (pH, fracturability, firmness, cohesiveness), and flavor intensity
(%~ by sensory evaluation.
The variance homogeneity was verified by the Bartlett test, and Duncan's multiple range test used
to determine significant differences (p c 0.05) among treatments (10).
The results of the experimental design for cheesemaking showed that the proxirnate analysis
(moisture, fat, proteinl 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 ripeniny period (Fig. 1). This increase was likely due to the presence of the
bacterial and enzyme additives. l~he pH va!ues of experimental cheeses were lower than the
control cheese (Fig. 2), mainly due to the production of lactic acid by live lactobacilli. Where
added, Neutrase liberated neutral peptides that partly neutralized the lactic acid.
Significant difference in LAB and lactobacilii counts were observed between the control and
experimental cheeses up to 2 months, except for the beginning of rnaturation (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
- ~A21621 81
experimental cheeses had an average cour,t of 6.0-6.7 log/g. Cheeses supplemented with either
1.0 to 2.0% of heat-shocked ceils showed comparable counts because of the rapid growth ot
residual live lleat-shocked ceils. 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 classified as first class and many of them would qualify as premium
class (Table 3). The pH values were between 5.01 and 5.13. pl I values above 5.2 or below 4.85
are considered undesirable (12). Low pH is known to influence cheese texture by decreasing the
fracturation force (13, 14), but the effect of pH was not clearly demonstrated (l~ig. 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
maturation period, with relative stabilization during the last 3 months. These changes are due to
the degradation of ~5,-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 10 5 Au/g of cheese were more
fracturable and less cohesive than those with Neutrase at 2.0 or l.0 X 105 Au/g, thereby
confirming 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
rheological measurements is often caused by the non-homogeneity of cheese (17, 18, l9).
Statistical analysis of rheological parameters showed that the addition of Neutrase appears to be
the most effective 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 (l) flavor intensity, (2) heat-
shocked cells (HS) and (3) Neutrase concentration.
~ .
~ ~, 'A 2 l ~ 1 8 ~ ; ~7~
The order of efficiency in acceleraUng ripening at 6 months was: HS (2.0%) i Neutrase B > HS
(~.G%) ~ Neutrase C ~ HS (1%) + Neutrase A, B, C. Aithough % increases varied from 100% to
50%, HS (2.0%) plus Neutrase C showed a significant.y greater increase in flavor intensity as
cornpared to the other l-eal~nenls throughout maturation, except at 6 months. These results
compared favorably with the reduction of 30 to 50% in ripening time claimed by many authors (9,
21, 22, 23, 24).
The quali~y of control cheese and cheeses supplemented with live (LL) and heat-shocked ~HS)
cells remained excellent (class 1) throughout maturation. However, total cheese quality (flavor &
texture) was strongly influenced by pitte~ess development in cheeses with added Neutrase.
Cheeses which received live + ~IS celis and Neutrase showed a good quali~ (class ll) after 6 and
9 months. However, bittemess developed significantly with increased PJeutrase concentration.
Tex~ure defects were not directly associa.ed with intensity of treatment but parUy with ptl of the
cheese.
Based on total quality score and % increase in flavs~r, this novei process recommends the addition
of 1.0C~o HS cells, along with Neutrase (1.0 X 105 Aulg of cheese). To control the undesirable
microflora, addition of live Lactobacillus casei-sut~sp-caseJ (~A) cells is also suggested at a
concentration not higher than 4.0 (Log,0 CFU/ml of milk) to maintain the desired pH.
This novel process does not appear to be expensive, ~iven the ~ow c~ncentrations of additives
necessary, and the simple apparatus required for the heat-shock treatment. When we combine
the effect of elevated storage temperature, as shown in the presentstudy, ~his process represents
an important technolo~y for accelerating Cheddar cheese ripening.
., . , . ,, . ~
CA~ l 6~
REFERENCES CITED
1. Olson, N. 1990. FEMS Microbiology Reviews 87:131-148.
2. Law, B.A. Flavor developrnent in cheese. In: advances in the microbiology andbiochemistry of cheese and fermented milk (Davies, F.L. and B.A. Law, eds), pp 209-228,
Academic Press, London, 1984.
3. Arora, G. and Lee, ~.H. 1990. J. Dairy Sci. 73:274-279.
4. Lee. S.Y. and Lee, B.H. 1989. J. Foods Sci. 54:119-122 & 126.
5. Lee, B.H., Haché, S. and Simard, R.E. 1986. J. Ind. Microbiol. 1:209-217.
6. Laleye, L.C., Simard, R.E., Le~,~B.H. and Holley, R.A. J. Dairy Sci. 72:3134-3142.
7. Lee, B.H., Laleye, L.C., Simard, R.E., Munsch, M.H. and ~oliey, R.A. 1990. J. Food Sci.
55:391 -3g7.
8. Law, B.A. and Wigmore, A.S. 1g82. J. Dairy Res. 49:137.
9. APHA. Standard Methods For the Examination of Dairy Products (Richardson, G.H. ed),
15th edition. American Publich Health Association, Washington, D.C., U.S.A. 1985.
10. Steel, R.G.D. and Torrie, J.H. Principles and Procedure of Statistics. A Biometrical
approach, 2nd edition, McGraw-Hill Book Cornpany, New-York, N.Y. 1980.
11. Lee, B.H., Laleye, L.C., Simard, R.E., Holley, R.;~., Emmons, D.B. and Giroux, R.N.1989.
J. Food Sci. 55:386-390.
12. Gilles, J. and Lawrence, R.C. 1973. New Zeal. J. Dairy Sci. & Technol. 8:148-
13. Cream~r, L.K. and Olson, N.F. 1982. J. Food Sci. 47:631-
14. Creamer, L.K., J. Gilles and Lawrencel R.C.1988. New Zeal. J. Dairy Sci. Technol.23:23-
15. Fedrick, l.A. and Dulley, T.R. 1984. New Zeal. J.Dairy Sci. Technol. 19:141-
16. El Abboudi, M., Pandian, S., Trépanier, G., Simard, R.E. and Lee, B.H.1992. J. Food Sci.
17. Lee, C.H., lmoto, E.M. and Rha, C. 1978. J. Food Sci. 43:1600.
18. Walstra, P. and Van Vliet, T. 1982. Int. Dairy Fed. Bull. Doc. 153:22.
19. Trépanier, G., Simard, R.E. and Lee, B.H. 1991. J. Food Sci. 53:696-700.
20. El-Soda, M., Desmazeaud, M.J., Aboudonia, S. and Badvan, A. 19~2. Michwissenschaft
37:325-
21. ~I-Soda, M., Ezzart, N., El Deeb, S., Mashly, R.l. and Moustapha, F.1986. Le Lait 66:177-
22. Abdelbaky, A.A., El-Neshawy, A.A., Rabie, A.M. and Ashour, M.M. lg86. Food Chem.
21 :301-
- l,A2162181 o,
23. Fedrick, I.A., Cromie, S.J., Dulley, J.R. and Gilks, J.E. 1986. New Zeal. i. Dairy Sci.
Technol. 21:191.
. ~
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T~ 4:1'ERCENTAGElNCl~EASElN FLAVORINTENSITY'
TREATMENT ,~ MATURAliON TlME(months)
2 4 6 9
LIVELACTO
+ o n12.5 50.() 3.1
i~SLACTO1.(1~
+ NF,l)'rRASE
LIVELACTO A 33012.5 62.5 (1.0
+ B 50.0 375 5().0 (1.0
HSLACTO1.n',~ C 33.0 ~37 5 fi2.: fi.25
LIVELACTO B 17.025.1) lUU.O 0.0
HSLACTO2.0i~ C X3.05U() ~7.5 12.5
a: ~ vori!lt(~ it~ p~rim~llt~ cc X 100~
~ Q~vorin~ y~f ~ntrol c~
qb
CA2 1 62 1 8 1
Table 3: SEr~SiOKY EVALUATION OF CHEESE L)UKING MATURATI- N
TREATMENT MATlJKA l lC)N TIME ~months)
~ ! 2 ¢ fi 9
T S' TEXTb T S TEXT T S T EX1 T S TEXT T S TEXT
CONTROL 92.() 1.0 92.(1 1.0 92() 1.() 92.0 1.0 93.0 0.0
LIVE LACTO
+ Y2 5 ().0 92.0 0.0 '33 () ().() 93.0 ().0 92.2 0.3
HS LAC'TO l.O~o
+ NEUTRASE
LIVE LACTO A 92.0 0.5 92.4 0.5 92.5 0.5 92.0 0.5 91.0 0.5
+ B 92.() 0.5 92.5 1.0, 92 5 ().() 9().9 1.5 91.0 0.5
HS LACTO 1 0~ C 92.() 0.5 92.5 1.0 92.0 1.() 89.5 1.5 88.0 1.0
Ll~'E l AC-rO B 92.() 0.~s 91 0 05 ~2.5 U.0 8'3.0 1.5 90.0 0.0
HS LACTO 2.()~. C 92 0 1.0 9().0 0.5 '3~.() 0.5 88 0 2.0 89.0 0.0
a: Total quality (flavor and texture) score; mcan val-le (n = I to 2); scal~ of I to 100: Class 1: 2 92
{'I.lSS 11: 91 to ~7
Cl~-iss ]11: < 86
h: Texture score; mean value (n = I to 2), scale of 0 to 3 (normal texture to w~k or acid texture).
~.
r ~-
CA2 1 62 1 8 1
Tablc 2: I'ROXIMATE Al\IAl.YSIS OF ONE-MON1 1~-OL,D CHEESES'
T}~EATMENT MOISIlJRE FAT l'~OTEIN SALT pH
% c~o % ~/u
CONTKOL 34.43b 35.0()c29.6&1 1.37f 5.27
LIVE LACTO
+ 34.34b 33.fi2c25.42e 1.55f 5.01h
HS LACrO 1.()'7u
+ NEIJTI~ASE
LrVE LACTO A 33.76b 34.~3c 25.9()e 1.32f 5.14i
t 1~ 33.t~b 34.75c25.fifi~ 1.50f 5.13i
HS LACrO 10% C 34.12b 34.62c 25.35e 1.55f 5.13i
LIVE LACTO B 34.31b 35.25c 25.54e 1.53f 5.10j
+
HS LACTO 2.0% C 33.82b 34.25c 25.fi4c 1.33f 5.13j
;3: mcan valuc (n = 1 to 2). MCIII5 in tlle SdnlC COlllllln IlOt follow~1 by a COlnrllOII letler
are significantly diffcrcnt (P < 0.05~.
i
(e~-g~-) a~ou ql~ pue .~ Jo ~e~e (~cj~j a~ F~ l F: r. ~ u:sr~ u!u c I U! C o ;o S~ ase.~aU! :e ~noqe ~u~q o~ ~-a.r ~a~ a~i'~,J.t ,0 ~uno:ue =nV ~ x ~, n uosu~ :~
OGt Y ~ !!'U 30 au ~ u/~ o~ auru!nllo~ ;~inili~ e 3~ aurlG~)
~'Z i iaSe~-i;)Se~ sr~ tqo;ae-;,
2U i;eS 2~ -,V 5.0l X GG ~ se 3na~ 1
zaul~auua~T ~6C~O Z + ove- SH ~=
du~;auua~ ~6l0C ~ ove~
t 3S~iJ !~.
9u~ ec ~/!lV S.~l X ooz~ ase:;ni~N l a
z~uli~uuay ~G~ Z + o ~e I sH I
2u~;auuad ~6100 + o~el I
2U~ J v/~l~v o- x G~ ;r~
- 2ui;-u::aY ~ .01 ~ . ci_e-l ST~) . ac~v I C3~ 0HS-l -H
~u-;~ uua~.: ~6 i û G . o- e~ ~_
21 i~-~s ~ s ~ ~ .~e n.~
~ z8uilauu.~ o;ai--l SH l
2 u~.u. aY -~ .G ~ e~
7~uiuec ~ OG I V asEI~ni~
7aUna'i'J~'Ci '~ I . o'ai'~; SH I IllT~V~0~V1 3Nl D
2UilaUUa~Y ~Q~ + c3ai~, 1
2uilauuay ~GO I o: i-. SH .(o~ae~i sHi ~ 'V~0s--~1 a3~1X'HS-'V~H
Zaullauu i~T ~b IG ~G o~ (olar l~ T:~VSO I ~Vl 3.~i
~V N ~ V N ;e~ pJrp~r~S lO~TLl~i'OZ) oo
3 'VlSNO"~ VY~ H ~NO~ _
S IVZ)lld3~NoiLTaav 3,Ullcav .NriT~l~_iS3a 3/~T.. 'iaiTV lN3W1~3~1
N '!S~(l lVlt\,'3Wii3dX3 :i alCel
5)
