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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 1 To improve the quality of meat Support muscle Breeding

  1. Meat tenderization by novel bacterial collagenases Masahiro OGAWA, PhD Food Science Division Faculty of Agriculture Kagawa University The Great Seto Bridge , Kagawa, Japan 1

  2. To improve the quality of meat Support muscle  Breeding  Fattening  Meat aging  Enz or mech. treatment Locomotive muscle 2

  3. Tough meats are underutilized Beef Chicken meat Beef cattle Milk cow Broiler Egg-laying hen shank sinewy meat 3

  4. 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

  5. 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

  6. Tough meat has high content of collagen Table. Collagen and elastin contents in cattle muscles Collagen content Elastin (%) Muscle type (%) Psoas major muscle 2.24 0.08 (tenderloin) Longissimus dorsi muscle 2.76 0.07 (rib roast) 4.75 1.82 Semitendinosus muscle Bailey, A. J., and N. D. Light, 1989. Connective Tissue in Meat and Meat Products. Elsevier Science Publishers, Ltd., Essex, U.K. Fillet Outside leg Rib roast Shank 6

  7. Uniq Un ique st structure e of f coll llag agen  33% Glycine and 20% Proline+Hyp 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-............  Hydroxylation of Pro and Lys 4-Hydroxyproline 4-  Triple helix  Layered structure and crosslinks 7

  8. 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 2) Active even at 70 ℃ Papain Rel. activity (%) Rel. activity (%) Papain Temperature (  C ） Temperature (  C ） 8

  9. Action of commercial methods on meat Background toughness Connective tissue A bundle of Actomyosin toughness . myofibrils . Muscle cells . . Muscle fibers Connective tissue Collagen fibers  Loss of meat juice  Crumbly texture 9

  10. 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) 10

  11. Outline of Today’s talk  Exploring cold-active collagenolytic enzyme  Exploring salt-resistant collagenolytic enzyme 11

  12. Feat atures of f cold ld-ac activ ive enzym yme Cold-active enzyme Mesophilic enzyme Enzymatic activity (Mammals) 5ºC 37ºC 60ºC Temperature （ ℃ ） Effect of temperature on enzyme activity 12

  13. Co Coll llectin ing o of f cold ld-ad adap apted ba d bacteria ia Soil and water of high mountain Deep sea water (height 2500m) (Depth of 200m) Foods in a Refrigerator 13

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

  15. Summary of screening results Number of Positive Samples Origin bacteria 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 Foods in Fridge ⑥ 5 14 0 Total 55 692 10 C35 15

  16. Identi tifi ficat atio ion of of st stra rain in C3 C35 ★ Gram-negative ★ Bacillary, motility ★ growth at 4 ℃ 3 μm × 1 μm Gram-Stain Shewanella sp. strain C35 Phylogenetic tree based on 16S rRNA gene sequence analysis 16

  17. Temp mpera ratu ture re eff ffect t on on th the gr grow owth th of of C3 C35 Relative growth (%) 120 28 ℃ 100 80 60 40 20 0 0 5 10 15 20 25 30 35 40 Temperature (℃) Psychrophiles: cannot grow above 20ºC, Opt. growth temp. < 15ºC Psychrotrophs: can grow < 5ºC 17

  18. En Enzym yme su subs bstrat ate Fluorescence MOCAc MOCAc Pro Pro Leu Leu Quenched Gly Dnp Gly Leu Dnp Protease Leu Ala Asp Ala Asp MOCAc-PLGL(Dnp)AR Measure Fluorescence Intensity (Ex 328nm, Em 393nm) 18

  19. Te Tempe perature eff ffects o of f coll llagenase fr from S S. . C35 Rel. activity (%) Papain Rel. activity (%) Papain Temperature (  C ） Temperature (  C ） Remaining activity (%) Relative activity (%) 120 120 100 100 80 80 60 60 40 40 20 20 0 0 0 10 20 30 40 50 60 0 10 20 30 40 50 60 Temperature (℃) Temperature (℃) Enzymatic activity at each temperature Thermal stability 19

  20. Optimum pH of C35 collagenase 120 Relative activity (%) 100 80 60 40 20 0 4 5 6 7 8 9 10 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) 20

  21. Subs bstrat ate spe pecif ific icit ity hrs 0 12 24 36 48 0 12 24 36 48 0 12 24 36 48 0 12 24 36 48 Collagen Myofibril Serum albumin 21

  22. Evaluation of toughness Lump of meat was immersed in 1.0 μ g/mL enzyme solution at 4 ℃ for 5days 22

  23. Breaking strength test using a blade plunger Load (x10 gf) C35 Papain Ct Strain (x10%) 23

  24. Tenderness of enzyme treated meat (raw) Protein concentration of Load value at 15% Collagen hydrolyzing Enzyme enzyme activity （ U/mL ） Strain （ gf ） （ μg /mL ） 586 ± 169(n=12) Ct 628 ± 256(n=6) 0.1 0.335 C35 256 ± 104(n=6) 1.0 3.35 425 ± 123(n=12) 0.1 1.12 Papain 339 ± 97(n=12) 1.0 11.2 Meat was immersed in enzyme solution at 4  C for 5 days 24

  25. C35 enzyme application Collagenolytic activity (U) Works Roast meats Not work Fig. C35 enzyme activity along with salt Cured products Injector Pickle solution containing salt, sugar, and nitrite 25

  26. 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

  27. Screening of salt-tolerant collagenolytic enzyme The number of The number The name of Fish species bacteria of positive positive strains strains strains YT1 ～ 57 1 Yellowtail 224 57 AJ1 ～ 16 2 Amberjack 96 16 3 Sea bream 132 0 4 Jpn. seaperch 325 1 JS51 5 Pacific saury 29 0 6 Black porgy 34 1 BP8 7 Mackerel 159 0 8 Mullet 8 0 9 Young yellowtail 28 0 10 Walleye Pollack 96 2 WP1, 2 SF1 ～ 12 11 Slime flounder 192 12 1323 89 27

  28. Salt-resistance of enzymes 0.120 0.180 NaCl － N 0.160 NaCl ＋ (1.5M) Enzymatic activity （ U/10 μ L ） 0.100 y （ U/10 μ L ） 0.140 0.080 0.120 ity 0.100 ivit 0.060 ic activ 0.080 matic Enzymat 0.040 0.060 0.040 0.020 0.020 0.000 0.000 C35 YT3 BP8 JS51 WP1 WP2 SF9 SF10 SF11 SF12 C35 YT3 BP8 JS51 SF10 Collagen activity with1.5M NaCl Salt-resistance of collagenolytic enzymes 28

  29. Identification of collagenolytic bacteria BP8 JS51 SF10 （ Black porgy ） （ Jpn. seaperch ） （ Slime flounder ） BP8 and JS51 SF10 Rod-shaped （ 3μm x 1μm ） Rod-shaped （ 5μm x 2μm ） Gram negative bacteria 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

  30. Cost reduction of enzyme production Culture medium Synthetic medium Natural medium (Minimal medium) (Tryptic soy broth) Ingredients (g/L) Ingredients Casein Digest Peptone………… … 17.0 Glucose Papaic Digest of Soybean Meal .... 3.0 Ammonium sulfate Disodium Phosphate ..................... 2.5 Vitamins Dextrose ......................................... 2.5 Minerals Sodium Chloride ............................. 5.0  Reduction of materials’ cost  Reduction of Impurity 30

  31. Optimization of minimal medium Amino acids M Ⅰ standard medium Balch’s Vitamin Solution Ala Arg 15mM Glucose Asn 15mM Phosphate buffer(pH7.4) Asp Cys 10mM CaCl 2 Gln 10mM Ammonium Sulfate Glu 10mM Amino acids(10mM) Gly Ile Leu Lys Pro Ser Trp Val 31

  32. Effect of amino acids on bacterial growth Pseudomonas SF10 Shewanella JS51 10 10 9 9 8 8 7 7 6 6 OD 600 OD 600 600 5 5 OD OD 4 4 3 3 2 2 1 1 0 0 TSB Ct Ala- Arg- Asn- Asp- Cys- Gly- Ile- Lys- Pro- Val- TSB Ct Ala- Arg- Asn- Asp- Cys- Gly- Ile- Lys- Pro- Val- Gln- Glu- Leu- Ser- Trp- Gln- Glu- Leu- Ser- Trp- Optical density of culture incubated at 4  C and 110rpm for 7days 32

  33. Treatment with enzyme-containing pickle using a meat injector Pickle containing enzyme １ cm 5days incubation at 4  C Cooked at 65  C, 30min 1.5cm 3.0cm 33

  34. The genus Shewanella YT3 C35 Yellowtail Cold soil BP8 JS51 Present Black porgy Jpn. seaperch The genus Pseudomonas Future SF10 10 Slime flounder 34

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