collagenases Masahiro OGAWA, PhD Food Science Division Faculty of - - PowerPoint PPT Presentation

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Meat tenderization by novel bacterial collagenases

Masahiro OGAWA, PhD Food Science Division Faculty of Agriculture Kagawa University

The Great Seto Bridge , Kagawa, Japan

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To improve the quality of meat

Locomotive muscle Support muscle

• Breeding
• Fattening
• Meat aging
• Enz or mech. treatment

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Tough meats are underutilized

Beef Chicken meat

Beef cattle Milk cow Broiler Egg-laying hen

shank sinewy meat

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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

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Two factors to affect meat toughness

Muscle fibers in meat

Fibroblast cell

Collagen fiber

Muscle fiber Perimysium Unwrapped Elastic fiber

Collagen fibrils

A collagen molecule

Unwrapped perimysium (connective tissue)

Actomyosin toughness Background toughness

Inside

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• Table. Collagen and elastin contents in cattle muscles

Bailey, A. J., and N. D. Light, 1989. Connective Tissue in Meat and Meat Products. Elsevier Science Publishers, Ltd., Essex, U.K.

Muscle type 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

Shank Fillet Rib roast Outside leg

Tough meat has high content of collagen

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• 33% Glycine and 20% Proline+Hyp
• Hydroxylation of Pro and Lys
• Triple helix
• Layered structure and crosslinks

Gly-Pro-Met-Gly-Pro-Ser-Gly-Pro-Arg-Gly-Leu-Hyp- Gly-Pro-Hyp-Gly-Ala-Hyp-Gly-Pro-Gln-Gly-Phe-Gln- Gly-Pro-Hyp-Gly-Glu-Hyp-Gly-Glu-Hyp-Gly-Ala-Ser- Gly-Pro-Met-Gly-Pro-Arg-Gly-Pro-Hyp-............ 4- 4-Hydroxyproline

Un Uniq ique st structure e of f coll llag agen

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Conventional methods to tenderize meat

Drawback : Degraded texture and loss of juiciness Drawback: 1) Loss of juiciness after cooking 2) Active even at 70℃

• 2. The usage of Protease －Papain and bromelain
• 1. The usage of a meat tenderizer

Papain Papain

Temperature (C） Temperature (C）

• Rel. activity (%)
• Rel. activity (%)

9 Collagen fibers Muscle cells Connective tissue Connective tissue A bundle of myofibrils Muscle fibers

. . . .

Action of commercial methods on meat

Loss of meat juice Crumbly texture

Actomyosin toughness Background toughness

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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, 201-

214 (1986)

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Outline of Today’s talk

• Exploring cold-active collagenolytic enzyme
• Exploring salt-resistant collagenolytic enzyme

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Temperature（℃） Enzymatic activity Mesophilic enzyme (Mammals) Cold-active enzyme 5ºC 37ºC 60ºC

Feat atures of f cold ld-ac activ ive enzym yme

Effect of temperature on enzyme activity

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Co Coll llectin ing o

• f

f cold ld-ad adap apted ba d bacteria ia

Soil and water of high mountain (height 2500m) Deep sea water (Depth of 200m) Foods in a Refrigerator

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Screenin ing of f coll llag agenoly lytic ic ba bacteria ia

Incubate samples on TSA at 4℃ for 10 days Apply isolated bacteria on collagen gel plate containing TSA Incubate the plate at 4℃ for 3~5 days

Positive Bacteria Degradation of collagen gel (4C，4days)

Native Collagen Gel

Screen cold-adapted bacteria Screen collagenolytic bacteria

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10 692 126

Cold-region ⑤

2 233

Cold-region ④

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Cold-region ③

120

Cold-region ②

8 169

Cold-region ① Positive bacteria Number of bacteria

Total 11 7 5 10 17

Samples

Origin

C35

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Foods in Fridge ⑥

5 55

Summary of screening results

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Identi tifi ficat atio ion of

• f st

stra rain in C3 C35

3 μm×1 μm

★ Gram-negative ★ Bacillary, motility ★ growth at 4℃

Gram-Stain

Phylogenetic tree based on 16S rRNA gene sequence analysis

Shewanella sp. strain C35

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20 40 60 80 100 120 5 10 15 20 25 30 35 40

Relative growth (%) Temperature (℃)

Temp mpera ratu ture re eff ffect t on

• n th

the gr grow

• wth

th of

• f C3

C35

28℃ Psychrophiles: cannot grow above 20ºC, Opt. growth temp. < 15ºC Psychrotrophs: can grow < 5ºC

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MOCAc Dnp Quenched Pro Leu Leu Gly Ala Asp Protease

En Enzym yme su subs bstrat ate

Measure Fluorescence Intensity (Ex 328nm, Em 393nm) MOCAc-PLGL(Dnp)AR

Dnp Pro Leu Leu Gly Ala Asp MOCAc Fluorescence

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Te Tempe perature eff ffects o

• f

f coll llagenase fr from S S. . C35

20 40 60 80 100 120 10 20 30 40 50 60

Relative activity (%) Temperature (℃)

20 40 60 80 100 120 10 20 30 40 50 60

Remaining activity (%) Temperature (℃)

Enzymatic activity at each temperature Thermal stability

Papain

Temperature (C）

• Rel. activity (%)

Papain

Temperature (C）

• Rel. activity (%)

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Optimum pH of C35 collagenase

20 40 60 80 100 120 4 5 6 7 8 9 10

Relative activity (%) pH

0.1 M Sodium-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)

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Subs bstrat ate spe pecif ific icit ity

0 12 24 36 48 0 12 24 36 48 0 12 24 36 48 0 12 24 36 48

Collagen Myofibril Serum albumin

hrs

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Evaluation of toughness

Lump of meat was immersed in 1.0 μg/mL enzyme solution at 4℃ for 5days

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Breaking strength test using a blade plunger

Load (x10 gf) Strain (x10%) C35 Ct Papain

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Tenderness of enzyme treated meat (raw)

Enzyme

Protein concentration of enzyme （μg/mL） Collagen hydrolyzing activity（U/mL）

Load value at 15% Strain（gf）

Ct 586±169(n=12) C35 0.1 0.335 628±256(n=6) 1.0 3.35 256±104(n=6) Papain 0.1 1.12 425±123(n=12) 1.0 11.2 339±97(n=12)

Meat was immersed in enzyme solution at 4 C for 5 days

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C35 enzyme application

Works Not work

Roast meats Cured products

Pickle solution containing salt, sugar, and nitrite Injector

Collagenolytic activity (U)

• Fig. C35 enzyme activity along with salt

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• 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

Screening of salt-tolerant collagenolytic bacteria

Collagen gel plate

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Screening of salt-tolerant collagenolytic enzyme

Fish species The number of bacteria strains The number

• f positive

strains The name of positive strains 1 Yellowtail 224 57 YT1～57 2 Amberjack 96 16 AJ1～16 3 Sea bream 132 4

• Jpn. seaperch

325 1 JS51 5 Pacific saury 29 6 Black porgy 34 1 BP8 7 Mackerel 159 8 Mullet 8 9 Young yellowtail 28 10 Walleye Pollack 96 2 WP1, 2 11 Slime flounder 192 12 SF1～12 1323 89

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0.000 0.020 0.040 0.060 0.080 0.100 0.120 0.140 0.160 0.180 C35 YT3 BP8 JS51 SF10

Enzymat matic ic activ ivit ity y （U/10μ L）

0.000 0.020 0.040 0.060 0.080 0.100 0.120 C35 YT3 BP8 JS51 WP1 WP2 SF9 SF10 SF11 SF12

Enzymatic activity （U/10μ L）

N

NaCl－ NaCl＋(1.5M)

Salt-resistance of collagenolytic enzymes Collagen activity with1.5M NaCl

Salt-resistance of enzymes

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Identification of collagenolytic bacteria

SF10 BP8 JS51

BP8 and JS51 Rod-shaped （3μm x 1μm） Gram negative bacteria 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

（ Black porgy） （ Jpn. seaperch ） （Slime flounder）

Bacteria identification by 16S rRNA analysis

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Cost reduction of enzyme production

• Reduction of materials’ cost
• Reduction of Impurity

Synthetic medium

(Minimal medium)

Culture medium Natural medium

(Tryptic soy broth) 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

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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)

Ala Arg Asn Asp Cys Gln Glu Gly Ile Leu Lys Pro Ser Trp Val Amino acids

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1 2 3 4 5 6 7 8 9 10 TSB Ct Ala- Arg- Asn- Asp- Cys- Gln- Glu- Gly- Ile- Leu- Lys- Pro- Ser- Trp- Val-

OD OD600

Effect of amino acids on bacterial growth

1 2 3 4 5 6 7 8 9 10 TSB Ct Ala- Arg- Asn- Asp- Cys- Gln- Glu- Gly- Ile- Leu- Lys- Pro- Ser- Trp- Val-

OD OD600

600

Shewanella JS51 Pseudomonas SF10

Optical density of culture incubated at 4C and 110rpm for 7days

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Treatment with enzyme-containing pickle using a meat injector

Pickle containing enzyme

5days incubation at 4C Cooked at 65C, 30min

1.5cm 3.0cm １cm

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BP8 JS51 The genus Shewanella

Black porgy

• Jpn. seaperch

C35

Cold soil

YT3

Yellowtail

SF10 10

Slime flounder

The genus Pseudomonas Present Future

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Eating habits have changed!

Quoted from HP of the Ministry of Agriculture, Forestry, and Fisheries

Increase Decrease No change

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Ch Chewin ing g po power of

• f eld

lderly ly pe peop

• ple

le

The number of times of chewing

Old Young

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Population change in Japan

10161 9776 9165 8292 7343 6204 5347 1407 1519 1733 1469 1490 1594 1260 1160 1422 1645 2167 2235 2247 2387

2000 4000 6000 8000 10000 12000 14000 2005年 2010年 2015年 2025年 2035年 2045年 2055年 0～64歳 65～74歳 75歳以上

Population (104)

Quoted from the HP of Japan cabinet office

0-64y.o. 65-74y.o.

• ver 75y.o.

2005 2010 2015 2025 2035 2045 2055