Formation and Degradation of Beta-casomorphins in Dairy Processing

Figures & data

Figure 1. The primary structure of β-casein A2 indicating amino acid substitution of the variants (adapted from Swaigood, 2003).

Figure 2. Release of BCM7 β-casein variant (adapted from Woodford, 2009).

Table 1 BCMs released from bovine milk

Beta-casomorphins	Structure	References
BCM 4, β-CN f(60–63)	Tyr-Pro-Phe-Pro	EFSA (2009), Kamiński et al. (2007)
BCM 5, β-CN f(60–64)	Tyr-Pro-Phe-Pro-Gly	EFSA (2009), Kamiński et al. (2007)
BCM 6, β-CNf(60–65)	Tyr-Pro-Phe-Pro-Gly-Pro	Kamiński et al. (2007)
BCM 7, β-CN f(60–66)	Tyr-Pro-Phe-Pro-Gly-Pro-Ile	EFSA (2009), Kamiński et al. (2007)
BCM 8, β-CN f(60–67)	Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro	EFSA (2009), Kamiński et al. (2007)
BCM 9, β-CN f(60–68)	Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn	Saito et al. (2000)
BCM 10, β-CN f(60–69)	Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn-Ser	Toelstede and Hofmann (2008)
BCM 11, β-CN f(60–70)	Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn-Ser-Leu	Kamiński et al. (2007)

Table 2 BCMs in cheeses

Cheese	β-casomorphins	Content (μg/g)	References
Brie	BCM7	5–15	Jarmolowska et al. (1999)
		0.15	De Noni and Cattaneo (2010)
		6.48	Sienkiewicz-Szłapka et al. (2009)
	BCM5	3.14	Sienkiewicz-Szłapka et al. (2009)
Cheddar	BCM7	0.11	De Noni and Cattaneo (2010)
	BCM5	Not reported	De Noni and Cattaneo (2010)
	BCM9	Identified	Singh et al. (1997)
Gorgonzola	BCM7	0.01	De Noni and Cattaneo (2010)
	BCM5	Not reported	De Noni and Cattaneo (2010)
Fontina	BCM7	0.04	De Noni and Cattaneo (2010)
	BCM5	Not reported	De Noni and Cattaneo (2010)
Gouda	BCM7	0.06	Sienkiewicz-Szłapka et al. (2009)
		0.1	De Noni and Cattaneo (2010)
	BCM5	0.05	Sienkiewicz-Szłapka et al. (2009)
	BCM9	Identified	Saito et al. (2000); Toelstede and Hofmann (2008)
	BCM10	Identified	Toelstede and Hofmann (2008)
Rokpol	BCM7	1.66	Sienkiewicz-Szłapka et al. (2009)
	BCM5	2.57	Sienkiewicz-Szłapka et al. (2009)
Edamski	BCM7	1	Sienkiewicz-Szłapka et al. (2009)
	BCM5	0.46	Sienkiewicz-Szłapka et al. (2009)
Kasztelan	BCM7	0.04	Sienkiewicz-Szłapka et al. (2009)
	BCM5	0.14	Sienkiewicz-Szłapka et al. (2009)
Taleggio, Caprino and Grana Padano	BCM7	No detected	De Noni and Cattaneo (2010)
	BCM5	Not reported	De Noni and Cattaneo (2010)
	BCM5	Not reported	De Noni and Cattaneo (2010)

Table 3 BCMs released by different bacteria

β-casomorphins	Starter culture	Origin	References
β-casomorphin 4	A mutant of Lactobacillus helveticus	Bovine milk	Matar and Goulet (1996)
β-casomorphin 5	A mixture of Lactococcus lactis subsp. lactis and Streptococcus thermophilus^a	Bovine milk	Sienkiewicz-Szłapka et al. (2009)
β-casomorphin 7	A mixture of Streptococcus salivarius ssp. thermophilus, Lc. lactis ssp. lactis and Lc. lactis ssp. cremoris^b	Bovine milk	De Noni and Cattaneo (2010) Sienkiewicz-Szłapka et al. (2009) Jarmolowska et al. (1999)
β-casomorphin 9	A mixture of Lactococcus lactis spp. Lactis and Leuconostoc mesenteroides ssp. dextranicum	Bovine milk	Gómez-Ruiz et al. (2004)

^aStarter culture used for Gouda cheese making (Messens et al., 1999).

^bStarter culture used for Brie cheese making (Lefier et al., 2000).

Table 4 Methods for analysis of BCMs

Beta-casomorphines	Matrix	Sample preparation	Analytical technique	Limits of detection	References
BCM7	Cow milk	Cieślińska et al., 2007	ELISA	Not reported	Cieślińska et al. (2012)
BCM5 BCM7	Cheese	Extraction chloroform/ methanol, centrifugation, fat removal, and lyophilization and purification on C18T SPE cartridge	ELISA	11.10^−3 μg/mL BCM-5 13.10^−3 μg/mL BCM-7	Sienkiewicz-Sz1apka et al. (2009)
BCM7	Cheese	Extraction chloroform/methanol, gel filtration	Ion exchange chromatography	Not reported	Jarmołowska et al., (1999)
BCM3 BCM4 BCM5 BCM6 BCM7	Cheese	Extraction chloroform/ methanol, centrifugation, fat removal, and lyophilization	HPLC-UV (220, 254, 280 nm)	2 μg/mL (BCMs)	Muehlenkamp and Warthesen (1996)
BCM5 BCM7	Human colostrum, milk	Extraction methanol/chloroform followed by shaking, centrifugation, lyophilization, and purification on C18 SPE cartridge	HPLC-UV (280 nm)	0.8 μg/mL BCM-5 0.6 μg/mL BCM-7	Jarmołowska et al. (2007)
BCM7 BCM9 BCM13 BCM21	Cow milk	Enzymatic digestion Chromatographic fractionation/purification	HPLC-UV (280 nm)	Not reported	Jinsmaa and Yoshikawa (1999)
BCM4	Fermented milk	Extraction chloroform/ methanol, absorption/desorption on charcoal/evaporation	HPLC-UV (220 nm) LC-MS MS scan (200–800 m/z)	Not reported	Matar and Goulet (1996)
BCM5 BCM7	Milk-based infant formulas	milk coagulation, centrifugation, filtration and lyophilization	HPLC-MS/MS MRM transitions (m/z) BCM-5 580.3→408.2; 286.0 BCM-7 790.3→381.1; 530.2	Not reported	De Noni (2008)
BCM5 BCM7	Cheese, milk, yoghurt, infant formulas, dried milk	Parrot et al. (2003); Donkor et al. (2007)	HPLC-MS/MS MRM transitions (m/z) BCM-5 580.3→408.2; 286.0 BCM-7 790.3→381.1; 530.2	Not reported	De Noni and Cattaneo, (2010)
BCM5 BCM7 BCM10 BCM11	Cheese and milk	Extraction, fat removal, purification on polymeric SPE cartridge	HPLC-QIT-MS Q-IT in full scan and MS^n	Not reported	Juan-Garcia et al. (2009)
BCM7	Enzyme modified cheese	Centrifuge, methylene chloride and water, concentrated by Speed Vac	HPLC-API-MS	Not reported	Haileselassie et al. (1999)
BCM11	Cheese	Kuchroo and Fox (1982)	HPLC-QIT-MS operated in MS scan and MS/MS full scan	Not reported	Rizzello et al. (2005)
BCM9	Cheese	Homogenization with water following by centrifuge, dialyzation, lyophilization	HPLC-GPC Fast atom bombardment-MS	Not reported	Saito et al. (2000)
BCM9 BCM10	Cheese	Extraction with deionized water, centrifugation, paper filtration, lyophilization	HPLC TOF-MS LC-MS/MS	Not reported	Toelstede et al. (2008)
BCM9	Yoghurt	Homogenization, remove sugar and lactic acid on cation exchanger columns	LC-MS/MS	Not reported	Sabeena Farvin et al. (2010)
BCM13	Yoghurt	Extraction, trisodium citrate dehydrate and DTT, centrifuge, purification on SPE cartridge	microLC-TOF-MS	Not reported	Kunda et al. (2012)

EFSA. (2009). Review of the potential health impact of β-casomorphins and related peptides. EFSA Science Report 231:1–107, Available from http://www.efsa.europa.eu/en/efsajournal/pub/231r.htm

 Google Scholar

Kamiński, S., Cieślińska, A. and Kostyra, E. (2007). Polymorphism of bovine beta-casein and its potential effect on human health. J. Appl. Genet. 48(3):189–198.

 PubMed Web of Science ®Google Scholar

Saito, T., Nakamura, T., Kitazawa, H., Kawai, Y. and Itoh, T. (2000). Isolation and structural analysis of antihypertensive peptides that exist naturally in gouda cheese. J. Dairy Sci. 83(7):1434–1440.

 PubMed Web of Science ®Google Scholar

Toelstede, S. and Hofmann, T. (2008). Sensomics mapping and identification of the key bitter metabolites in gouda cheese. J. Agr. Food Chem. 56(8):2795–2804.

 PubMed Web of Science ®Google Scholar

De Noni, I. and Cattaneo, S. (2010). Occurrence of [beta]-casomorphins 5 and 7 in commercial dairy products and in their digests following in vitro simulated gastro-intestinal digestion. Food Chem. 119(2):560–566.

 Web of Science ®Google Scholar

Sienkiewicz-Szłapka, E., Jarmołowska, B., Krawczuk, S., Kostyra, E., Kostyra, H. and Iwan, M. (2009). Contents of agonistic and antagonistic opioid peptides in different cheese varieties. Int. Dairy J. 19(4):258–263.

 Web of Science ®Google Scholar

Singh, T. K., Fox, P. F. and Healy, A. (1997). Isolation and identification of further peptides in the diafiltration retentate of the water-soluble fraction of Cheddar cheese. J. Dairy Res. 64(03):433–443.

 PubMed Web of Science ®Google Scholar

Matar, C. and Goulet, J. (1996). β-casomorphin 4 from milk fermented by a mutant of Lactobacillus helveticus. Int. Dairy J. 6(4):383–397.

 Web of Science ®Google Scholar

Jarmolowska, B., Kostyra, E., Krawczuk, S. and Kostyra, H. (1999). Beta-casomorphin-7 isolated from Brie cheese. J. Sci. Food Agr. 79(13):1788.

 Web of Science ®Google Scholar

Gómez-Ruiz, J. Á., Ramos, M. and Recio, I. (2004). Identification and formation of angiotensin-converting enzyme-inhibitory peptides in Manchego cheese by high-performance liquid chromatography—tandem mass spectrometry. J. Chromatogr. A 1054(1–2):269–277.

 PubMed Web of Science ®Google Scholar

Messens, W., Estepar-Garcia, J., Dewettinck, K. and Huyghebaert, A. (1999). Proteolysis of high-pressure-treated Gouda cheese. Int. Dairy J. 9(11):775–782.

 Web of Science ®Google Scholar

Lefier, D., Lamprell, H. and Mazerolles, G. (2000). Evolution of Lactococcus strains during ripening in Brie cheese using Fourier transform infrared spectroscopy. Lait 80(2):247–254.

 Google Scholar

Cieślińska, A., Kaminski, S., Kostyyra, E. and Sienkiewicz-Szłapka, E. (2007). Beta-casomorphin 7 in raw and hydrolyzed milk derived from cows of alternative β-casein genotypes. Milchwissenschaft 62(2):125–127.

 Web of Science ®Google Scholar

Cieślińska, A., Kostyra, E., Kostyra, H., Oleński, K., Fiedorowicz, E. and Kamiński, S. (2012). Milk from cows of different β-casein genotypes as a source of β-casomorphin-7. Int. J. Food Sci. Nutr. 63(4):426–430.

 PubMed Web of Science ®Google Scholar

Muehlenkamp, M. R. and Warthesen, J. J. (1996). [beta]-Casomorphins: Analysis in cheese and susceptibility to proteolytic enzymes from Lactococcus lactis ssp. cremoris. J. Dairy Sci. 79(1):20–26.

 PubMed Web of Science ®Google Scholar

Jarmołowska, B., Sidor, K., Iwan, M., Bielikowicz, K., Kaczmarski, M., Kostyra, E. and Kostyra, H. (2007). Changes of β-casomorphin content in human milk during lactation. Peptides 28(10):1982–1986.

 PubMed Web of Science ®Google Scholar

Jinsmaa, Y. and Yoshikawa, M. (1999). Enzymatic release of neocasomorphin and [beta]-casomorphin from bovine [beta]-casein. Peptides 20(8):957–962.

 PubMed Web of Science ®Google Scholar

Parrot, S., Degraeve, P., Curia, C. and Martial-Gros, A. (2003). In vitro study on digestion of peptides in Emmental cheese: Analytical evaluation and influence on angiotensin I converting enzyme inhibitory peptides. Die Nahrung 47(2):87–94.

 PubMedGoogle Scholar

Donkor, O. N., Henriksson, A., Singh, T. K., Vasiljevic, T. and Shah, N. P. (2007). ACE-inhibitory activity of probiotic yoghurt. Int. Dairy J. 17(11):1321–1331.

 Web of Science ®Google Scholar

Juan-García, A., Font, G., Juan, C. and Picó, Y. (2009). Nanoelectrospray with ion-trap mass spectrometry for the determination of beta-casomorphins in derived milk products. Talanta 80(1):294–306.

 PubMed Web of Science ®Google Scholar

Haileselassie, S. S., Lee, B. H. and Gibbs, B. F. (1999). Purification and identification of potentially bioactive peptides from enzyme-modified cheese. J. Dairy Sci. 82(8):1612–1617.

 PubMed Web of Science ®Google Scholar

Kuchroo, C. N. and Fox, P. F. (1982). Soluble nitrogen in Cheddar cheese: Comparison of extraction procedures. Milchwissenschaft 37(6):331–335.

 Web of Science ®Google Scholar

Rizzello, C. G., Losito, I., Gobbetti, M., Carbonara, T., De Bari, M. D. and Zambonin, P. G. (2005). Antibacterial activities of peptides from the water-soluble extracts of Italian cheese varieties. J. Dairy Sci. 88(7):2348–2360.

 PubMed Web of Science ®Google Scholar

Sabeena Farvin, K. H., Baron, C. P., Nielsen, N. S., Otte, J. and Jacobsen, C. (2010). Antioxidant activity of yoghurt peptides: Part 2—Characterisation of peptide fractions. Food Chem. 123(4):1090–1097.

 Web of Science ®Google Scholar

Kunda, P. B., Benavente, F., Catalá-Clariana, S., Giménez, E., Barbosa, J. and Sanz-Nebot, V. (2012). Identification of bioactive peptides in a functional yogurt by micro liquid chromatography time-of-flight mass spectrometry assisted by retention time prediction. J. Chromatogr. A 1229(0):121–128.

 PubMed Web of Science ®Google Scholar