Meat tenderization by novel bacterial collagenases

Meat tenderization by novel bacterial
collagenases
Masahiro OGAWA, PhD
Food Science Division
Faculty of Agriculture
Kagawa University
The Great Seto Bridge , Kagawa, Japan 1
To improve the quality of meat
Support muscle
Breeding
Fattening
Meat aging
Enz or mech. treatment
Locomotive muscle
2
Tough meats are underutilized
Beef
Chicken meat
Beef cattle
Milk cow
shank
sinewy meat
Broiler
Egg-laying hen
3
Beef is the most costly protein
To produce 1 kilogram’s animal products
Beef
Pork
Chicken
meat
Hen egg
Quoted from HP of the Ministry of Agriculture, Forestry, and Fisheries
4
Two factors to affect meat toughness
Actomyosin toughness
Background toughness
A collagen molecule
Collagen fibrils
Inside
Muscle fiber
Collagen fiber
Unwrapped
Perimysium
Muscle fibers in meat
Elastic fiber
Fibroblast cell
Unwrapped perimysium (connective tissue)
5
Tough meat has high content of collagen
Table. Collagen and elastin contents in cattle muscles
Collagen content
(%)
Elastin (%)
Psoas major muscle
(tenderloin)
2.24
0.08
Longissimus dorsi muscle
(rib roast)
2.76
0.07
Semitendinosus muscle
4.75
1.82
Muscle type
Bailey, A. J., and N. D. Light, 1989. Connective Tissue in Meat and Meat Products. Elsevier Science Publishers, Ltd., Essex, U.K.
Fillet
Rib roast
Outside leg
Shank
6
Unique structure of collagen
 33% Glycine and 20% Proline+Hyp
Gly-Pro-Met-Gly-Pro-Ser-Gly-Pro-Arg-Gly-Leu-HypGly-Pro-Hyp-Gly-Ala-Hyp-Gly-Pro-Gln-Gly-Phe-GlnGly-Pro-Hyp-Gly-Glu-Hyp-Gly-Glu-Hyp-Gly-Ala-SerGly-Pro-Met-Gly-Pro-Arg-Gly-Pro-Hyp-............
 Hydroxylation of Pro and Lys
 Triple helix
44-Hydroxyproline
 Layered structure and crosslinks
7
Conventional methods to tenderize meat
1. The usage of a meat tenderizer
Drawback : Degraded texture and loss of
juiciness
2. The usage of Protease －Papain and bromelain
Drawback:
1) Loss of juiciness after cooking
Papain
Temperature (C）
Rel. activity (%)
Rel. activity (%)
2) Active even at 70℃
Papain
Temperature (C）
8
Action of commercial methods on meat
Background toughness
A bundle of
myofibrils
Muscle
fibers
..
..
Connective tissue
Actomyosin toughness
Muscle cells
Connective tissue
Collagen fibers
 Loss of meat juice
 Crumbly texture
9
To alter tough meat to tenderized meat
with high quality
Enzymes which can preferentially hydrolyze
collagen fibers are most probable
Previous report:
Tenderization of beef with bacterial collagenase
(Clostridium histolyticum collagenase)
A. Foegeding E. and Larick DK. Meat Sci., 18, 201214 (1986)
10
Outline of Today’s talk
 Exploring cold-active collagenolytic enzyme
 Exploring salt-resistant collagenolytic enzyme
11
Features of cold-active enzyme
Cold-active enzyme
Enzymatic activity
Mesophilic enzyme
(Mammals)
5ºC
37ºC
60ºC
Temperature（℃）
Effect of temperature on enzyme activity
12
Collecting of cold-adapted bacteria
Soil and water of high mountain
(height 2500m)
Deep sea water
(Depth of 200m)
Foods in a Refrigerator
13
Screening of collagenolytic bacteria
Incubate samples on TSA at 4℃ for 10 days
Screen cold-adapted bacteria
Apply isolated bacteria on collagen gel plate containing TSA
Screen collagenolytic bacteria
Incubate the plate at 4℃ for 3~5 days
Native Collagen Gel
Positive Bacteria
Degradation of collagen gel (4C，4days)
14
Summary of screening results
Samples
Origin
Number of
bacteria
Positive
bacteria
Cold-region ①
17
169
8
Cold-region ②
10
120
0
Cold-region ③
5
30
0
Cold-region ④
7
233
2
Cold-region ⑤
11
126
0
5
14
0
55
692
10
Foods in Fridge ⑥
Total
C35
15
Identification of strain C35
★ Gram-negative
★ Bacillary, motility
★ growth at 4℃
Gram-Stain
3 μm×1 μm
Shewanella sp. strain C35
Phylogenetic tree based on 16S rRNA gene sequence analysis
16
Relative growth (%)
Temperature effect on the growth of C35
120
28℃
100
80
60
40
20
0
0
5
10
15
20
25
30
35
40
Temperature (℃)
B
Psychrophiles: cannot grow above 20ºC, Opt.
growth temp. < 15ºC
Psychrotrophs: can grow < 5ºC
17
Enzyme substrate
Fluorescence
MOCAc
Pro
Leu
Quenched
Gly
Dnp Leu
Protease
Ala
Asp
MOCAc-PLGL(Dnp)AR
MOCAc
Dnp
Pro
Leu
Gly
Leu
Ala
Asp
Measure Fluorescence Intensity
(Ex 328nm, Em 393nm)
18
Papain
Rel. activity (%)
Rel. activity (%)
Temperature effects of collagenase from S. C35
Papain
Temperature (C）
120
100
80
60
40
20
0
0
10
20
30
40
50
60
Remaining activity (%)
Relative activity (%)
Temperature (C）
120
100
80
60
40
20
0
Temperature (℃)
Enzymatic activity at each temperature
B
0
10
20
30
40
50
60
Temperature (℃)
Thermal stability
19
Relative activity (%)
Optimum pH of C35 collagenase
120
100
80
60
40
20
0
4
5
6
7
8
9
10
pH
B
0.1 MCDSodium-Acetate buffer (pH 5.0, 6.0)
0.1 M PIPES buffer (pH 6.0, 7.0)
0.1 M Tris-HCl buffer (pH 7.0, 8.0, 9.0)
20
Substrate specificity
0 12 24 36 48 0 12 24 36 48 0 12 24 36 48 0 12 24 36 48 hrs
Collagen
Myofibril
Serum albumin
21
Evaluation of toughness
Lump of meat was immersed in 1.0 μg/mL enzyme solution at 4℃ for 5days
22
Load (x10 gf)
Breaking strength test using a blade plunger
C35
Papain
Ct
Strain (x10%)
23
Tenderness of enzyme treated meat (raw)
Enzyme
Protein concentration of
enzyme
（μg/mL）
Collagen hydrolyzing
activity（U/mL）
Ct
Load value at 15%
Strain（gf）
586±169(n=12)
0.1
0.335
628±256(n=6)
1.0
3.35
256±104(n=6)
0.1
1.12
425±123(n=12)
1.0
11.2
339±97(n=12)
C35
Papain
Meat was immersed in enzyme solution at 4 C for 5 days
24
Works
Roast meats
Not work
Collagenolytic activity (U)
C35 enzyme application
Fig. C35 enzyme activity along with salt
Cured products
Injector
Pickle solution
containing salt, sugar, and nitrite
25
Screening of salt-tolerant collagenolytic bacteria
 Bacteria isolation of fish body surface using 0.5M NaCl
 Culturing bacteria on tryptic soy agar
 Screening collagenelytic bacteria on collagen gel plates
 Halo formation
Collagen gel plate
26
Screening of salt-tolerant collagenolytic
enzyme
Fish species
The number of
bacteria
strains
The number
of positive
strains
The name of
positive
strains
1
Yellowtail
224
57
YT1～57
2
Amberjack
96
16
AJ1～16
3
Sea bream
132
0
4
Jpn. seaperch
325
1
5
Pacific saury
29
0
6
Black porgy
34
1
7
Mackerel
159
0
8
Mullet
8
0
9
Young yellowtail
28
0
10
Walleye Pollack
96
2
WP1, 2
11
Slime flounder
192
12
SF1～12
1323
89
JS51
BP8
27
Salt-resistance of enzymes
0.120
0.180
NaCl－
NaCl＋(1.5M)
N
0.100
Enzymatic activity （U/10μ L）
Enzymatic activity （U/10μ L）
0.160
0.080
0.060
0.040
0.020
0.140
0.120
0.100
0.080
0.060
0.040
0.020
0.000
C35 YT3 BP8 JS51 WP1 WP2 SF9 SF10 SF11 SF12
Collagen activity with1.5M NaCl
0.000
C35
YT3
BP8
JS51
SF10
Salt-resistance of collagenolytic enzymes
28
Identification of collagenolytic bacteria
BP8
JS51
（ Black porgy）
（ Jpn. seaperch ）
BP8 and JS51
Rod-shaped （3μm x 1μm）
Gram negative bacteria
SF10
（Slime flounder）
SF10
Rod-shaped （5μm x 2μm）
Gram negative bacteria
Characteristics common to the three strains
★ Rod-shaped bacterium with motility
★ Gram stain negative
★ Grow at 4 degree C
Bacteria identification by 16S rRNA analysis
29
Cost reduction of enzyme production
Culture medium
Natural medium
Synthetic medium
(Tryptic soy broth)
(Minimal medium)
Ingredients (g/L)
Casein Digest Peptone……………17.0
Papaic Digest of Soybean Meal .... 3.0
Disodium Phosphate ..................... 2.5
Dextrose ......................................... 2.5
Sodium Chloride ............................. 5.0
Ingredients
Glucose
Ammonium sulfate
Vitamins
Minerals
 Reduction of materials’ cost
 Reduction of Impurity
30
Optimization of minimal medium
MⅠ standard medium
Balch’s Vitamin Solution
15mM Glucose
15mM Phosphate buffer(pH7.4)
10mM CaCl2
10mM Ammonium Sulfate
10mM Amino acids(10mM)
Amino acids
Ala
Arg
Asn
Asp
Cys
Gln
Glu
Gly
Ile
Leu
Lys
Pro
Ser
Trp
Val
31
Effect of amino acids on bacterial growth
Pseudomonas SF10
10
9
9
8
8
7
7
6
6
OD600
10
5
4
5
Val-
Trp-
Ser-
Pro-
Lys-
Leu-
Ile-
Gly-
Glu-
Gln-
Cys-
Asp-
Asn-
Arg-
Ala-
Ct
Val-
Trp-
Ser-
Pro-
Lys-
Leu-
Ile-
Gly-
Glu-
0
Gln-
0
Cys-
1
Asp-
1
Asn-
2
Arg-
2
Ala-
3
Ct
3
TSB
4
TSB
OD600
Shewanella JS51
Optical density of culture incubated at 4C and 110rpm for 7days
32
Treatment with enzyme-containing pickle using
a meat injector
Pickle containing enzyme
１cm
5days incubation at 4C
Cooked at 65C, 30min
1.5cm
3.0cm
33
The genus Shewanella
YT3
C35
Yellowtail
Cold soil
BP8
JS51
Black porgy
Jpn. seaperch
Present
The genus Pseudomonas
Future
SF10
Slime flounder
34
Eating habits have changed!
Decrease
No change
Increase
Quoted from HP of the Ministry of Agriculture, Forestry, and Fisheries
35
The number of times of chewing
Chewing power of elderly people
Old
Young
36
Population change in Japan
0～64歳
0-64y.o.
65～74歳
65-74y.o.
75歳以上
over 75y.o.
14000
Population (104)
12000
1160
1407
10000
1422
1519
1645
2167
1733
2235
1469
8000
2247
1490
2387
1594
6000
1260
10161
4000
2000
9776
9165
8292
7343
6204
5347
0
2005年
2005 2010年
2010 2015年
2015 2025年
2025 2035年
2035 2045年
2045 2055年
2055
Quoted from the HP of Japan cabinet office
37

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