SCIENTIFIC PAPERS SUPPORTING THE USE OF PRESERFOOD TM BIO ACTIVE INGREDIENTS IN HEALTH RELATED ISSUES: ACRYLAMIDE REDUCTION,

1. Research Scientificpapersaboutthe use and benefits of theaddition of preserfoodtmbioactivecomponentstofood and drinks 1 PreserFood ™

2. Acrylamide is a substance that may be formed in foods, typically starchy products including crisps, French fries, bread and crispbreads, during cooking processes including frying, baking and roasting at temperatures of 120 °C or higher. Acrylamide is a known carcinogen in experimental animals hence efforts should be made to minimise exposure from all sources including diet. 2 Acrylamide I

3. exposure from all sources including diet. A substantial body of international research has been carried out to build greater understanding of acrylamide, how it is formed in foods, what the risks are for consumers and how to reduce occurrence levels. The European Commission has funded research projects to this end and the former Scientific Committee on Food issued an opinion on acrylamide in 2002 shortly after the first study on acrylamide in foods was published. 3 Acrylamide II

4. Since the discovery of acrylamide in foods in 2002, industry has sought to identify ways to reduce its formation in foods. As acrylamide is formed in food by common cooking practices, it is likely that people have been exposed to acrylamide in their diet for some considerable time. Choosing a balanced and varied diet, and avoiding overcooking of foods, will contribute to reducing acrylamide intake levels. 4 Acrylamide III

5. Acrylamide levels appear to rise as food is heated for longer periods of time. Though researchers are still unsure of the precise mechanisms by which acrylamide forms in foods, many believe it is a byproduct of the Maillard reaction. In fried or baked goods, acrylamide may be produced by the reaction between asparagine and reducing sugars (fructose, glucose, etc.) or reactive carbonyls at temperatures above 120 °C (248 °F). 5

6. Based on current stage of knowledge, acrylamide is a natural byproduct that forms when certain carbohydrate-rich foods are fried, baked, or roasted at temperatures above 120 °C. Acrylamide causes cancer in rats when administered orally in high dose experiments, increasing tumors in the nervous system, oral cavity, peritoneum, thyroid gland, mammary gland, uterus, and clitoris. 6

7. J Agric Food Chem. 2005 Apr 20;53(8):3259-64. Addition of glycine reduces the content of acrylamide in cereal and potato products. Bråthen E, Kita A, Knutsen SH, Wicklund T. Matforsk, NorwegianFoodResearchInstitute, Osloveien 1, N-1430 As, Norway. erland.brathen@matforsk.no The effects of adding amino acids on the content of acrylamide in potato crisps, French fries, flat breads, and bread crusts were investigated. Addition of glycine or glutamine during blanching of crisps reduced the amount of acrylamide by approximately 30% compared to no addition. No effect was found in French fries. Addition of glycine during doughmakingsignificantly reduced acrylamide in both flat breads and bread crusts. In bread crusts the reduction of acrylamide ranged from 50 to >90% depending on the baking condition. In flat breads the reduction varied between 60 and >95%. 7

8. Research Innovation Discovery Humanhealth Food safety 8 ScientificPapers

9. Effect of Citric Acid and Glycine Addition on Acrylamide and Flavor in a Potato Model System MEI YIN LOW, GEORGIOS KOUTSIDIS, JANE K. PARKER, J. STEPHEN ELMORE,ANDREW T. DODSON, AND DONALD S. MOTTRAM* School of Food Biosciences, The University of Reading, Whiteknights, Reading, RG6 6AP, UnitedKingdom Acrylamide levels in cooked/processed food can be reduced by treatment with citric acid or glycine. In a potato model system cooked at 180 °C for 10-60 min, these treatments affected the volatile profiles. Streckeraldehydes and alkylpyrazines, key flavor compounds of cooked potato, were monitored. Citric acid limited the generation of volatiles, particularly the alkylpyrazines. Glycine increased the total volatile yield by promoting the formation of certain alkylpyrazines, namely, 2,3- dimethylpyrazine, trimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, tetramethylpyrazine, and 2,5-diethyl-3-methylpyrazine. However, the formation of other pyrazines and Streckeraldehydes was suppressed. It was proposed that the opposing effects of these treatments on total volatile yield may be used to best advantage by employing a combined treatment at lower concentrations, especially as both treatments were found to have an additive effect in reducing acrylamide. This would minimize the impact on flavor but still achieve the desired reduction in acrylamide levels. J. Agric. Food Chem. 2006, 54, 5976-5983 9

10. Arthrosisis the most common osteoarticulary problem in our society: more than 50% of the population suffer from it after the age of 65, and 80% of people over 75. It consists of a degeneration of the articulary cartilage which disappears until it leaves the subchondral bone exposed. Arthrosis has no cure at present and the most widely used treatments are pain-killers and NSAID, (non-steroidal anti-inflammatory drugs), which only relieve pain but do not repair the damage in the cartilage or influence the development of the disease. 10

11. Taking a supplement of glycine, a food additive, helps to prevent degenerative diseases such as arthrosis or osteoporosis The study, carried out at the Cellular Metabolism Institute in Tenerife and at the Department of Biochemistry and Molecular Biology of the University of Granada by Doctor Patricia de Paz Lugo and supervised by Doctors Enrique MeléndezHevia, David Meléndez Morales and José Antonio Lupiáñez Cara, established that the direct intake of this substance as a food additive helps to prevent arthrosis and other degenerative diseases, in addition to other diseases related to a weakness in the mechanical structure of the organism, including the difficulty of repairing physical injuries. 11

12. The work carried out by the scientist from the CMI shows that collagen has a unique structure with a right-handed triple superhelix in which the glycine represents a third of its residues. Mathematical analysis of the metabolic route of the synthesis of the glycine, developed by the research group to which Patricia de Paz belongs, demonstrated that this amino acid should be considered an essential amino acid. The doctoral thesis carried out at the CMI and the UGR [http://www.ugr.es] has shown that the capacity of the metabolism to synthesiseglycine is very limited. The conclusion of this study is that glycine, administered in daily doses of 10 grams divided into two doses of 5 grams, one in the morning and one at night, leads to a general improvement in these problems over a period of time which, in most cases, is between two weeks and four months. 12

13. Evaluation and control of the risk of food borne pathogens and spoilage bacteria present in "Awa-Uirou", a sticky rice cake containing sweet red bean paste Okahisa, Naoki,Inatsu, Yasuhiro, Juneja, Vijay, Kawamoto, S. One of the most common types of food poisoning in Japan is caused by the consumption of steamed sticky rice cake contaminated with Bacillus cereus, a common foodborne pathogen. Illnesses have been traditionally associated with B. cereus spores contaminating this product. Also, the product can also be spoiled due to the growth of B. subtilis, a spoilage bacterium. Accordingly, there is a need to explore the potential use of a preservative in such foods. We determined that the addition of 0.5% glycine before the steaming process could serve as a hurdle to growth of bacteria in steamed rice confection and prevent the risk of food poisoning and quality loss. These findings will be of immediate use to retail food service operations and regulatory agencies to ensure the microbiological safety of sticky rice cake and related foods. Okahisa, N., Inatsu, Y., Juneja, V.K., Kawamoto, S. 2008. Evaluation and control of therisk of food borne pathogens and spoilage bacteria present in "Awa-Uirou", a sticky rice cake containingsweet red bean paste. FoodbornePathogens and Disease. 5(3):351-359 13

14. The effect of glycine in the production of toxic volatile aldehydes from heated corn oil Carlos Macku and Takayuki Shibamoto The fatty aldehydesgenerated from heated corn oil and from several corn oil/glycine mixtures were collected by a dynamic headspace sampling method and subsequently reacted with cysteamine to yield corresponding thiazolidines. Derivatizedaldehydes were analyzed by a capillary gas chromatograph with flame photometric detector. Six fatty aldehydes, including formaldehyde and acetaldehyde, decreased in concentration in relation to increasing amounts of glycine in the oil. Journal of the American Oil Chemists' Society Volume 68, Number 11, 884-885, DOI: 10.1007/BF02660607 14

15. 15 Dietary glycineprevents the development of liver tumors caused by the peroxisomeproliferator WY-14,643 Michelle L. Rose, Russell C. Cattley, Corrie Dunn1, Victoria Wong1, Xiang Li and Ronald G. Thurman Previous studies demonstrated that dietary glycine prevents elevated rates of cell proliferation following treatment with the peroxisomeproliferator and liver carcinogen WY-14,643. Since increased cell replication is associated with the development of hepatic cancer caused by peroxisome proliferators, glycine may have anti-cancer properties. Therefore, experiments were designed to test the hypothesis that dietary glycine would inhibit the hepatocarcinogenic effect of WY-14,643. Male F344 rats were fed four different NIH 07-based diets: 5% glycine; 5% valine for nitrogen balance (control); 0.1% WY-14,643 + 5% valine (WY-14,643); 0.1% WY-14,643 + 5% glycine (WY-14,643 + glycine).

16. 16 Food consumption did not differ among the groups, but WY-14,643-fed rats weighed 10–25% less than expected based on previous studies. Serum glycine levels were elevated 4–5-fold by glycine-containing diets; however, the 10-fold increase in peroxisomal enzyme activity caused by WY-14,643 was unaffected by the addition of 5% glycine to the diet. After 22 weeks, livers from rats fed WY-14,643 had a similar incidence and multiplicity of proliferative lesions (foci and adenomas) to those fed WY-14,643 + glycine. Moreover, cell proliferation in the surrounding `normal' parenchyma (labeling index ≈ 4%) and foci (labeling index ≈ 50%) did not differ between WY-14,643 and WY-14,643 + glycine-fed rats. However, after 51 weeks of dietary exposure to WY-14,643, glycine prevented formation of small (0–5 mm diameter) tumors by 23% and inhibited the development of medium size (5–10 mm) tumors by 64%.

17. 17 Furthermore, glycine prevented the formation of the largest tumors (>10 mm) by nearly 80%. Thus, glycine did not inhibit early foci formation; however, it significantly decreased their ability to progress to tumors. Moreover, the inhibitory effect of glycine was greater with increasing tumor size. These studies demonstrate that dietary glycine prevents the development of hepatic tumors caused by the peroxisomeproliferator WY-14,643 consistent with the idea that it may be an effective chemopreventive agent.

18. 18 Reducing Acrylamide in Fried Snack Products by Adding Amino Acids CHEONG TAE KIM, EUN-SUN HWANG, AND HYONG JOO LEE The aims of this study were to develop commercial methods for reducing the acrylamide content in processed foods and apply them to commercial snacks. The formation of acrylamide in fried foods was found to depend on the composition of raw materials as well as the frying time and temperature. In potato chips, acrylamide was rapidly formed at over 160 °C, with the amount proportional to the heating duration and temperature. Free amino acids were used to reduce acrylamide, with lysine, glycine, and cysteine having the greatest effects in the aqueous system. Lysine and glycine were effective at inhibiting the formation of acrylamide in wheat-flour snacks. In potato snacks, the addition of 0.5% glycine to pallets reduced acrylamide by more than 70%. Soaking potato slices in a 3% solution of either lysine or glycine reduced the formation of acrylamide by more than 80% in potato chips fried for 1.5 min at 185 °C. These results indicate that the addition of certain amino acids by soaking the uncooked products in appropriate solutions is an effective way of reducing acrylamide in processed foods. JFS C: Food Chemistry and Toxicology

19. 19 Glycineintakedecreases plasma free fattyacids, adiposecellsize, and bloodpressure in sucrose-fedrats Mohammed El Hafidi, Israel Perez, Jose Zamora, Virgilia Soto,Guillermo Carvajal-Sandoval, and Guadalupe Banos Am J PhysiolRegulIntegr Comp Physiol 287: R1387–R1393, 2004. First published August 26, 2004; doi:10.1152/ajpregu.00159.2004. In SFR treated with glycine, mitochondrial respiration, as an indicator of the rate of fat oxidation, showed an increase in the state IV oxidation rate of the -oxidation substrates octanoic acid and palmitoylcarnitine. This suggests an enhancement of hepatic fatty acid metabolism, i.e., in their transport, activation, or -oxidation. …Thesefindingsimplythatthe protection by glycine against elevated BP might be attributed to itseffect in increasing fatty acid oxidation, reducing intra-abdominal fataccumulation and circulating NEFA, which have been proposed aslinks between obesity and hypertension.

20. 20 High-DoseGlycineTreatment of RefractoryObsessive-CompulsiveDisorder and BodyDysmorphicDisorder in a 5-Year Period W. Louis Cleveland, Robert L. DeLaPaz, Rashid A. Fawwaz and Roger S. Challop This paper describes an individual who was diagnosed with obsessive-compulsive disorder (OCD) and body dysmorphic disorder (BDD) at age 17 when education was discontinued. By age 19, he was housebound without social contacts except for parents. Adequate trials of three selective serotonin reuptake inhibitors, two with atypical neuroleptics, were ineffective. Major exacerbations following ear infections involving Group A β-hemolytic streptococcus at ages 19 and 20 led to intravenous immune globulin therapy, which was also ineffective. At age 22, another severe exacerbation followed antibiotic treatment for H. pylori. This led to a hypothesis that postulates deficient signal transduction by the N-methyl-D-aspartate receptor (NMDAR). Treatment with glycine, an NMDAR coagonist, over 5 years led to robust reduction of OCD/BDD signs and symptoms except for partial relapses during treatment cessation. Education and social life were resumed and evidence suggests improved cognition. Our findings motivate further study of glycine treatment of OCD and BDD.

21. 21 Mutagenicity of CookedHamburgerisReducedby Addition of Ascorbateand ErythorbatetoGroundBeef Tetsuta Kato, Kazuyuki Hiramoto, and KiyomiKikugawa* School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432–1 Horinouchi, Hachioji, Tokyo 192–0392, Japan (Received June 2, 2000; Accepted June 20, 2000) The addition of phenolic antioxidants, cysteine, Nacetylcysteine, unsaturated fatty acids, ascorbateand erythorbatehas been evaluated in the reduction of free radical Maillard intermediates, the pyrazinecation radicals, and also the mutagenicity of a heated model system composed of glucose/glycine/creatinine. The aim of the present study was to determine whether these components were useful to reduce the mutagenicity of cooked hamburger. The effect of these components, added at low concentrations to ground beef, on the generation of the mutagenicity of cooked hamburger was examined. Mutagenicityof hamburger was assayed by the Ames test using Salmonella typhimuriumTA98 strain with metabolic activation after the mutagens were purified by the use of blue rayon. Mutagenicity of hamburger was reduced to a half by the addition of ascorbate or erythorbate at 0.33%, whereas the mutagenicity was not reduced by epigallocatechingallate(EGCG), cysteine, N-acetylcysteine, soybeanoil or lard at the low concentrations.

22. 22 Neuroprotective effects of glycine in rats ESCAMILLA-ALFARO et al. Journal of Theoretical and Experimental Pharmacology 2010 Vol.1 N.2 ay-Aug72-75 The aim of this study was to evaluate the neuroprotective effects of glycinein an experimental animal model of permanent brain ischemic injury.Glycine is an inhibitory neurotransmitter amino acid that acts as neuromodulator of N-methyl-D-aspartate (NMDA) receptors, critically involved in the process of ischemic brain injury.Conclusions: Glycine administered i.p. to rats with permanent left carotid occlusion limited the ischemic brain damage probably by increasing the neurological availability of glycine concentration enough to prevent the desensitization of NMDA receptors and consequently altering the cascade of events that lead to cellular death.

23. 23 Reduction of acrylamide in fried foods by addition of amino acids and vacuumfrying CheongTae Kim, HyongJooLee SeoulNationalUniversity CONCLUSIONS The effects of lysine, glycine, cysteine and garlic on reduction rate of acrylamide in aqueous model systems were good. √ The formation of acrylamide in fried foods depends on the frying time and temperature. √ The acrylamide was neither destroyed in water nor oils. √ The effects of lysine and glycine on reduction rates of acrylamide in fried foods were particularly predominant. √ Reducing the formation of acrylamide in potato-chips was successfully conducted by vacuum frying and by dipping glycine and lysine solutions.

24. 24 FURTHER READING Reduction of Acrylamide Formation in Potato Chips by Low ... Vol. 69, Nr. 8, 2004—JOURNAL OF FOOD SCIENCEE 405 Published on Web 10/7/2004 © 2004 Institute of Food Technologists Further reproduction without permission is prohibited E: Food ...; CODE OF PRACTICE FOR THE REDUCTION OF ACRYLAMIDE IN FOODS (CAC/RCP ... CAC/RCP 67 Page 1 of 8 CODE OF PRACTICE FOR THE REDUCTION OF ACRYLAMIDE IN FOODS (CAC/RCP 67-2009) INTRODUCTION 1. Recent concern over the presence of acrylamide in food dates from 2002.; A "Toolbox" for the Reduction of Acrylamide in Bread Products AcrylamideAcrylamide is a substance that is produced naturally in foods as a result of high-temperature cooking, e.g., baking, grilling, or frying.;

25. 25 Acrylamide Risk Rediuction Strategy Risk Reduction Strategy and Analysis of Advantages and Drawbacks for AcrylamideAcrylamide Risk Rediuction Strategy; Acrylamide in cereal products: A review Strategies for acrylamide reduction in cereal products ..... 122 5.1. Impact of raw material ...; MEDICION DE HOLDUP EN COLUMNA DE EXTRACCION SPRAY acrylamide reduction in potato chips by using commercial asparaginasemedicion de holdup en columna de extraccion spray; A "Toolbox" for the Reduction of Acrylamide in Biscuits,Crackers ... AcrylamideAcrylamide is a substance that is produced naturally in foods as a result of high-temperature cooking, e. g., baking, grilling, or frying.; AcrylAmide conference SFA AcrylAmide conference December 2-3, 2008 The Blackwell Hotel & Conference Center The Ohio State University, Fisher College of Business Campus Columbus, Ohio Acrylamide Reduction ...;

26. 26 Two Years Acrylamide – A Stocktaking from the Risk Assessment Angle Furthermore, innovative production processes for instance for crisps are under discussion whose (reproducible) efficacy in conjunction with acrylamide reduction has not yet been ...; Wellness Foods Europe Table 2: Current methods for acrylamide reduction in cereal and potato based foods Product type Method Disadvantage Potato product Soaking potatoe slices in glycine or glutamine ...; Heat-Generated Food Toxicants; Identification, Characterisation ... Distribution of Margin of Exposure (MoE) of the acrylamide reduction scenarios over the percentiles. Managing the Risk The scenario analyses performed indicates a possible maximum ...; Reduction of Acrylamide Formation in Fried Potato Strips by Differe Reduction of Acrylamide Formation in Fried Potato Strips by Different Pre-frying Treatments Reduction of Acrylamide Formation in Fried Potato Strips by Differe;

27. 27 Effect of Added Asparagine and Glycine on Acrylamide Content in ... When glycine was applied on the surface of the fermented dough, there was also a significant reduction of acrylamide content in the bread. Addition of glycine but not asparagine ...; Further Development of the CIAA “Toolbox” Concept Rev 9 4 Appropriate sampling and statistically relevant numbers of analyses are therefore essential to determine acrylamide amounts in products, and to assess the actual reduction ...; ACRYLAMIDE IN FOOD But to date there is no single method for getting rid of acrylamide in foods; reduction must be done on a case-by-case basis. Exposure assessments are a critical component in ...;

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