Antioxidant and Antihypertensive Natural Peptides in Dry-Cured Ham By-Products Good morning, my name is Leticia Mora. I would like to thank organizers to give me the opportunity to describe one of the lines that I am developing in the Institute of Agrochemistry and Food Technology here in Valencia, Spain. My talk is going to be about the … Dr. Leticia Mora Institute of Agrochemistry and Food Technology (IATA-CSIC)

Meat Industry By-products By-products represent a cost for the meat processing sector as well as an important environmental problem. Meat production sector is one of the most contaminants due to the high generation of: Waste water, air pollution, solid waste. Meat industry annually produces tons of by-products that represent a cost for the meat processing sector as well as an important environmental problem. In fact, the sector of meat production one of the most contaminants due to the generation of high amounts of: Waste water Air pollution Solid waste Regarding this, meat production is more contaminant in terms of waste water generated and air pollution than the contaminants generated in the production of household energy or the use of cars and is responsible for the emission of the highest amounts of CO2, related with the current global warming problem. For this reason, the efficient use of meat by-products… reduce/compensate Waste water Air pollution (CO2 emissions) The efficient use of meat by-products is very important to reduce the environmental impact of this sector as well as an economical source.

Meat Industry By-products Most of the solid waste in the meat industry is produced during slaughtering. Live animal Manure Slaughtered Meat By-products Non-edible Edible Intestinal contents The majority of the waste in the meat industry is produced during slaughtering. Nowadays, industries are making a strong effort converting by-products and wastes into useful sources of both, edible and non-edible products, producing valuable products and functional ingredients with a significant added-value and/or a strong economic potential. Edible products depending on tradition, culture, and religion of the production countries. Pharmaceuticals Hide Bone Blood Human food Animal feed Fertilizers Insulin Leather Soups Industries are making a strong effort converting by-products into useful sources of edible and non-edible products, producing valuable products and functional ingredients with added-value and economic potential.

Meat Industry By-products Dry-cured ham slicering process for its commercialization Commercial slices Animal feed industries Solid waste Human food-related uses: Household broths and soups. Fried rinds as appetizer. However, the generation of by-products is not only during slaughtering as the processing of meat products for its commercialization such as the slicering of dry-cured ham generates a high amount of wastes rich in proteins. The removal of bones and rinds is still done by specialists in a traditional way and it generates solid waste. This waste is frequently sold to animal feed industries or used as human food in the form of fried rinses as appetizer or concentrated soups.

Bioactive peptides generation Small peptides (2-20 amino acids). Inactive within the sequence of the parent proteins. When liberated by the action of enzymes can act as regulatory compounds. Natural Under controlled enzymatic conditions Food processing Gastrointestinal digestion By-products Bioactive peptides are usually small peptides between 2 and 20 amino acids that are inactive in the sequence of the parent protein but when liberated by the action of enzymes can act as regulatory peptides in the biological system. The generation of bioactive peptides can be natural if they are generated… …during the food processing such as fermentation or curing proceses by the action of microorganisms and endogenous enzymes …during the digestion by the action of gastrointestinal enzymes On the other hand, the generation of bioactive peptides from by-products such as olivemill wastewater, cheese whey, winery sludge, citrus peel, etc is done under controlled enzymatic conditions which permits the control of the hydrolisis and the generated peptides

Dry-cured ham as a source of bioactive products Dry-cured ham is a high-quality product very well valorated by consumers around the world. During the processing of dry-cured ham occurs an intense proteolysis responsible for its characteristic flavour and texture that results in the generation of small peptides and free amino acids. Proteolysis Dry-cured ham is a high-quality product very well valorated by consumers around the world. During its processing occurs an intense proteolysis responsible for its characteristic flavour and texture. At the end of proteolysis, due to the action of endogenous enzymes, a lot of small peptides and free amino acids are generated, and we wonder why not these small peptides could be acting as bioactive peptides by exerting a function in the organism?. Intact protein Naturally generated peptides Why not potential bioactive peptides??

Dry-cured ham as a source of bioactive products High content in salt (5-6%) of dry-cured ham makes it non-recommended for consumers with CVDs. Synthetic drugs cause side effects. Search for non-toxic and naturally generated peptides to control the factors influencing CVDs as alternative to synthetic drugs is of great interest among researchers. Captopril Enalapril A clinical study to evaluate the effect of dry-cured ham ingestion on CVDs in 13293 individuals resulted on no association between the higher levels of consumption and the incidence of CVDs. Medicina Clinica (2009) 133, 754-580 One of the most relevant challenges of dry-cured ham consists of the high content in sodium chloride (5-6%) that makes it non recommended for consumers having cardiovascular troubles, especially hypertension. At present there is a wide range of synthetic drugs on the market for the treatment of hypertension including captopril and enalapril but they are believed to cause side effects. For this reason, the search for non-toxic and naturally generated antihypertensive peptides as alternative to synthetic drugs is of great interest among researchers. In 2009, … This fact could be due to a balance (compensation) between the amount of salt present in the ham that would produce hypertension and the presence of antihypertensive peptide sequences naturally generated during the processing. HYPOTESIS: Dry-cured ham as a source of bioactive peptides

Antihypertensive peptides in dry-cured ham ACE I inhibitory peptides role in the regulation of blood pressure. ACE Inhibitory peptides Angiotensin I ACE I Angiotensin II ACE I plays an important role in the regulation of blood pressure. The enzyme ACE-I converts an inactive angiotensin-I to angiotensin-II which is a potent vasoconstrictor that produces an increase in blood pressure. For this reason, the presence of ACE-inhibitory peptides would regulate blood pressure. Inhibitors of ACE activity reduce systolic blood pressure. Potent vasoconstrictor Increase of blood pressure

Antihypertensive peptides in dry-cured ham In vivo test In vitro test DRY-CURED HAM ANTIHYPERTENSIVE ACTIVITY ACE INHIBITORY ACTIVITY IN A DRY-CURED HAM EXTRACT After 8h of ingestion: Decrease in the SBP of -8.3 ± 0.5 mmHg The presence of antihypertensive peptides in dry-cured ham was studied in vitro using the ACE inhibitory activity assay. It was observed that there was an increase in the ACE inhibitory activity with the amount of peptides tested. Same extract of peptides was tested in SHR obtaining a decrease in SBP of 8 mmHg after 8h of ingestion. The obtained results confirmed the potential of dry-cured ham as a source of antihypertensive peptides so next objective was the identification of the peptide sequences. Dry-cured ham is a source of naturally generated antihypertensive peptides. Food Research International, (2015), 78, 71-78.

SIZE-EXCLUSION CHROMATOGRAPHY Extraction Pretreatment Fractionation Identification Quantitation SIZE-EXCLUSION CHROMATOGRAPHY Fractionation according to the molecular mass So a peptidomics strategy was used for the ident… After the extraction and pretreatment of the peptides, we started with the fractionation using two different types of chromatography. The next step after the extraction of the peptides, is the peptide fractionation. Peptide fractionation use to be done by combining different types of chromatographies. For a primary separation, we use size-exclusion chromatography that fractionates the peptides according to their molecular mass.

SIZE-EXCLUSION CHROMATOGRAPHY Extraction Pretreatment Fractionation Identification Quantitation SIZE-EXCLUSION CHROMATOGRAPHY In vitro ACE inhibitory assay 82% of ACE inhibitory activity The next step after the extraction of the peptides, is the peptide fractionation. Peptide fractionation use to be done by combining different types of chromatographies. For a primary separation, we use size-exclusion chromatography that fractionates the peptides according to their molecular mass.

SIZE-EXCLUSION CHROMATOGRAPHY Extraction Pretreatment Fractionation Identification Quantitation SIZE-EXCLUSION CHROMATOGRAPHY In vivo antihypertensive assay (2, 4, 6, 8, and 24h) The next step after the extraction of the peptides, is the peptide fractionation. Peptide fractionation use to be done by combining different types of chromatographies. For a primary separation, we use size-exclusion chromatography that fractionates the peptides according to their molecular mass.

SIZE-EXCLUSION CHROMATOGRAPHY Extraction Pretreatment Fractionation Identification Quantitation SIZE-EXCLUSION CHROMATOGRAPHY In vivo antihypertensive assay (2, 4, 6, 8, and 24h) So this fraction was concentrated and analysed by HPLC. After 8h ingestion: reduction of SBP in -38.0 ± 0.5 mmHg

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY Extraction Pretreatment Fractionation Identification Quantitation HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY The secondary separation was done using HPLC and by collecting the eluted fractions for the ACEI inhibitory assay.

Highest ACE inhibitory activity Extraction Pretreatment Fractionation Identification Quantitation Highest ACE inhibitory activity The next step of this strategy is the use of mass spectrometry in tandem which is crucial to know the identify of the antihypertensive peptides. The obtained sequences were

nLC-ESI-MS/MS Extraction Pretreatment Fractionation Identification Quantitation Sequence IC50(mM) PAPPK 199,58 KAAAAP 19,79 AMNPP 304,50 IKLPP 193,90 AAPLAP 14,38 KPVAAP 12,37 KPGRP 67,08 PSNPP 192,27 IAGRP 25,94 KVLPG 265,44 TGLKP 51,57 AAATP 100,00 KAAAATP 25,64 nLC-ESI-MS/MS The next step of this strategy is the use of mass spectrometry in tandem which is crucial to know the identify of the antihypertensive peptides. The obtained sequences were Q/ToF Mass spectrometer

Decrease in the SBP of -25.62 ± 4.5 mmHg Antihypertensive peptides in dry-cured ham Peptide AAATP IC50=100 mM After 8h of ingestion: Decrease in the SBP of -25.62 ± 4.5 mmHg The petides contained in the extract were identified by MS in tandem. From the identified peptides, AAATP was the sequence showing the lowest IC50 value and was assayed in SHR obtaining… The presence of antihypertensive peptides in dry- cured ham could counteract the negative effect of salt and contribute for a better possibility of consumption. J Proteomics, (2013), 78, 499-507.

Antioxidant peptides in dry-cured ham Peptide SNAAC IC50=75.2 mM Cooking resistance and maintenance of antioxidant activity a a a a Simulated gastrointestinal digestion a Also several sequences have been identified in DCH as antioxidant such as the peptide SNACC that showed a similar antioxidant capacity to the synthetic BHT that was confirmed using different antioxidant assays. This antioxidant peptide showed good stability during cooking maintaining its antioxidant capacity. Also it was tested with an in vitro simulated GI digestion… The observed increase in the antioxidant activity after the gastrointestinal digestion could be due to the basic conditions of the media together with the positio of the cisteine in the terminal position of the peptide contribute the formation of dimers which increase the antioxidant ability. a b Escudero et al (2013), Food Chemistry, 138, 1282-1288. Mora et al (2016), Food Chemistry, (under review).

Natural antioxidant peptides in dry-cured ham by-products Effect of cooking on the antioxidant capacity of natural peptides Cooking increases the antioxidant activity in all the assayed antioxidant methodologies. Effect of simulated gastrointestinal digestion In vitro digestion affected the sequence of many of the natural peptides, resulting in differences in the antioxidant capacity of digested broths.

Peptidomic aproach to identify those peptides responsible for the main differences in antioxidant activity between types of cooking Finally, in order to identify those peptide sequences responsible for the main differences in antioxidant activity between types of cooking, a peptidomic approach based on the relatively quantification of the peptides by comparison with a control was done using a label-free mass spectrometry methodology. This loading plot shows the protein of origin of all the identified peptides in control and samples cooked for 20 and 60 min. The highest amount of the observed peptides were from collagen protein which resulted to be main responsible for the observed differences between samples.

The analysis of the peptides derived from collagen protein using a Principal Component Analysis resulted in three main clusters showing significant differences in the peptides obtained according to the type of cooking and after GI digestion. The loading plot will permit the identification of those peptides responsible for main differences between groups and thus potentially responsible for the antioxidant capacity observed.

Conclusion The intense proteolysis occurred during the dry-curing process is responsible for the generation of small peptides, many of them showing antihypertensive or antioxidant activities. The cooking process and in vitro gastrointestinal digestion of dry-cured ham by-products positively affect the sequence of natural peptides and the observed antioxidant activity of broths. The identification of those peptides responsible for main bioactivity is possible through the development of novel approaches using peptidomics and modern bioinformatic tools.

HIGHVALFOOD Acknowledgments This work has received funding from the European Unions’ Seventh Framework Programme for research, technological development and demonstration: Grant Agreement No. PCIG13-GA-2013-614281 HIGHVALFOOD Grant from Generalitat Valenciana in Spain Grant No. GV/2015/138 Acknowledgements:

THANK YOU FOR YOUR ATTENTION Dr. Leticia Mora lemoso@iata.csic.es