Lipolytic enzyme derived from a aspergillus microorganism having an accelerating effect on cheese flavor development

The present invention relates to a novel lipolytic enzyme derived from a novel Aspergillus microorganism. Cheese aged in the presence of a low concentration of this lipolytic enzymes ripens faster than with conventional lipolytic enzymes and without any associated rancidity.

TECHNICAL FIELD 
The present invention relates to a novel lipolytic enzyme derived from a 
noval Aspergillus microorganism. Cheese aged in the presence of a low 
concentration of this lipolytic enzyme ripens faster than with 
conventional lipolytic enzymes and without any lipolytic enzyme-associated 
rancidity. 
BACKGROUND ART 
Accelerating of cheese aging to improve cost efficiency through a reduction 
of storage space is becoming more important to the cheese industry. 
Currently, lipases from two different sources, animal pregastric glands, 
and animal pancreases, are being added to cheese curd as accelerators. A 
third source, microbial lipases, has been used in commercial production of 
strongly flavored cheeses, but not mild cheeses. 
Lipases derived from the pregastric gland of kid, calf and lamb are 
currently being added to accelerate ripening in cheese of Italian type 
(Provolone, Romano, Parmesan) where the characteristic rancid flavor of 
low molecular weight free fatty acids (e.g. butyric acid) is desirable. 
However, when these pregastric lipases are used to accelerate mild 
flavored cheese, e.g. Cheddar, too much of the low molecular weight fatty 
acids are produced and a rancid flavor develops. When the animal pancreas 
lipases are used in a high concentration, excessive amounts of lauric acid 
is produced in the cheese, making a soapy taste. Moreover, unless highly 
purified, these lipases can contain an abundance of protease which 
although in small amounts aids cheese softening, in larger amounts it 
produces bitter off-flavors. 
In the search for lipases showing desirable ripening characteristics, 
numerous organisms such as Pseudomonas fragii, P. fluorescens, 
Staphylococcus aureus, Clostridium lipolytica, Geotrichium candidum, 
Penicillium roqueforti, Aspergillus niger, A. flavus, A. oryzae, Candida 
cylindracea and Rhizopus oligosporous, have been used in laboratory 
studies. Many of these microbial lipases are not yet available 
commercially. In a study of various lipases for cheese ripening, Harper, 
W. J., [J. Dairy Science 40 556 (1957)], the following data has been 
developed with respect to selective liberation of free fatty acids from 
20% butterfat cream. 
TABLE 1 
______________________________________ 
Release of Free Fatty Acid from 20% Butterfat Cream 
Mol. % Produced in 3 hrs. @35 C. 
Ca- Ca- 
pro- pryl- 
Lipase Source 
Butyric ic ic Capric 
Lauric 
______________________________________ 
Imported kid rennet 
32.8 11.3 7.1 11.8 33.6 
(paste) 
Pregastric esterases 
kid 44.4 15.2 7.6 12.3 21.5 
lamb 48.1 8.6 14.2 9.3 19.3 
calf 36.7 8.9 14.8 10.7 39.0 
Domestic calf rennet 
10.7 3.1 trace trace 86.5 
Milk lipase 13.5 8.2 10.2 8.7 60.0 
Pancreatic lipase 
8.4 2.1 trace trace 89.1 
A. niger lipase 
43.1 18.9 20.2 17.5 trace 
______________________________________ 
Little information is available on the relationship between specific free 
fatty acids and flavor intensity in cheese although the presence of 
butyric acid usually is associated with strong rancidity development in 
strong cheese and the presence of large amounts of lauric may lead to a 
soapy flavor. Longer chain fatty acids have been linked to fruity flavors. 
It is generally noted that all of the even numbered free fatty acids are 
present but the concentration of the specific fatty acids varies in 
different types of cheese. 
Disclosure of the Invention 
The present invention relates to isolated cultures of a selected mutant 
strain of Aspergillus fungus. When cultured in a suitable nutrient medium, 
under proper aeration and agitation, it produces a novel lipolytic enzyme. 
For the purposes of this disclosure, this type of enzyme shall be referred 
to as a lipase although esterases are included in this term. lipase. 
Fungus 
The instant fungus has been identified as a novel strain of Aspergillus. 
The present fungus has been deposited in the American Type Culture 
Collection and given the Accession Number 20719. 
Enzyme 
The present Aspergillus organism makes a novel and especially useful lipase 
when cultured in a oil-based medium the same manner as known Aspergillus 
organisms. However, the lipase from the present Aspergillus. organism 
hydrolyzes longer chain C.sub.6 and C.sub.8 triglycerides such as 
tricaproin and tricaprylin more efficiently than short chain C.sub.4 
triglycerides such as tributyrin. 
Lipase having this characteristic appears to be useful as a ripening 
accelerator in mild flavored cheese production, such as Cheddar cheeses. 
The less efficient hydrolysis of the shorter C.sub.4 triglycerides 
suppresses the development of rancid flavors which are unwanted in mild 
cheeses. 
When isolated from the present Aspergillus organism under proper dilution, 
agitation, pH, and salt concentration conditions, the present lipase is 
associated with low levels of protease which soften the cheese without 
producing bitter flavors.

MODES FOR CARRYING OUT THE INVENTION 
Culturing the Organism 
The Aspergillus organism is stored on sterile soil under refrigeration. 
Portions of the soil stock are transferred to potato dextrose agar (PDA) 
and grown 4-5 days at 20.degree.-35.degree. C., 30.degree. C. preferably, 
to use as primary inoculum for shake flask and fermenter growth. 
Lipase can be made in a shaker flask by inoculating directly from the PDA 
slant into 100 ml. of sterile medium at pH 5.1 (5.0-5.2) containing 6% soy 
bean meal, 5% monobasic ammonium phosphate, 0.5% Magnesium sulfate 
(crystal), 2% soy oil, 1 L Erlenmeyer flasks. After 3-5 days at 30.degree. 
C. and an agitation of 265 RPM, the culture is filtered. Preferred ranges 
for culture ingredients are: 1-10% oil; &gt;0.1-1% sulfate; 1-5% phosphate; 
and 3-8% soybean meal. The concentrations of MgSO.sub.4, (NH.sub.4)H.sub.2 
PO.sub.4 and oil is important to optimum production of lipase activity. 
The initial pH must not be below 5.0. 
Production in a fermenter requires some modifications. The medium 
compoonents are as described for shake flasks, but it is necessary to use 
an intermediate step of inoculum to reduce the growing time to the 
interval of 3-5 days. The inoculum volume used is 5% (1 to 10) of a 
nutrient containing 4% whole wheat flour and 8% corn steep water at pH 
5.5. The sterile inoculum nutrient is inoculated with the PDA slant and 
grown 30-50 hrs. at 30.degree. C. and then transferred to the growing 
medium. Here, the oil may be corn or soy oil and the concentration may be 
from 1-6%. All of the oil can be present initially or added to increments 
after growth has started. 
Preparing the Lipase 
Filtering the fungal mycelium effectively isolates the lipase. The lipase 
activity of the crude culture when harvested (pH 4.5-5.0) is bound to the 
mycelium. The mycelium-free culture filtrate contains essentially no 
lipase activity (&lt;5%), and all soluble extracellular components which may 
have a negative effect on cheese flavor. 
Release of lipase activity from the mycelium into a second extraction 
filtrate depends on the concentration of the mycelium, the pH, the buffer 
salt concentration of the extraction filtrate (0.5-3.0% PO.sub.4 at 
optimum pH), the concentration of surfactant (preferably &lt;1%), and the 
treatment time. 
FIG. 1 illustrates how the amount of lipase released to extraction filtrate 
using Ethofot 242/25 surfactants varies according to the concentration of 
mycelium. All of these data was obtained at pH 6.2. Optimal recovery of 
lipase from mycelium is achieved normally at pH 6.2 because the lipase 
(free from mycelium) shows excellent stability for long periods. As the pH 
increases the lipase is less stable, and it is actively destroyed above pH 
7.5. For example, after 42 hours at 4.degree.-5.degree. C. filtrate showed 
94%, 87% and 74% activity retention respectively at pH 6.3, 6.8 and 7.4. 
After treatment under optimal conditions, about 80-90% of the lipase 
activity can be recovered from the mycelium filtrate. 
The lipase in the extraction filtrate can be used directly for cheese 
flavor modification or alternatively concentrated in vacuo or by 
ultrafiltration before cheese addition. The filtrate or concentrate can be 
dried in vacuum at 0.degree.-35.degree. C. or spray dried to produce a dry 
concentrate which can be blended with other components, e.g. sodium 
chloride, phosphates, etc., used routinely in cheese manufacture. The 
methods of concentration and drying are conventional techniques known to 
those of ordinary skill in the art. 
Assaying Lipase Activity 
Two different assays have been used for lipase activity. In the first, 
potentiometric titration is performed so as to determine lipase 
forestomach units (LFUs). One LFU equals the activity that releases 
1.5.mu. mol of butryric acid per minutes, Food Chemical Codex 3rd Ed., 
National Acadamic Press, 1981. 
The tiration substrate is prepared by dispersing an amount of sodium 
caseinate equivalent to 600 mg of casein, in 95 ml of water contained in a 
one-half pint freezer jar that fits the head of a suitable high-speed 
blender. This is mixed with 0.5 gm of hydroxylated lecithin. Finally 5.0 
ml of Tri-n-butyrin is added and mixed for 60 sec. at low speed. This 
substrate must be held to 33.degree. C. and used within 4 hours. 
The sample is prepared by suspending or dissolving an accurately weighed 
amount of enzyme in water. 
To measure, the titrator is filled with 0.05N sodium hydroxide, and the 
instrument is calibrated following the manufacturer's instructions. The 
substrate is mixed for about 15 sec with a magnetic stirrer, then 20.0 ml 
is pippetted into the reaction vessel of the titrator. One ml of the 
sample is added and equilibrated for 15 min. The rate, in ml per min., at 
which the titrant was delivered during the titration, is determined and 
recorded as R. The activity of the enzyme is calculated by the formula: 
EQU LFU/g=R.times.0.025.times.10.sup.3 /(W.times.1.25), 
in which W is the weight, in g, of the enzyme preparation contained in the 
1.0 ml of Sample taken for analysis. 
It should be noted that the enzyme's ability to hydrolyze tricaproin (C6), 
tricaprylin (C8), tricaprin (C10), can be measured simply by replacing 
tri-n-butyrin with the desired substrate. 
A second assay is the cream-acid titration method. An aliquot (20.0 ml) of 
10.5% butterfat Half and Half is placed into 2 oz. glass jars and brought 
to 33.degree. C. in thermostated water bath. After temperature equilibrium 
has been established (about 10 minutes), 1.0 ml of enzyme solution is 
added and incubated for 2 hours. Then 10 ml of ethanol (denatured formula 
2A is suitable) is added to stop the reaction before titrating with 0.05N 
NaOH to pH 9.5. The titration should be carried out with a glass electrode 
and the incubation mixture plus alcohol should be stirred with a magnetic 
bar. 
A substrate control (20 ml half & half plus 10 ml water) is run to correct 
for the amount of alkali required to neutralize the salts present in the 
cream. 
An enzyme control is also run to correct for the amount of alkali required 
to neutralize the salts present in the enzyme. 
The increase in acidity corresonding to a 1% hydrolysis of the butterfat is 
equivalent to a titration difference of 1.65 ml 0.05N NaOH. Activity is 
calculated from the following relationship: 
##EQU1## 
Effect of pH, Temperature and Time on Lipase Activity 
The effects of temperature and pH on tributryin hydrolysis by the present 
lipase is noted in FIGS. 2-4. As seen in FIG. 2, the optimum temperature 
for enzyme activity is approximately 42.5.degree. C., while FIG. 3 shows 
that the optimum pH is closed to 7.0. A sharp decrease in activity as the 
temperature is raised can be seen in FIG. 4, the effect being more 
dramatic with an increase in pH. The plots given in FIGS. 5a-f also 
indicate that an increase in time accentuates the adverse effect of pH 
values above 6.2, although pH 7.0 is clearly optimal at temperatures below 
60.degree. C. 
Triglyceride Hydrolysis 
The present lipase has been used to hydrolyze specific triglycerides 
(tricaprylin, tricaproin, and tributyrin) into their respective free fatty 
acids (FFAs) (caprylic, caproic, and butyric acids. Conventional gas 
chromatography techniques can be used to measure these FFAs. 
Hydrolysis has been measured in the following manner. Five gm of 
triglyceride is blended with 100 ml phosphate buffer (pH 6.5, 0.05 m) 
containing 1% gum arabic to form an emulsion, which is shaken with 2.0 ml 
of fungal lipase (100 mg) for 4 hours at 33.degree. C. To stop the 
reactions, 10 ml of ethanol is added, and the resulting free fatty acids 
are measured by gas chromatography. 
The free triglyceride hydrolysis results are as follows: 
TABLE 2 
______________________________________ 
Enzyme Treated Ratio 
Substrate % Free Fatty Acid 
(C.DELTA.)/C.sub.4 
______________________________________ 
Tributyrin (C4) 
3.39 1.00 
Tricaproin (C6) 
3.76 1.11 
Tricaprylin (C8) 
5.26 1.55 
______________________________________ 
The present lipase hydrolyzes both tricaproin and tricaprylin more 
efficiently than it hydrolyses tributyrin. This type of hydrolysis can be 
demonstrated by taking a ratio of the two numbers (C/C.sub.4). Increased 
rate of hydrolysis can be shown by the ratio TC/TB. Typically, the 
increased rate of activity results in equivalent C.sub.8 /C.sub.4 
production. This specificity is unique for the present lipase. 
The following table shows the tricaprylin (TC) to tributyrin (TB) 
hydrolysis ratio (TC/TB) for known lipases, as cited by known authors: 
TABLE 3 
__________________________________________________________________________ 
RELATIVE ACTIVITY OF LIPASE PREATIONS AS CITED IN 
LITERATURE 
__________________________________________________________________________ 
Activity vs. 
Activity vs. 
Preparation Tributyrin 
Tricaprylin 
TC/TB Cite 
__________________________________________________________________________ 
Hog pancreas 100 37 0.37 Enzymologia 
Human milk 100 59 0.57 11 178 (1944) 
Bovine pancreas 
100 41 0.41 
__________________________________________________________________________ 
Release of FFA from Milk Fat 
mol % mol % 
C.sub.4 
C.sub.8 
__________________________________________________________________________ 
Milk Lipase 13.9 1.8 0.13 Nelson J. H. 
Steapsin 10.7 1.5 0.14 J Oil 
Pancreatic Lipase 
14.4 1.4 0.09 Amer Chem Soc 
Calf Esterase 
35.0 1.3 0.04 49 559 1972 
Esterase pancreatin 
15.83 
3.0 0.19 
Calf pregastric esterase 
40.8 8.6 0.21 Farnham 
Kid pregastric esterase 
50.0 8.4 0.17 et al 
Lamb pregastric esterase 
44.8 8.3 0.19 1956 
__________________________________________________________________________ 
% of Total Free Fatty Acids (microequivalent basis) 
after 3 hrs. at 35.degree. C. on milk fat 
__________________________________________________________________________ 
Imported crude kid 
32.8 7.1 0.22 Harper W. J. 
rennet paste J. Dairy Sci 
Domestic purified calf 
10.7 trace .00 40 556 (1957) 
rennet paste 
Calf oral lipase 
36.7 4.8 0.13 
Kid oral lipase 
44.4 7.6 0.17 
Lamb oral lipase 
48.1 14.2 0.30 
Aspergillus lipase 
43.1 20.2 0.47 
Milk lipase 13.5 10.2 0.76 
Pancreatic lipase 
8.4 trace .00 
__________________________________________________________________________ 
u moles FFA liberated/ml enzyme 
__________________________________________________________________________ 
Syncephalastrum 
11.2 7.6 0.68 Chopra et al 
racemosum J. Dairy SC 65 
1890 (1982) 
__________________________________________________________________________ 
ml 0.1 N Acid Produced in 2 hrs. 30.degree. C. 
__________________________________________________________________________ 
Penicillum roqueforti 
1.45 0.56 0.39 
Aspergillus niger 
1.20 2.18 1.82 Shipe, W. F. 
Arch. Bioch. 30 
165 (1951) 
__________________________________________________________________________ 
FIGS. 6 and 7 illustrate the significantly better TC hydrolysis versus TB 
hydrolysis of the present lipase and how it can be increased with 
temperature. At 40.degree. C. and pH 7.0 the TC/TB ratio is 1.73, while at 
50.degree. C. and pH 7.0 it is an even greater 2.49. 
Milk Triglyceride Hydrolysis 
The present lipase has been tested for hydrolysis of those triglycerides 
present naturally in milk. To test for hydrolysis, 180 ml of 10.5% butter 
fat cream is treated with 0.36 g (0.2%) and 0.90 g (0.5%) of either the 
present lipase or calf lipase. Samples are held for 15 days at 
0.degree.-3.degree. C. before free fatty acid (C4 to C12) is determined by 
gas chromatography. A control cream sample without enzyme is carried also 
through the experiment. 
The results of the milk test are as follows. 
TABLE 4 
______________________________________ 
Mg FFA/ml cream 
0.2% 0.5% 0.2% 0.5% 
Specific Present Present 
Calf Calf 
FFA Control Lipase Lipase Lipase Lipase 
______________________________________ 
C4 0.18 0.37 0.52 0.38 0.53 
C6 0.09 0.32 0.43 0.13 0.20 
C8 0.10 0.20 0.27 0.12 0.15 
C10 0.14 0.30 0.41 0.20 0.16 
C12 &lt;0.05 &lt;0.05 0.09 0.05 0.05 
______________________________________ 
TABLE 5 
______________________________________ 
% Hydrolysis of Specific Glyceride 
0.2% 0.5% 0.2% 
Specific Present Present 
Calf 0.5% Calf 
FFA Control Lipase Lipase Lipase Lipase 
______________________________________ 
C4 6.0 12.3 17.3 12.7 17.7 
C6 4.5 16.0 21.5 6.5 10.0 
C8 11.9 23.8 32.1 14.5 17.8 
C10 8.3 17.9 24.4 11.9 9.5 
C12 &lt;0.8 &lt;0.8 1.5 0.8 0.8 
______________________________________ 
TABLE 6 
______________________________________ 
% Hydrolysis of Specific Glyceride 
(Corrected for FFA present in Control) 
Specific 
0.2% Present 
0.5% Present 
0.2% Calf 
0.5% Calf 
FFA Lipase Lipase Lipase Lipase 
______________________________________ 
C4 6.3 11.3 6.7 11.7 
C6 11.5 17.0 2.0 6.0 
C8 11.9 20.2 2.6 5.9 
C10 9.6 16.1 3.6 1.2 
C12 0 (0.7) 0 0 
______________________________________ 
Although FFA production of the present lipase is nearly identical to that 
of calf lipase for C4 production, all of the other FFA data show that the 
present lipase hydrolyzes the glycerides of C6, and C8, and C10 to a much 
greater extent. For example the present lipase shows, respectively, 5.8x, 
4.6x, 2.7x more C6, C8, C10 than the calf lipase at 0.20% concentration, 
and 2.8x, 3.4x and 13.4x at 0.50% concentration. 
Triglyceride Hydrolysis in Oils 
The present lipase has been tested also for production of 5% emulsions of 
soy, butter, and coconut oils. Chromatography of the treated emulsions (4 
hours at 33.degree. C.) reveals the ability of this lipase to hydrolyze a 
whole range of triglycerides. The level of certain triglycerides in the 
mixtures and the ability to fully emulsify the substrates can alter the 
rate of specific free fatty acid released as illustrated in Table 7. 
______________________________________ 
Fatty Acid 
Soy Oil Butter Oil 
Coconut Oil 
Determined 
% FFA % FFA % FFA 
______________________________________ 
C2 **ND ND ND 
C4 ND 0.16 ND 
C6 ND 0.066 0.029 
C8 ND 0.014 0.19 
C10 ND 0.062 0.042 
C12 ND 0.19 0.22 
C14 ND 0.062 0.047 
C16 
C18 0.13 0.57 0.57 
C18-1 0.97 0.072 0.072 
C18-2 3.25 0.24 1.61 
______________________________________ 
**ND = none detected 
Cheese Preparation 
Cheese has been made with the present lipase in the following manner. Milk 
is obtained from a diary farm, batch pasteurized at 145.degree. F. for 30 
minutes, and then held at 36.degree. F. until the following day. (Colby 
cheese is made using Hansen CH 60Y.TM. as a starter). Equivalent activity 
amounts of the present lipase and Miles 600 calf lipase (by cream assay) 
are preweighed and mixed with salt to give 2% salt by weight. Eight 
treatments of the cheese are made using 20 pound blocks. Control blocks 
with salt but without enzyme are made as well. 
The resulting cheeses are sampled at 1, 28, 72, 102, and 198 days. They are 
evaluated organoleptically and analyzed for free fatty acids by gas 
chromatography, known acid degree value (ADV) tests, and also by 12% TCA 
soluble nitrogen tests. 
After 72 hours, the cheeses having 0.048% present lipase and 0.128 calf 
lipase have about the same ADV and total FFA, which is 50-70% greater than 
that of the control. However, at 102 and 198 days, the concentrations of 
enzyme used (and for which data has been tabulated) are too high for good 
flavor in that too much free fatty acid has developed. The calf lipase 
produces too much butyric acid, and the present lipase, too much total 
free fatty acid C14-C18. 
At lower concentrations (0.012 and 0.024%) the present lipase gives good 
aged flavor without rancidity associated with butyric acid at 102 days. 
The control has weak flavor at this point, while the calf lipase showed 
the typical rancidity associated with butyric acid, even with low levels 
of the enzyme preparation (0.064 and 0.032%). 
FIGS. 8 and 9 illustrates the triglyceride hydrolysis data for the control 
cheese and cheeses prepared with 0.048% present lipase and 0.128% calf 
lipase. It is readily apparent that at 72days and 102 days, the butyric 
acid content in cheese made from the calf lipase is very elevated over 
that of the control. On the other hand, cheese made with the present 
lipase is more like the control cheese as far as butyric acid levels. 
The present lipase reduces the mild cheese aging process by 2-5 times. At 
102 days the present lipase-added cheese shows greater C16, C18, C18-1 and 
C18-2 content than the control cheese, indicating an acceleration (i.e. 
more rapid flavor development) of the usual ripening process. Thus, 
cheeses manufactured with the present lipase develop a Cheddar like flavor 
while those of the calf lipase produces an Italian type flavor. It is 
apparent that the lipase derived from the present Aspergillus organism is 
unique in shortening the storage time needed for Cheddar and other mild 
flavored cheeses. 
Having described the invention with particular reference to preferred form, 
it will be obvious to those skilled in the art to which the invention 
pertain, that, after understanding the invention, various changes and 
modifications may be made without departing from the spirit and scope of 
the invention as defined by the appended claims.