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C19) United States C12) Patent Application Publication C10) Pub 1111111111111111 IIIIII IIIII 111111111111111 11111 lllll 111111111111111 111111111111111 11111111 US 20190045803Al c19) United States c12) Patent Application Publication c10) Pub. No.: US 2019/0045803 Al RICHARDS et al. (43) Pub. Date: Feb. 14, 2019 (54) ROSEMARY/PHOSPHOLIPASE Related U.S. Application Data COMPOSITIONS AND METHODS OF (60) Provisional application No. 62/325,744, filed on Apr. PRESERVING MUSCLE TISSUE 21, 2016. (71) Applicant: Wisconsin Alumni Research Publication Classification Foundation, Madison, WI (US) (51) Int. Cl. (72) Inventors: Mark P. RICHARDS, Madison, WI A23B 4122 (2006.01) (US); Jie YIN, Madison, WI (US); A23L 3/3472 (2006.01) Wenjing ZHANG, Madison, WI (US) A23L 3/3571 (2006.01) (52) U.S. Cl. CPC .............. A23B 4122 (2013.01); A23L 3/3472 (73) Assignee: Wisconsin Alumni Research (2013.01); A23V 2250/54 (2013.01); A23V Foundation, Madison, WI (US) 2002/00 (2013.01); A23V 2250/21 (2013.01); A23L 3/3571 (2013.01) (21) Appl. No.: 15/568,784 (57) ABSTRACT (22) PCT Filed: Apr. 17, 2017 The disclosure provides for compositions and methods for the preservation of meat tissues, including fish, beef, poultry (86) PCT No.: PCT/USl 7 /27942 and pork, and meat analogs containing added heme protein, § 371 (c)(l), using very low amounts of phospholipase A2 (PLA2) (2) Date: Oct. 23, 2017 enzymes in a combination with rosemary. 10 weeks at ·20C Day1.4 w R Patent Application Publication Feb. 14, 2019 Sheet 1 of 4 US 2019/0045803 Al a: CL+ T"'" ■ C) LL Patent Application Publication Feb. 14, 2019 Sheet 2 of 4 US 2019/0045803 Al N 0~ 00 ■ ;.t) c:i C'l C) M N -LL ~ 1"-f > i3 wdrmttt,,., ...,.,,,,,,,,,,. Patent Application Publication Feb. 14, 2019 Sheet 3 of 4 US 2019/0045803 Al ■ ■ C) C) -LL -LL Patent Application Publication Feb. 14, 2019 Sheet 4 of 4 US 2019/0045803 Al ■ -u. US 2019/0045803 Al Feb. 14,2019 1 ROSEMARY/PHOSPHOLIPASE concentration of no more than about 175 ppm, at a concen­ COMPOSITIONS AND METHODS OF tration of no more than about 200 ppm, at a concentration of PRESERVING MUSCLE TISSUE no more than about 225 ppm, at a concentration of no more than about 250 ppm, at a concentration of about 175 ppm to PRIORITY CLAIM about 225 ppm, at a concentration of about 190 ppm to about 210 ppm, at a concentration of of about 180 ppm to about [0001] This application is a national phase application under 35 U.S.C. § 371 of International Application No. 220 ppm, at a concentration of of about 195 ppm to about PCT/US2017/027942, filed Apr. 17, 2017, which claims 205 ppm, or at a concentration of of about 200 ppm. The benefit of priority to U.S. Provisional Application Ser. No. PLA2 enzyme may be contacted at a concentration of about 62/325,744, filed Apr. 21, 2016, the entire contents of each 50 or about 60 U/kg, at a concentration of no more than of which are hereby incorporated by reference. about 63 U/kg, at a concentration ofno more than about 100 U/kg, at a concentration ofno more than about 350 U/kg, at BACKGROUND OF THE DISCLOSURE a concentration of no more than about 525 U/kg, at a concentration of about 63 U/kg to about 450 U/kg, at a 1. Field of the Disclosure concentration of about 100 U/kg to about 350 U/kg, at a concentration of between about 200 U/kg to about 300 U/kg, [0002] This disclosure relates to composition and methods at a concentration of between about 225 U/kg to about 275 for the preservation of meat products including fish, fowl, U/kg ppm, or at a concentration of about 250 U/kg. red meat, and meat analogues containing added heme pro­ tein. In particular, phospholipase A2 enzymes are used at [0007] The muscle tissue may be avian tissue, fish, shell­ very low concentrations to reduct spoilage and preserve fish tissue, reptile tissue or amphibian tissue, mammalian storage of such meat products and meat analogs containing tissue, red meat, beef, elk, deer or bison meat, pork tissue, added heme proteins. rabbit tissue, mutton tissue, cooked or cured muscle tissue, or uncooked and uncured muscle tissue. The meat analog 2. Related Art may contain added heme protein and may be treated with bacterial PLA2. The method may further comprise freezing [0003] Food preservation is a complicated process that said muscle tissue. The muscle tissue or meat analog may be requires both a means of preventing microbial contamina­ treated at O to 6° C. The muscle tissue or meat analog may tion and a means of preventing the development of off-colors be treated substantially in the absence of exogenous cal­ or off-flavors rendering the food unpalatable. Indeed, off­ cium. The muscle tissue may contain hemoglobin at levels odor and off-flavor development during refrigerated and that are 80% of fresh unstored tissue for 2, 3, 4, 5, 6, 7, 8, frozen storage of fish products is a major obstacle to 9 or 10 days following treatment with said PLA2 enzyme consumer acceptance. The USDA estimates that more than and rosemary extract. The muscle tissue or meat analog may 96 billion pounds of food in the U.S. were lost by retailers, remain palatable at 0.6° C. for 2, 3, 4, 5, 6, 7, 8, 9 or 10 days foodservice, and consumers in 1995, and meat, poultry and beyond the date upon which untreated muscle tissue or meat fish made up 8.5% of that number-over 8 billion pounds. analog would no longer be palatable. The muscle tissue or [0004] Lipid oxidation is the process that causes the meat analog may remain palatable at -10.0° C. for 2, 3, 4, formation of stale and rancid odors/flavors that are undesir­ 5, 6, 7, 8, 9 or 10 month beyond the date upon which able. Lipid oxidation is more problemtic in fish compared to untreated muscle tissue or meat analog would no longer be beef, pork and poultry, in part due to the higher content of palatable. highly unsaturated fatty acids in fish muscle. Heme proteins [0008] Also provided is a storage-stable muscle tissue or in fish muscle also promote lipid oxidation much more meat analog containing added heme protein comprising rapidly compared to those in the terrestrial animals. Any about 50 or about 60 U/kg to about 525 U/kg phospholipase process or food additive that can improve the shelf life of A2 enzyme (PLA2) and rosemary extract at about 150 ppm meat, particularly fish, by only two days ( during refrigerated to about 250 ppm. The rosemary extract may be present at storage) is of great commercial interest. a concentration of about 150 ppm, at a concentration of no [0005] Previously, the inventors tested a commercial more than about 17 5 ppm, at a concentration of no more than source of porcine phospholipase A2 (PLA2) as an inhibitor about 200 ppm, at a concentration of no more than about 225 of lipid oxidation in washed cod muscle containing added ppm, at a concentration of no more than about 250 ppm, at hemoglobin as an oxidant. A usage level of0.00007% PLA2 a concentration of about 175 ppm to about 225 ppm, at a (0.7 ppm, 245 Units/kg) prevented lipid oxidation during 7 concentration of about 190 ppm to about 210 ppm, at a days of iced storage in washed cod muscle containing added concentration of of about 180 ppm to about 220 ppm, at a hemoglobin as an oxidant. This is equivalent to 700 mg concentration of of about 195 ppm to about 205 ppm, or at protecting 1000 kilograms of muscle food. The enzyme a concentration of of about 200 ppm. The PLA2 enzyme activity was 350 Units/mg of PLA2. may be contacted at a concentration of about 50 or 60 U/kg, at a concentration of no more than about 63 U/kg, at a SUMMARY OF THE DISCLOSURE concentration of no more than about 100 U/kg, at a concen­ [0006] Thus, in accordance with the present disclosure, tration of no more than about 350 U/kg, at a concentration there is provided a method of improving storage life of (a) of no more than about 525 U/kg, at a concentration of about comminuted or intact muscle tissue or (b) meat analog 63 U/kg to about 450 U/kg, at a concentration of about 100 containing added heme protein, comprising contacting said U/kg to about 350 U/kg, at a concentration of between about tissue with about 50 or about 60 U/kg to about 500 U/kg 200 U/kg to about 300 U/kg, at a concentration of between phospholipase A2 enzyme (PLA2) and rosemary extract at about 225 U/kg to about 275 U/kg ppm, or at a concentration about 150 ppm to about 525 ppm. The rosemary extract may of about 250 U/kg. The muscle tissue may be selected from be contacted at a concentration of about 150 ppm, at a avian tissue, fish tissue, shellfish tissue, pork tissue, beef US 2019/0045803 Al Feb. 14,2019 2 tissue, bison tissue, mutton tissue, pork tissue, elk tissue, (rosemary 200 ppm), P+R (exPLA2 1 ppm plus rosemary deer tissue, rabbit tissue, reptile tissue or amphibian tissue. 200 ppm). 1 ppm PLA2 was equivalent to 126 Units/kg The meat analog may contain added heme protein. sausage. [0009] In still another embodiment, there is provided a [0019] FIG.
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