Meat tenderizing enzyme…
Myofibril hydrolysis
culated based on this and adding enzyme to diluted marinade
before transferring to meat in a tumbler. After tumbling for 1 h at
5 °C, meat and marinade were sealed in bags as above and kept
under refrigeration prior to cooking on an electric skillet at specified
temperatures. Samples were cored after allowing to cool, and ten-
derness was measured as before.
Myofibrillar proteins were extracted according to the method of
Olson and others (1976) and the protein content determined using
a Leco Protein analyzer (Leco Corp., Warrendale, Pa., U.S.A.). Myo-
fibrillar protein breakdown was estimated by the method of Tsai
and others (1983). Fifty L enzyme (1 mg/mL) was added to 1.95 mL
of myofibril extract and incubated at 37 °C for 1 h. The reaction was
stopped by adding 2 mL of 15% trichloroacetic acid (TCA), and the
mixture was allowed to stand at room temperature for 15 min, fol-
lowed by centrifugation. The absorbance of supernatant was mea-
sured spectrophotometrically at 280 nm. The activities of the aspar-
tic protease were compared to that of papain.
Residual activity
For residual activity determinations, 0.1 Anson unit (AU) of ei-
ther AP or papain was added to 100 g ground meat; this was shaped
into patties. Controls were also set up by forming patties with
equivalent weight of distilled water instead of enzyme. These were
cooked to internal temperatures of 55 °C, 65 °C, and 75 °C. The
meat was allowed to cool to room temperature, and 10 g was sus-
pended in 40 mL 0.1 M Tris-maleate buffer (pH 5.5). The suspen-
sion was homogenized for 1 min using a polytron (Polyscience,
Niles, Ill., U.S.A. - model X520) followed by centrifugation at 10000
rpm for 10 min. Residual activity in supernatant was measured by
the modified Anson (1939) method.
1.0 mL supernatant was added to 2.0 mL hemoglobin substrate
and incubated at room temperature for 10 min. The reaction was
stopped by adding 5.0 mL of 0.3 M TCA, and the suspension al-
lowed to stand for 10 min at room temperature. This was filtered and
1.5 mL filtrate added to 2.8 mL of 0.5 M NaOH followed by 1.0 mL
Folin–Ciocalteu phenol reagent. After standing for 10 min, absor-
bance was spectrophotometrically (Hewlett-Packard, Waldbronn,
Germany; model HP8453E) measured at 750 nm.
Collagen breakdown
Hydrolysis of collagen was measured according to the method of
Cronlund and Woychik (1987). We added 0.2 mL enzyme (1 mg/mL)
to 20 mg bovine tendon collagen (Sigma Chemical Co.) suspended
in 3.8 mL Tris buffer (0.02 M Tris, 0.005 M calcium chloride, pH 7.4);
this was incubated at 40 °C for 3 h or 70 °C for 30 min. After this pe-
riod, the reaction mixtures were spun in a microfuge for 10 min at
14000 rpm. Supernatant (1.5 mL) was mixed with 4.5 mL of 5 N HCl
and kept in a drying oven at 110 °C for 16 h for complete hydrolysis
of soluble peptides. The hydrolysate obtained was analyzed for hy-
droxyproline content. Collagenolytic activity of AP was compared to
that of papain.
Hydrolysate was diluted 25 times with distilled water and 1 mL
chloramine T solution (1.41 g chloramine T dissolved in 100 mL ci-
trate-acetate buffer, pH 6.0) added to 1 mL diluted hydrolysate. The
solution was allowed to stand at room temperature for 20 min, 1.0
mL color reagent was added, and it was transferred to a 60 °C water
bath where it was further incubated for 15 min. Tubes were allowed
to cool, and absorbance was measured at 560 nm.
Statistical analysis
Differences between means of treatments were analyzed using
JMP statistical software with significance level set at p ꢀ 0.05 (SAS
2000)
Results and Discussion
Sample treatment and evaluation of tenderness
Post-rigor beef top rounds and briskets were randomly injected
with varying concentrations (0.002 to 0.05 AU/100 g meat) of AP or
papain using a single needle injector (Dayton Electrical Manufac-
turing Co., Niles, Ill., U.S.A.) until there was a 5% increase in meat
weight. The injected meat was tumbled at 5 rpm under vacuum (8
Pa) and 2 to 4 °C to ensure adequate distribution of enzyme within
the muscle tissue. The meat was cooked on an electric skillet to in-
ternal temperatures of 55 °C, 65 °C, and 75 °C. After cooling to room
temperature, tenderness was objectively measured using a Warn-
er-Bratzler shear test on a TAX-T2 texture analyzer (Texture Tech-
nologies Corp, Scarsdale, N.Y., U.S.A.). The blade was set to move at
a speed of 2.0 mm/s through a fixed distance of 30 mm. The maxi-
mum force required to shear the cored sample gives an indication
of meat tenderness (inversely related to maximum shear force).
Samples were also evaluated for effect of marinade treatment
on tenderness. Beef (top rounds) were sliced 15 mm thick across
the length of the fibers, weighed and placed in a vacuum tumbler.
355 g marinade (Lawry’s Herb and Garlic with Lemon Juice 30
Minute Marinade) was poured into a graduated cylinder and di-
luted to 1000 g with distilled water. Percent uptake of marinade by
meat was established by adding marinade to a known weight of
meat and tumbling for an h at 5 °C. Both meat and marinade were
transferred into re-sealable bags and held overnight at 5 °C, after
which the meat was re-weighed. Percent uptake was estimated as:
Meat protein hydrolysis
AP did not hydrolyze collagen and showed very limited hydrol-
ysis of myofibrillar proteins (Figure 1). However, papain showed
significant hydrolysis of collagen and about 7-fold activity toward
myofibrillar proteins, as compared to AP. Indiscriminate break-
down of meat proteins by papain confirmed reports by other stud-
ies (Foegeding and Larrick 1986; Miller and others 1989; Takagi and
others 1992). The selective breakdown of meat proteins by AP is
also similar to effects of an alkaline elastase isolated from Bacillus
species (Takagi and others 1992). Unlike AP, however, the alkaline
protease had strong activity for selectively cleaving and degrading
elastin and collagen, but it had no effect on myofibrillar proteins.
Effect on meat tenderness
Enzymatic hydrolysis of meat proteins is generally known to in-
crease solubilization of free amino groups and hydroxyproline,
which may result in loss of muscle integrity and reduced shear force
or increased tenderness (Fogle and others 1982). Evaluation of the
relative effects of injected AP and papain on tenderness of beef
briskets and top rounds showed that maximum shear force was
reduced by both enzymes (Figure 2). Papain continued to increase
tenderness of both meat portions as its dose was increased. At doses
beyond 0.01 AU/100 g meat, papain-treated meat was mushy and
could no longer be cored for tenderness evaluation. On the other
hand, AP reduced maximum shear force by about 25 to 30% and did
not show any further increase in tenderness beyond an enzyme
dose of 0.01 AU/100 g meat. Similar effects were observed with
marinated beef, but higher doses were required in this case, likely
% uptake ϭ [(weight of marinated sample –
weight before marination)weight before marination] ϫ 100
This was found to be 12%; enzyme dose for marinating was cal-
Vol. 67, Nr. 6, 2002—JOURNAL OF FOOD SCIENCE 2139