Effects of sugar- free chocolate containing s1-casein peptide on stress: a survey based on questionnaire data and salivary cortisol concentration measurement

  • Samira Yeganehzad
  • Abolfazl Pahlevanloo
  • Maryam Kiumarsi
  • Azadeh Zayerzadeh
  • Seyed-Alireza Sadjadi
  • Mostafa Shahidi
  • Narjes Nadali
Keywords: bioactive peptide, stress reduction, sugar-free chocolate, cortisol measurement, stress questionnaire


Stress reduction, plays an important role in the prevention of different disorders such as blood pressure, obesity, ulcers, nervousness, problems with memory and etc. Proper lifestyle and healthy diet habits could keep stress under control. Some preliminary researches, demonstrated the effect of food-derived bioactive peptides on stress reduction. The aim of this study was to investigate the effect of daily consumption of 12 g sugar-free chocolate containing 150 mg peptide (aS1-casein (91-100)) on stress reduction in healthy normal-weighted participants. Clinical assessments included self-rated questionnaires and saliva cortisol level, which were recorded and analyzed before and after chocolate consumption for 2 weeks. Sixty participants completed the Cattle Inventory (CI), the Beck Depression Inventory (BAI) and the Depression Anxiety Stress Scales (DASS) questionnaires. To determine the thermal and mechanical stability of the peptide during chocolate processing, FTIR and HPLC were used. Results showed that consumption of peptide containing chocolate reduced anxiety and stress scores in questionnaire and cortisol level in saliva, significantly (P<0.05). Findings also demonstrated that peptide could resist chocolate processing condition and was significantly stable (P<0.05).  


Akyuz, S., Pince, S., & Hekin, N. (1996). Children's stress during a restorative dental treatment: assessment using salivary cortisol measurements. The Journal of clinical pediatric dentistry, 20(3), 219-223.

Alsayed, S., Elnagar, K., & Mousa, E. (2014). Influence of 7 weeks self management education on the BAI and 6MWD of COPD stable patients. Egyptian Journal of Chest Diseases and Tuberculosis, 63(3), 603-609.

Alvandi A.1988. Validity and reliability of Cattle inventory for Iranians. Tehran University; 1988.

Beckett, S. T., Yorkreco, Nestec, York. (2009). Industrial Chocolate Manufacture and Use. Second edition, Springer Science+Business Media Dordrecht.

Carlson NR (1994). Physiology of Behavior. Allyn and Bacon: Massachusetts. 359–361.

Crichton, G. E., Elias, M. F., & Alkerwi, A. A. (2016). Chocolate intake is associated with better cognitive function: The Maine-Syracuse Longitudinal Study. Appetite, 100, 126-132.

Daley, R. J., Rebuck, J. A., Welage, L. S., & Rogers, F. B. (2004). Prevention of stress ulceration: current trends in critical care. Critical care medicine, 32(10), 2008-2013.

D’Hondt, M., Demaré, W., Van Dorpe, S., Wynendaele, E., Burvenich, C., Peremans, K., & De Spiegeleer, B. (2011). Dry heat stress stability evaluation of casein peptide mixture. Food chemistry, 128(1), 114-122.

Dong, A., Huang, P., & Caughey, W. S. (1990). Protein secondary structures in water from second-derivative amide I infrared spectra. Biochemistry, 29(13), 3303-3308.

Guesdon, B., Messaoudi, M., Lefranc-Millot, C., Fromentin, G., Tomé, D., & Even, P. C. (2006). A tryptic hydrolysate from bovine milk α s1-casein improves sleep in rats subjected to chronic mild stress. Peptides, 27(6), 1476-1482.

Kim, J.H., Desor, D., Kim, Y.T., Yoon, W.J., Kim, K.S., Jun, J.S., Pyun, K.H. and Shim, I. (2007). Efficacy of [alpha] s1-casein hydrolysate on stress-related symptoms in women. European Journal of Clinical Nutrition, 61(4), 536.

Kim, Y. J., Kang, S., Kim, D. H., Kim, Y. J., Kim, W. R., Kim, Y. M., & Park, S. (2017). Calorie reduction of chocolate ganache through substitution of whipped cream. Journal of Ethnic Foods, 4(1), 51-57.

Krimm, S., & Bandekar, J. (1986). Vibrational spectroscopy and conformation of peptides, polypeptides, and proteins. Advances in protein chemistry, 38, 181-364.

Kroger, M., Meister, K., & Kava, R. (2006). Low‐calorie sweeteners and other sugar substitutes: a review of the safety issues. Comprehensive reviews in food science and food safety, 5(2), 35-47.

Martin, F.P.J., Rezzi, S., Peré-Trepat, E., Kamlage, B., Collino, S., Leibold, E., Kastler, J., Rein, D., Fay, L.B. and Kochhar, S., (2009). Metabolic effects of dark chocolate consumption on energy, gut microbiota, and stress-related metabolism in free-living subjects. Journal of proteome research, 8(12), 5568-5579.

Masih, T., Dimmock, J. A., Epel, E. S., & Guelfi, K. J. (2017). Stress-induced eating and the relaxation response as a potential antidote: A review and hypothesis. Appetite, 118, 136-143.

Messaoudi, M., Lefranc-Millot, C., Desor, D., Demagny, B., & Bourdon, L. (2004). Effects of a tryptic hydrolysate from bovine milk alpha S1-casein on hemodynamic responses in healthy human volunteers facing successive mental and physical stress situations. European Journal of Nutrition, 44: 128–132.

Miclo, L., Perrin, E., Driou, A., Papadopoulos, V., Boujrad, N. & Vanderesse R. (2001). Characterization of alpha-casozepine, a tryptic peptide from bovine alpha (s1)-casein with benzodiazepine-like activity. FASEB Journal, 15: (10), 1780–1782.

Mohanty, D. P., Mohapatra, S., Misra, S., & Sahu, P. S. (2016). Milk derived bioactive peptides and their impact on human health–A review. Saudi journal of biological sciences, 23(5), 577-583.

Nejtek, V. A. (2002). High and low emotion events influence emotional stress perceptions and are associated with salivary cortisol response changes in a consecutive stress paradigm. Psychoneuroendocrinology, 27(3), 337-352.

Pacak, K., & Palkovits, M. (2001). Stressor specificity of central neuroendocrine responses: implications for stress-related disorders. Endocrine reviews, 22(4), 502-548.

Ramezanzadeh, F., Aghssa, M. M., Abedinia, N., Zayeri, F., Khanafshar, N., Shariat, M., & Jafarabadi, M. (2004). A survey of relationship between anxiety, depression and duration of infertility. BMC women's health, 4(1), 9.

Ryan, M., McEvoy, E., Duignan, S., Crowley, C., Fenelon, M., O’Callaghan, D. M., & FitzGerald, R. J. (2008). Thermal stability of soy protein isolate and hydrolysate ingredients. Food chemistry, 108(2), 503-510.

Sapolsky, R. M., Romero, L. M., & Munck, A. U. (2000). How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocrine reviews, 21(1), 55-89.

Sauer, A., & Moraru, C. I. (2012). Heat stability of micellar casein concentrates as affected by temperature and pH. Journal of dairy science, 95(11), 6339-6350.

Sheih, I. C., Fang, T. J., & Wu, T. K. (2009). Isolation and characterisation of a novel angiotensin I-converting enzyme (ACE) inhibitory peptide from the algae protein waste. Food Chemistry, 115(1), 279-284.

Susi, H., & Byler, D. M. (1986). [13] Resolution-enhanced fourier transform infrared spectroscopy of enzymes. Methods in enzymology, 130, 290-311.

Szabó, M. (2010). The short version of the Depression Anxiety Stress Scales (DASS-21): Factor structure in a young adolescent sample. Journal of adolescence, 33(1), 1-8.

Takai, N., Yamaguchi, M., Aragaki, T., Eto, K., Uchihashi, K., & Nishikawa, Y. (2004). Effect of psychological stress on the salivary cortisol and amylase levels in healthy young adults. Archives of oral biology, 49(12), 963-968.

Torres, S. J., & Nowson, C. A. (2007). Relationship between stress, eating behavior, and obesity. Nutrition, 23(11), 887-894.

Urison R (2000). Ulceration gastric. In: Fink G (ed). Encyclopedia of stress. San Diego: Academic Press, 3, 631–634.

Violle, N., Messaoudi, M., Lefranc-Millot, C., Desor, D., Nejdi, A., Demagny, B., & Schroeder, H. (2006). Ethological comparison of the effects of a bovine S1-casein tryptic hydrolysate and diazepam on the behaviour of rats in two models of anxiety. Pharmacology Biochemistry and Behavior, 84(3), 517-523.

Zhu, Y. S., & FitzGerald, R. J. (2010). Direct nanoHPLC-ESI-QTOF MS/MS analysis of tryptic caseinophosphopeptides. Food chemistry, 123(3), 753-759.

How to Cite
Yeganehzad S, Pahlevanloo A, Kiumarsi M, Zayerzadeh A, Sadjadi S-A, Shahidi M, Nadali N. Effects of sugar- free chocolate containing s1-casein peptide on stress: a survey based on questionnaire data and salivary cortisol concentration measurement. JNSD. 4(2).
Original Article(s)