Journal of Functional Foods 38 (2017) 160–167 Contents lists available at ScienceDirect Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff Effects of dry-cured ham rich in bioactive peptides on cardiovascular health: A randomized controlled trial Silvia Montoro-García a,⇑, María Pilar Zafrilla-Rentero a, Francisco Miguel Celdrán-de Haro a, Juan José Piñero-de Armas b, Fidel Toldrá c, Luis Tejada-Portero a, José Abellán-Alemán a a Cátedra de Riesgo Cardiovascular y Departamento de Nutrición, Facultad de Ciencias de la Salud, UCAM Universidad Católica San Antonio de Murcia, Campus de los Jerónimos, s/n, Guadalupe 30107, Murcia, Spain b Cátedra de Estadística ‘‘Big data”, UCAM Universidad Católica San Antonio de Murcia, Spain c Instituto de Agroquímica y Tecnología de Alimentos, CSIC, Av Agustín Escardino 7, 46980 Paterna, Valencia, Spain a r t i c l e i n f o Article history: Received 19 April 2017 Received in revised form 5 September 2017 Accepted 8 September 2017 Keywords: Dry-cured ham Bioactive peptides Hypertension ACE inhibition Cardiovascular risk factors a b s t r a c t Establishing health effects of bioactive compounds from dry-cured meat is an active area of clinical research. The present study aims to investigate whether consuming dry-cured ham with biopeptides, among other bioactive compounds, modifies blood pressure (BP) and improves other risk factors for cardiovascular disease in humans. This two-arm, cross-over, randomised controlled trial involved 38 healthy subjects with pre-hypertension. Participants received 80 g/day dry-cured pork ham or 100 g/day cooked ham (control product). A daily intake of 80 g dry-cured ham did not impair BP or 24 h sodium excretion. Total cholesterol, LDL and basal glucose levels dropped after dry-cured ham consumption (p = 0.00019, p = 0.021 and p = 0.014, respectively). Cooked ham did not affect any of the clinical and biochemical markers. Dry-cured ham components could exert a plethora of activities over the cardiovascular system including lipid and glucose metabolism. Additional studies are needed to confirm the effects of dry-cured meat biopeptides on diverse risk factors in pathological conditions. Ó 2017 Elsevier Ltd. All rights reserved. 1. Introduction Nutrition is often considered one of the most ‘‘modifiable” risk factors in cardiovascular primary prevention, which is why it is frequently used. The scientific community has increased its interest in different food bioactives, from polyphenols to biopeptides (Bahadoran, Mirmiran, & Azizi, 2013; Chakrabarti, Jahandideh, & Wu, 2014). In fact, a large number of dietary supplements and nutraceuticals (of both vegetal and animal origin) have been tested in the development of natural therapies for cardiovascular disease (CVD) prevention such as antihypertensive agents (Alexander, 2014; Kawasaki et al., 2000; Seppo, Jauhiainen, Poussa, & Korpela, 2003). Abbreviations: ACE, Angiotensin I Converting Enzyme; BMI, Body Mass Index; BP, Blood Pressure; CVD, Cardiovascular Disease; DBP, Diastolic Blood Pressure; HDL, High Density Lipoprotein; LDL, Low Density Lipoprotein; RCT, Randomized Controlled Trial; SBP, Systolic Blood pressure; UCAM, Catholic University of Murcia. ⇑ Corresponding author at: Cátedra de Riesgo Cardiovascular, Facultad de Ciencias de la Salud, UCAM Universidad Católica San Antonio de Murcia, Campus de los Jerónimos, s/n, Guadalupe 30107, Murcia, Spain. E-mail address: email@example.com (S. Montoro-García). http://dx.doi.org/10.1016/j.jff.2017.09.012 1756-4646/Ó 2017 Elsevier Ltd. All rights reserved. Bioactive peptides are small compounds, comprising 2-20 amino acids and with a wide variety of pharmacological targets. Meat proteins offer huge potential as novel sources of bioactive peptides with a capacity to inhibit angiotensin I converting enzyme (ACE) and display antioxidant, antimicrobial and antiproliferative activities (da Cruz, Pimenta, de Melo, & Nascimento, 2016; Ryan, Ross, Bolton, Fitzgerald, & Stanton, 2011). Dry-cured ham is a traditional and ubiquitous Spanish food, its elaboration dates of hundreds of years and it is an important business in the country (Mora et al., 2015). Important biochemical changes occur during dry-curing, including intense proteolysis due to the action of endogenous muscle peptidases and resulting in the release of a variety of bioactive peptides. In fact, bioactive peptides have been found after hydrolysis (Arihara, Nakashima, Mukai, Ishikawa, & Itoh, 2001) and after in vitro simulated digestion of dry-cured ham (Escudero, Aristoy, Nishimura, Arihara, & Toldrá, 2012). Moreover, the in vitro antihypertensive capacity of bioactive peptides of Spanish dry-cured ham has been previously reported in rats (Escudero et al., 2012; Escudero et al., 2013). The antihypertensive activity of bioactive peptides was preserved even after the physiological digestion, which could be absorbed along the intestine and S. Montoro-García et al. / Journal of Functional Foods 38 (2017) 160–167 exert a decrease in systolic blood pressure (BP) in rats (Escudero et al., 2012). Nonetheless, pork leg is salted during ham curing, and the excessive dietary intake of sodium chloride is related with negative effects on human health, including hypertension, and, consequently, an increased risk of CVD (Morgan, Aubert, & Brunner, 2001). A prospective epidemiological study in 13,293 students analyzed the prevalence of CVD, weight gain and hypertension during a six-year follow up period, showing that regular consumption of dry-cured ham (>4 times per week) was not associated with any of these effects despite the higher dietary intake of salt (RuizCanela López et al., 2009). Whether bioactive peptides, among other compounds, from dry-cured ham counteract the salt intake has not been formally demonstrated, although clinical observations could be used to support this concept. The aim of the present randomized controlled trial (RCT) was to investigate whether the consumption of dry-cured pork ham rich bioactive peptides could modify BP and other cardiovascular risk factors. 2. Material and methods 2.1. Ethics statement The current study was registered in the Clinical Trials Database (ID: NCT02585089), performed in accordance with the Helsinki Declaration and approved by the Ethics Committee of the Catholic University of Murcia (UCAM, April 2015). All enrolled volunteers provided written informed consent. 161 ber 2015 to January 2016. Forty apparently healthy volunteers with untreated high-normal BP were recruited. One group (n = 21) received a controlled salt dry-cured ham of >11 months proteolysis (interventional product) while the other (n = 19) received cooked, uncured ham (control product), each for one month. After a two weeks wash out, the groups exchanged roles for another month. In this way, each group had consumed both meat products for one month (Fig. 1). The study arms were similar for age, gender, ethnicity, body mass index and BP, which allowed comparisons to be made between them (Table 2). The individuals did not know about the purpose of the study or which of the hams was supposed to be the interventional product. The volunteers were enrolled by simple randomisation by only one investigator and the randomization sequence was single-blind until the end of statistical analysis. Caucasian men and women from the University staff, aged 40– 55 years, in good general health and with prehypertension were screened in July-September 2015. An average systolic and diastolic arterial BP of >125 mmHg and >80 mmHg, respectively, was required to take part. Exclusion criteria were: smokers, Diabetes mellitus, diagnosed and treated hypertension, history of cardiovascular events (stroke, myocardial infarction or peripheral vascular disease), cancer and inflammation diseases. Medications - antihypertensives, antiaggregants, anticoagulants, antidepressants, anti-cholinergic or anti-spasmodic agents, the regular use of medications affecting intestinal motility, vasodilators, lipid lowering therapies and fish oil supplements also acted as exclusion criteria (all other supplements were assessed on a case by case basis). 2.2. Study design and subjects 2.3. Characteristics and dietary habits of volunteers In the context of the 7FP EU ‘‘Beneficial Effects of Bioactive Compounds in Humans (BACCHUS)” project, a two-arm crossover RCT with diet control was assessed at the UCAM from Septem- Information on weight, height, body mass index (BMI, kg/m2), body fat, water, and muscle was collected with bioelectrical impedance using a Tanita BC-541 (Illinois, USA) at the beginning and at Fig. 1. Study design and timetable. 162 S. Montoro-García et al. / Journal of Functional Foods 38 (2017) 160–167 Table 1 Physical characteristics of the meat products. Humidity, % Fat content, % Protein, % Salt, % Monounsaturated fatty acids, % Polyunsaturated fatty acids, % Saturated fatty acids, % Dry-cured ham Cooked ham 46.93 15.82 32.24 4.38 48.51 13.35 37.57 74.70 1.37 17.13 2.61 49.54 15.52 34.36 Table 2 Baseline characteristics of the volunteers. Age Gender (male) Ethnicity (Caucasian) BMI, (kg/m2) Fat content, % Systolic BP, mmHg Diastolic BP, mmHg Basal Glucose, (mg/dL) Cholesterol, (mg/dL) HDL, (mg/dL) LDL, (mg/dL) TG, (mg/dL) Creatinine, (mg/dL) Sodium excretion, (mEq/L/24 h) Group 1 (n = 18) Group 2 (n = 20) P value* 46.3 ± 5.0 79% 100% 25.7 ± 4.4 24.2 ± 7.7 136.0 ± 10.2 86.0 ± 11.5 81.5 ± 10.3 212.7 ± 40.1 57.5 ± 18.0 133.5 ± 35.7 104.5 ± 57.8 0.88 ± 0.19 158.8 ± 46.9 42.5 ± 5.0 85.7% 100% 27.0 ± 3.6 25.1 ± 7.0 134.5 ± 8.5 81.6 ± 7.3 91.4 ± 10.8 200.1 ± 41.8 52.4 ± 11.3 125.3 ± 35.4 112.2 ± 75.3 0.92 ± 0.18 154.95 ± 31.9 0.89 0.73 0.99 0.32 0.43 0.70 0.18 0.58 0.62 0.13 0.83 0.45 0.87 0.46 BMI: Body Mass index; HDL: High Density Lipoprotein; LDL: Low Density Lipoprotein; TG: Triacylglycerol. * T-Student test (mean-comparison tests). the end of the study by a qualified nurse. Standard positioning was used as described in the instruction manual in all measurements and skin-to skin contact was avoided. The procedure took approximately 60 s. A list of restricted foods (cold meat, chips, pickles, dry soups) was given at the beginning of the study in order to avoid the excessive consumption of salt and other cured meat products. The ham intake and food restriction was monitored in a weekly-visit to assess compliance to the study. All participants were asked to complete a survey designed to assess their exercise and dietary patterns at the beginning and at the end of the study to confirm no changes in dietary intake and lifestyle. The present survey was adapted from the validated ATTICA study survey (Trichopoulou, Costacou, Bamia, & Trichopoulos, 2003). The questionnaire included demographic characteristics (age, sex and education), detailed medical history, dietary and lifestyle habits, such as alcohol and physical activity. Since the questionnaire was designed for a Mediterranean population and the issues requested meet the criteria for clarity, simplicity and neutrality, divided into subject areas and avoiding double issues, and consisting of closed questions (with mutually exclusive answers), validation in the Spanish population was not considered necessary, and has already been used in a similar population (Abellán Alemán et al., 2016). 2.4. Meat products Spanish dry-cured ham with a controlled salt content after >11 months dry-curing process was the interventional product and cooked ham was the control product. Both hams were produced using raw material from 6-month old pigs (Landrace Large White). The average live weight at slaughter was 120 kg. Hams were bled according to traditional proce- dures. The different stages were as follows for dry-cured ham: pre-salting (0–2 °C and 80–95% relative humidity), incorporation of potassium nitrate, salting stage in which hams were completely covered with solid salt, stacked in without touching each other in a cold room (2–4 °C and 90–95% relative humidity for 12 days). This was followed by post-salting, whereby salted hams were kept at low temperatures (4–6 °C and 75–95% relative humidity for 60 days) and finally, the ripening-drying stage at 14–30 °C and a lower relative humidity (gradually reduced to 70%). The total length of the curing process was approximately 11.5 months. Table 1 displays the characteristics of the two meat products. The dry-cured ham contained 25% less salt than similar products on the market produced by the same company (4.38% vs. approx. 5.5%). As regard the cooked ham, it contained 2.61% salt. All meat products were manufactured and supplied by the local meat Company ElPozo Alimentación (Alhama de Murcia, Murcia, Spain) especially for the present study in daily vacuum bags and labelled with safety information (expiration date, nutritional composition and storage information). Volunteers ate 80 g of dry-cured ham daily for one month at any time of the day. In order to counteract the humidity, a higher amount of cooked ham (control) was given (100 g/day). The 100 g of cooked ham contained only 1 g salt more than the 3.6 g salt per 80 g of dry-cured ham. Meat products could be served at any time of the day, alone or with other foods but not cooked or heated. 2.5. Blood sampling and biochemical determinations For laboratory analysis, 8 h fasting peripheral venous blood samples were collected four times from all participants. Blood was taken before and after each period (interventional/control ham), following by a 2 weeks wash out period (total 4 time-points). The fasting blood lipid profile (total cholesterol, HDL, LDL and triacylglycerol); hemogram, plasma glucose (mg/dL) and creatinine levels were analysed. Participants were also asked to collect 24-h urine in standard containers (provided). Total volume of the collection was measured by the volunteers and checked by the analyst. Urine collections of less than 250 mL or those outside the range of 20–28 h were rejected. In accordance with the standard procedure, urinary sodium was measured using the ion selecting electrode method. Daily salt intake was estimated based on calculation of 24-h urinary sodium excretion on the assumption that all sodium ingested was in the form of sodium chloride. 2.6. Extraction and sequencing of biopeptides in hams A total of 20 grams of sample (dry-cured ham or cooked ham) were minced and homogenized with 80 mL of 0.01 N HCl in a Stomacher (IUL Instrument, Barcelona, Spain) for 8 min. Then it was centrifuged (10000g for 20 min at 4 °C), filtered through glass wool, and deproteinised with ethanol in ratio 1:3. Samples were kept at 4 °C overnight, centrifuged again (12000g for 20 min at 4 °C) and the supernatant dried in a rotary evaporator and finally lyophilised. Samples were resuspended in 100 mL of TFA 0.1%, diluted 1:10, and analysed using a Nano-LC Ultra 1 D Plus system (Eksigent of AB Sciex, CA, USA) coupled to a quadrupole/time-offlight (Q/ToF) TripleTOFÒ 5600+ system from AB Sciex Instruments (Framingham, MA, USA) that is equipped with a nanoelectrospray ionization source. Systems parameters were adjusted as described elsewhere (Escudero et al., 2013). The capillary column (3 mm, 75 mm 12.3 cm, C18) (Nikkyo Technos Co., Ltd., Japan) was eluted with mobile phases consisted of solvent A, 0.1% v/v formic acid in water, and solvent B, 0.1% v/v formic acid in 100% acetonitrile. Chromatographic conditions were a linear gradient from 5% to 35% of solvent B over 90 min, and 10 min from 35% to 65% of sol- 163 S. Montoro-García et al. / Journal of Functional Foods 38 (2017) 160–167 vent B, at a flow rate of 0.30 lL/min and running temperature of 30 °C. The Q/ToF was operated in positive polarity and information-dependent acquisition mode, in which a 250 ms ToF MS scan from 300 to 1250 m/z was performed, followed by 50 ms product ion scans from 100 to 1500 m/z on the 50 most intense 2 to 4 charged ions. Samples were treated and injected in a randomly. The identification of protein origin of peptides was done using Mascot Distiller v18.104.22.168 software (Matrix Science, Inc., Boston, MA). 2.7. Blood pressure monitoring During the screening phase (July-September 2015), BP was measured three times - about one minute apart and in the dominant arm - in a properly calibrated sphingomanometer (OMROM 705 CP) early in the morning on two consecutive days, according to ESH/ESC recommendations (O’Brien et al., 2005). Volunteers were recruited if the mean satisfied systolic and diastolic BPs above 125 and 80 mmHg, respectively. During the clinical study, a 24 h holter monitor recording was assessed for all the volunteers included in the study at four different time-points (before/after interventional and control products). Volunteers were appointed for a blood extraction and 24 h holter using a digital manometer device OMRON M24/7 BP5, properly calibrated. The systolic and diastolic BP readings were recorded. Measurements were performed every 15 and 30 min during the daytime and night time, respectively. Readings with <65% measurements were rejected. Average, daytime and night time BP were recorded. 2.8. Sample size and statistical analyses Continuous variables were tested for normal distribution by the Kolmogorov-Smirnov test. Data are expressed as mean ± standard deviation (SD) and confidence interval [CI 95%] for normally distributed data. Each categorical variable is expressed as frequency (percentage) of volunteers. The present analyses are focused on estimating the differences of the effect of the intake of dry-cured ham minus cooked ham after the first and second treatments (T1 and T2, respectively). For this objective, differences at different time points were assessed by linear random effect model assuming a different ‘‘baseline” value for each subject using the program R v3.2.4 and the lme4 package. A P-value of <0.05 was considered statistically significant. SPSS 18.0 software was used for the rest of statistical analyses (SPSS, Inc, Chicago, Illinois, USA). although high percentages of saturated fatty acids were also found for both products (37.57% and 34.36%) (Table 1). 3.2. Anthropometry, BP and biochemical parameters Among the 40 initial volunteers, only 38 completed the entire crossover study, with dropouts arising from the difficulty experienced in the daily ingestion of the products. Thirty-eight subjects (44.3 ± 5.3 years old; 82% males) were therefore included in the study. All variables showed the distribution pattern of normality. Mean anthropometric values were: body mass index (BMI) 26.4 ± 7.3 kg/m2, systolic and diastolic BP 135.1 ± 9.2 and 83.6 ± 9.6 mmHg, respectively (Table 2). Forty-five percent of the population presented a BMI of between 25 and 29.9 kg/m2 based on self-reported height and weight data, indicating a high percentage of overweight (n=18). In addition, >18% of individuals (n=7) were qualified as obese (BMI 30) (data not shown). As expected from random allocation to intervention groups, no significant differences (P>0.05) were observed at the beginning of the study between the two groups in terms of the anthropometric parameters, BP and biochemical parameters (Table 2). Volunteers were also asked not to change their dietary and lifestyle habits during the intervention period and to refrain from eating other cured meat products (sausages, etc.) (Table 3). 3.3. Dry-cured and cooked ham consumption As shown in Table 4, the intake of dry-cured ham during the first period decreases 16.67 points the total cholesterol levels (p = 0.00019), being less significant when consumed in the second period (p = 0.036). Low density lipoprotein levels were decreased in 7.5 points when dry-cured ham was consumed first (p = 0.021). A similar trend for LDL levels was also shown when it was consumed during the second period (p = 0.054). Similarly, dry-cured ham intake produced an important decrease of 26.36 points in TG levels only when consumed first (p = 0.010). High density lipoprotein levels and the TG/HDL-cholesterol and LDL/HDLcholesterol ratios did not show statistically significant change after any treatment (data not shown). Fasting glucose levels were strongly affected by the different treatments of the study. Glucose levels decreased in 9.0 points when dry-cured ham was consumed first (p = 0.001) but increased significantly when dry-cured ham was consumed after cooked ham (p = 0.043) (Table 4). The random Table 3 Dietary and life habits in the total population before and after the RCT. 3. Results 3.1. Identification of bioactive peptides and fat content in the tested meat products The detection and identification of peptides in the samples of cooked and dry-cured ham was necessary in order to verify the greater presence of ACE inhibitory peptides in the interventional product. Dry-cured ham presented higher content of the following ACE-inhibitory peptides: KAAAAP, AAPLAP, AAATP, KPVAAP, VPPAK, KPGRP and PAAPPK which were reported to be the most potent ACE-inhibitory peptides with IC50 values ranging from 12.37 to 25.94 mM (Escudero et al., 2013). None of the mentioned bioactive peptides were detected in the cooked ham samples, which were thus considered as an adequate control product (data not shown). Dry-cured ham was moderately low in fat (15.82%) but notoriously higher than cooked ham (1.37%). Fats were mainly monounsaturated in both products (48.5% and 49.54%, respectively), * Total (n = 38) Before RCT After RCT P value* Cereals, 3–4 day, % Fruits, 1–2 day, % Legumes, 1–2 week, % Red meat, 1–2 week, % Poultry, 1–2 week, % Eggs, 1–2 week, % Fish, 1–2 week, % Dairy products, daily, % Other cured meat, 3–5 week Red wine, 4–7 week Olive oil, daily Exercise Daily, % 4–5 times per week, % 2–3 times per week, % Never (sedentary), % Watching TV (h) <1,% 1–2, % 2, % 32.5 57.5 67.5 80 60 67.5 80 85 42.5 7.5 95 37.5 55 70 75 62.5 67.5 75 85 0.0 7.5 97.5 NS NS NS NS NS NS NS NS <0.05 NS NS 12.5 20 50 17.5 10 22.5 50 17.5 NS NS NS NS 37.5 35 27.5 32.5 37.5 32.5 NS NS NS Mean-comparison test, paired data. 164 S. Montoro-García et al. / Journal of Functional Foods 38 (2017) 160–167 Table 4 Difference of the effect of dry-cured ham minus cooked ham after the first (T1) and second treatment (T2) in biochemical parameters and blood pressure. Total (n=38) Total cholesterol (mg/dL) T1 T2 Estimate 16.673 7.575 Std. Error P-value 4.329 3.582 0.00019 0.03667 0.9322 1.0091 0.564 0.807 HDL (mg/dL) T1 T2 0.5395 0.2470 LDL (mg/dL) T1 T2 7.515 4.541 3.215 2.333 0.021 0.054 TG (mg/dL) T1 T2 26.36 2.412 10.08 11.645 0.010 0.836 Glucose (mg/dL) T1 T2 9.000 9.258 2.694 4.527 0.00114 0.0433 Average SBP (mmHg) T1 T2 1.668 0.546 1.081 1.251 0.125 0.664 Average DBP (mmHg) T1 T2 0.637 0.889 0.790 0.915 0.421 0.334 Daytime SBP (mmHg) T1 T2 2.729 1.508 1.232 1.420 0.261 0.290 Daytime DBP (mmHg) T1 T2 0.654 0.343 0.914 1.047 0.475 0.743 Night-time SBP (mmHg) T1 0.365 T2 3.115 2.180 2.699 0.867 0.251 Night-time DBP (mmHg) T1 0.229 T2 1.372 0.987 1.195 0.817 0.253 Na+ excretion (mEq/L/24 h) T1 8.442 T2 8.114 10.854 14.174 0.438 0.568 Creatinine (mg/dL) T1 T2 0.139 0.205 0.258 0.800 0.157 0.0521 HDL: High Density Lipoprotein; LDL: Low Density Lipoprotein; TG: Triacylglycerol; SBP: Systolic Blood pressure, DBP: Diastolic Blood Pressure. effect model did not find differences in BP, sodium excretion, creatinine levels after the dry-cured ham intake. Besides, chronic consumption of cooked ham in the first or second period did not alter anthropometric parameters, fasting glucose, serum lipid profile, blood pressure, sodium excretion or creatinine levels (data not shown). 3.4. Dry-cured ham consumption and lipid profile When the effect of both meat products was analysed independently in the two groups, the consumption of dry-cured pork ham during the first treatment produced a drop of 17.1 points (9.1%) in the total cholesterol levels (P = 0.000423). After the second treatment (cooked ham), cholesterol levels increased by 9.5 points (3.7%) back to basal levels (recovery effect) (p = 0.054) (Table 5). However, the total cholesterol levels of the volunteers who had cooked ham first (Group 1) were not statistically different from baseline levels (P = 0.48). Consuming dry-cured ham as the second treatment decreased the total cholesterol levels by 3.3 points (1.9%) but this was not statistically significant (P = 0.60). Again, Table 5 Effect of different treatments on total cholesterol levels. Cooked ham first treatment (group 1) Wash out after cooked ham (group1) Dry-cured ham second treatment (group 1) Dry-cured ham first treatment (group 2) Wash out after dry-cured ham (group2) Cooked ham second treatment (group 2) Estimate Std. Error P value 4.412 1.706 3.294 17.097 3.238 9.524 5.338 3.328 6.328 4.722 5.693 4.899 0.48716 0.78799 0.60372 0.000423 0.5707 0.054407 Random effect model. Table 6 Effect of dry-cured ham consumption on LDL levels. Cooked ham first treatment (group 1) Wash out after cooked ham (group1) Dry-cured ham second treatment (group 1) Dry-cured ham first treatment (group 2) Wash out after dry-cured ham (group2) Cooked ham second treatment (group 2) Estimate Std. Error P value 2.619 0.349 0.197 6.147 1.048 5.738 3.672 3.263 3.798 3.316 3.638 3.598 0.477 0.915 0.959 0.066 0.774 0.113 Random effect model. Table 7 Effect of different treatments on fasting glucose levels. Cooked ham first treatment (group 1) Wash out after cooked ham (group1) Dry-cured ham second treatment (group 1) Dry-cured ham first treatment (group 2) Wash out after dry-cured ham (group2) Cooked ham second treatment (group 2) Estimate Std. Error P value 6.471 5.294 4.941 6.571 5.905 9.000 3.958 2.742 2.948 2.653 2.481 4.236 0.03033 0.07535 0.09665 0.01479 0.02810 0.00096 Random effect model. as shown in Table 5, dry-cured ham consumption produced a higher reduction in total cholesterol levels than cooked ham alone. Low density lipoprotein levels were more affected by dry-cured ham consumption than by cooked ham. When dry-cured ham was consumed first, a decrease in 6.1 points was found but was not significant if consumed during the second treatment (Table 6). 3.5. Fasting blood glucose In both groups, dry-cured ham intake had a hypoglycaemic effect (only significant when consumed first, P = 0.0147). Cooked ham displayed contradictory effects, as its consumption increased and decreased glucose levels in group 1 and group 2, respectively (Table 7). The wash out phase produced recovering effects in both groups. 4. Discussion Dry-cured ham is a valuable source of bioactive compounds, which can be used in nutritional therapies and prevention. From a nutritional point of view, the curing of the pork muscle products leads to a complex matrix carrying proteins, vitamins, minerals, but also salt, cholesterol and triacylglycerides that provide calories to the diet. Furthermore, the processing technology also produces biochemical changes in the raw material, by modifying their lipid profile (free fatty acids) or naturally-occurring antioxidants and biopeptides. Recently, dry-cured ham extracts and specific pure peptides naturally generated during the processing of dry-cured S. Montoro-García et al. / Journal of Functional Foods 38 (2017) 160–167 ham, and showing ACE inhibitory activity, were studied for their stability during transepithelial transport in a Caco-2 cell monolayer (Marta Gallego et al., 2016). It was then concluded that intact dry-cured ham peptides as well as their hydrolysed fragments were able to be absorbed across the intestinal epithelium and reach the blood stream to exert an antihypertensive action (Marta Gallego et al., 2016). Nonetheless, this is the first clinical study to show the potential effect of dry-cured pork ham with bioactive peptides in humans, more specifically those with a relatively high-normal BP. In contrast to earlier in vitro findings, neither SBP nor DBP changed after dry-cured or control ham intake in the present data (Chen, Luo, Zhang, & Kong, 2016; Escudero et al., 2012; Escudero et al., 2013). A possible explanation for this might be that in in vivo conditions, digestion, genetic factors, microbiome, food interactions and even a higher dietary intake of salt could confound the results and contribute towards the BP levels of this trial. The total average daily sodium intake per capita in developed countries is 10–12 g (NaCl), which is 25 times higher than the minimum adult requirement (0.5 g of NaCl) (Bibbins-Domingo et al., 2010). The present study was carried out with a dry-cured ham controlled salt content. Besides, it is important to notice that the particular meat used in the current study is healthier than other processed meats (such as bacon or sausages) and participants took only 1 g/day of salt more than those eating the cooked ham. Therefore, these data cannot be extrapolated to all cured meats. The Dietary Approaches to Stop Hypertension (DASH) trial looked into BP reduction in participants who reduced 1 g/day their sodium intake over a 30-day period, thus one month of intervention should be enough to affect, if the case, BP (Sacks et al., 2001). The second major finding is that, despite increasing their daily salt intake, arterial BP and urine sodium excretion did not significantly increase over those obtained in basal levels. To explain the absence of adverse effects, antihypertensive mechanisms other than the inhibition of ACE should be considered. Homeostatic/renal mechanisms have been also regulating the BP and sodium excretion, perhaps in collaboration with the inhibitory activity of the biopeptides (Cicero, Aubin, Azais-Braesco, & Borghi, 2013; Nilsen, Pripp, Høstmark, Haug, & Skeie, 2016). Many trials investigating the BP-effects of foods have used extracts or synthetic foods/drinks containing active ingredients that appear naturally. In this line, our study highlights that, far from being a ‘‘restricted food” for highnormal BP individuals (Paik, Wendel, & Freeman, 2005), drycured ham consumption implying a higher salt intake (up to 3.6 g/day) did not negatively affect BP or sodium excretion, which may be regarded as a clinically relevant finding. On the other hand, experiments and clinical studies have linked bioactive compounds, to support cardiovascular-related health claims. In this light, the regular consumption of the interventional product has been reported to decrease platelet and monocyte activation and the levels of plasmatic P-selectin and interleukin-6 in healthy subjects with pre-hypertension (Martínez-Sánchez et al., 2017). Those observations suggest an intake-dependent improvement in the thrombogenic and inflammatory status too. Other major risks for CVD include elevated total cholesterol and lipoproteins levels (Miller et al., 2011). Interestingly, cholesterol levels only decreased after the intake of the interventional product and did recover after the wash out period. A direct implication of the dry-cured ham can therefore be assumed. Besides, LDL levels were also negatively affected by the dry-cured ham consumption, especially when consumed first. Some studies have also shown that including dry-cured ham in the diet of older adults has beneficial effects on some atherogenic risk factors such as lipid profile (Rebollo et al., 1998). These effects were not found after the con- 165 sumption of cooked ham with distinct lipid and biopeptides composition. There is evidence suggesting that the lowering of cholesterol may be explained by biopeptides that bound to bile acids and shield them from reabsorption (Nagaoka et al., 2001), but bioactive peptides could also interact directly with the biosynthesis/absorption of lipid cholesterol (Ruiz Ruiz, Betancur Ancona, & Segura Campos, 2014). Besides, we are not aware that any RCT have looked at the effect of bioactive compounds from meat on cholesterol levels, but some studies with cheese and soy proteins are consistent with our findings (‘‘Food labeling: health claims; soy protein and coronary heart disease. Food and Drug Administration, HHS. Final rule,” 1999; Nilsen, Høstmark, Haug, & Skeie, 2015; Raziani et al., 2016). In contrast to current recommendations (Micha, Michas, & Mozaffarian, 2012), dry-cured ham consumption did not raise cholesterol in this study, which is a finding of potential clinical and nutritional importance. Recently, there is increased awareness of the possibility that lipid-lowering agents may affect glucose control and insulin resistance which could also have important implications in the prevention of diabetes or metabolic syndrome (Zafrir and Jain, 2014). Bile acids sequestrants results in changes of TGR5 activity leading to higher release glucagon-like peptides and to increased insulin sensitivity and insulin secretion (L. Chen et al., 2010). Furthermore, some studies suggest that biopeptides could serve as dipeptidyl peptidase IV (DPP-IV) and a-glucosidase substrates and act as inhibitors (Lacroix, Meng, Cheung, & Li-Chan, 2016; Nongonierma, Mooney, Shields, & FitzGerald, 2014; Roskar et al., 2015). In fact, it was recently reported that dry-cured ham contains several peptides like KA and AAATP, with IC50 values of 6.27 and 6.47 mM, respectively, were strong inhibitors of DPP-IV while other peptides like AAAAG, ALGCA and LVSGM also showed inhibitory activity although at a lower rate (M. Gallego, Aristoy, & Toldrá, 2014). Diand tripeptides have not been analysed in the meat products. However, the presence of other of them was confirmed in the interventional dry-cured ham. The clinical evidences in this field are very scarce, but milk protein-derived hydrolysates studies in rats would support this concept (Uchida, Ohshiba, & Mogami, 2011; Uenishi, Kabuki, Seto, Serizawa, & Nakajima, 2012). Accordingly, the consumption of dry-cured meat has a lipid-lowering effect and a blood glucose regulatory capacity in humans, as the present data would suggest. Nonetheless, the data for total cholesterol and glucose blood levels were inconsistent for cooked ham because it produced opposite effects when consuming first and in the second treatment, this may be in part due to the dissimilar subject́s implication at the middle/end of the intervention because of the long observational period. Although the current evidence supports the involvement of biopeptides, it is also important to mention the complex nature of both meat products and it is uncertain to what extent other constituents (free fatty acids, minerals, antioxidants) could contribute to the physiological effects here described. Therefore, despite the promising results, we consider that these data must be interpreted with caution from the clinical point of view, in order to avoid a negative educational message. Specific RCT involving hyperlipidemic and/or high-glucose volunteers —without treatment— are mandatory before this can be confirmed. 4.1. Limitations The measurement of bioactive peptides in plasma is quite difficult due to the low concentration of such peptides and their short life, so, we were unable to confirm the presence of bioactive peptides in the blood of participants. The ideal quantity of any active compound that would provide beneficial effects in humans has 166 S. Montoro-García et al. / Journal of Functional Foods 38 (2017) 160–167 not been determined; nevertheless, the study was designed to involve the highest feasible daily intake of meat. 5. Conclusions Increasing attention has been paid to the development of risk models and the use of nutritional practices to reduce for premature CVD. The present findings illustrate for the first time that far from being a restricted food, the regular consumption of Spanish drycured ham positively impair modifiable risk factors. This study suggests that dry-cured ham rich in bioactive peptides may provide extremely interesting tools for nutritional therapy on glycaemia and cholesterolemia regulation in healthy subjects. Further in vivo studies in specific pathophysiological situations are still necessary to characterize and understand the true benefits of these health-protecting active compounds. Acknowledgement The authors are grateful to ElPozo Alimentación (Murcia, Spain) for the interventional and control products. Statement of authorship SMG drafted the manuscript, performed stats and controlled the recruitment. PZ designed the nutritional survey. FC performed the recruitment and translated the survey in a statistical form. JPA performed stats. LTP helped conduct the study. FT characterized the meat products. JA designed the study. Funding sources The research reported in this article was supported by the Projects BACCHUS (FP7-KBBE-2012-6-single stage, European Commission Grant Agreement 312090). SMG was supported by the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-2013) under REA grant agreement n° 608765. Conflict of interest All authors declare no conflict of interests. References Abellán Alemán, J., Zafrilla Rentero, M. P., Montoro-García, S., Mulero, J., Pérez Garrido, A., Leal, M., et al. (2016). 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