Food Research International 43 (2010) 15451548

Contents lists available at ScienceDirect

Food Research International

journal homepage: www.elsevier.com/locate/foodres

Short Communication

Identication of bioactive peptides in commercial Cheddar cheese

Stephanie Rae Pritchard a, Michael Phillips b, Kasipathy Kailasapathy b,*
a b

Centre for the Plants and the Environment, University of Western Sydney, Hawkesbury Campus, Locked Bag 1797, Penrith South DC, New South Wales 1797, Australia School of Natural Sciences, University of Western Sydney, Hawkesbury Campus, Locked Bag 1797, Penrith South DC, New South Wales 1797, Australia

a r t i c l e

i n f o

a b s t r a c t
This study examined the presence of antimicrobial, antioxidant and antihypertensive peptides in three commercially available Australian Cheddar cheeses. Peptide extracts as well as fractionated peptide extracts were examined. Commercial cheese A peptides exhibited the greatest inhibition against Bacillus cereus and also commercial cheese A fractionated peptides greater than 10 kDa showed the highest inhibition against B. cereus. Commercial cheese A peptides also showed the highest inhibition of 2,2-diphenyl-1-picrylhydrazyl (DPPH), a free radical used to measure antioxidant activity. All cheese fractionated peptides greater than 10 kDa demonstrated higher inhibition of DPPH after fractionation. Antihypertensive peptides were determined by inhibition of the angiotensin-converting enzyme (ACE). Overall, commercial cheese A had the lowest concentration required to inhibit ACE and commercial cheese A fractionated peptides lower than 5 kDa had the lowest inhibition after fractionation. These preliminary ndings suggest that peptide extracts of three commercial Australian Cheddar cheeses exhibit antimicrobial, antihypertensive and antioxidant properties. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 6 December 2009 Accepted 1 March 2010

Keywords: Cheddar cheese Antimicrobial Antihypertensive Antioxidant Bioactive peptide

1. Introduction Bioactive peptides are specic protein fragments that have a positive impact on body functions or conditions and may ultimately inuence health (Kitts & Weiler, 2003). They affect numerous biological processes including evoking behavioural, neurological, hormonal, gastrointestinal and nutritional responses (Clare & Swaisgood, 2000). They can be from 2 to 20 amino acids in size and many have multifunctional properties (Korhonen, 2009b; Rutherfurd-Markwick & Moughan, 2005). Bioactive peptides have been isolated from many dairy products including cheese, ker, milk, and yoghurt. They are inactive within protein molecules and can be released in three ways by enzymatic hydrolysis by digestive enzymes predominantly pepsin, trypsin and chymotrypsin, fermentation of milk with proteolytic starter cultures or proteolysis by enzymes derived from microorganisms or plants (Korhonen, 2009a). There are few reported studies on the bioactive properties of peptides isolated from commercial Cheddar cheeses (Dionysius et al., 2000; Gupta, Mann, Kumar, & Sangwan, 2009; Haileselassie, Lee, & Gibbs, 1999; Ong & Shah, 2008). In this study, three commercially available Australian Cheddar cheeses were investigated to determine if the peptides had antimicrobial, antihypertensive and antioxidant properties. New bioactive peptides that have more potent

activity than previous reported bioactive peptides could be discovered, which could provide greater health benets to consumers. 2. Materials and methods 2.1. Extraction of cheese peptides and SDSPAGE The water-soluble peptides were extracted as per Verdini, Zorrilla, and Rubiolo (2004). Three tubes each containing 100 g of grated cheese (Australian commercial cheese A, B and C) and 300 ml distilled water was homogenised (House and Home Multimixer). The tubes were placed in a 40 C water bath, shaking at 100 rpm for 1 h and then centrifuged 4250g at 4 C for 30 min (Sigma Laboratory Centrifuge 6K15, John Morris Scientic Pty Ltd., Chatswood, Australia). The pellet was discarded. The supernatant was re-centrifuged and then ltered through No. 42 Whatman lter paper (Interpath, Heidelberg West, Victoria, Australia) and 0.2 lm syringe membrane lter (Sartorius Australia, Melbourne, Australia). The ltrate was stored at 20 C until further use. The concentration of peptide in 100 ll was determined by weighing after freeze-drying (CHRIST Alpha 14, B. Braun Biotech International) overnight. A 12.5% SDSPAGE gel was run to conrm if the peptides had been extracted as per Laemmli, 1970 (data not shown). 2.2. Separation of cheese peptides by RP-HPLC

* Corresponding author. Tel.: +61 2 4570 1653; fax: +61 2 4570 1954. E-mail address: k.kailasapathy@uws.edu.au (K. Kailasapathy). 0963-9969/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2010.03.007

Extracted peptide samples were separated by an Alltima amino C18 column (Grace Davidson Discovery Science, Deereld, USA)

1546

S.R. Pritchard et al. / Food Research International 43 (2010) 15451548

using RP-HPLC (Shimadzu Scientic, Melbourne, Australia). Solvent A was 0.1% TFA in water and solvent B was 0.1% TFA in acetonitrile (LOMB Scientic, Taren Point, NSW, Australia). For each sample, 50 ll was injected and run with a linear gradient 0.260% of solvent B (0.1% TFA in acetonitrile) to 60 min followed by 0.2% of solvent B to 71 min at a ow rate of 1 ml/min at room temperature. The peptides were fractionated by Vivaspin 20 centrifugal concentrators with molecular weight cut-off (MWCO) PES membranes of 5 kDa and 10 kDa (Sartorius Australia, Melbourne, Australia) by centrifugation, 5580g, 5 C, 30 min and ran using the Alltima amino C18 column (Grace Davidson Discovery Science, Deereld, USA) using the same program as above. For each sample, 50 ll or 100 ll was injected. The ow rate was 1 ml/min. 2.3. Determination of antimicrobial activity Three bacteria Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 6538 and Bacillus cereus ATCC 11778 were obtained from the University of Western Sydney culture collection, Hawkesbury Campus, Australia. E. coli was subcultured on Difco Luria broth (BD, North Ryde, Australia) and both S. aureus and B. cereus were subcultured on Difco Brain Heart Infusion (BD, North Ryde, Australia). All strains were incubated aerobically at 37 C for 18 h. The 0.5 McFarland standard was prepared and used to approximate the concentration of the bacteria (1.5 108 cfu/ml). Negative controls contained tetracycline solution (16.12 mg/l) and positive controls contained tryptone water (0.052 mg/l). The samples contained 70 ll of the cheese peptide extract and 130 ll bacterial suspension. All experiments were carried out in triplicate in a 96-well plate and the absorbance at 595 nm (Bio-rad spectrophotometer, Biorad, Gladesville, Australia) was read after 24 h incubation. Percentage of inhibition was determined by:

percentage of inhibition

control inhibitor sample 100 control sample without ACE

The concentration required to inhibit 50% of ACE was calculated by the concentration of the peptide in the ACE mixture times fty divided by the percentage of inhibition. 2.5. Determination of antioxidant activity The free radical 1, 1-diphenyl-2-picrylhydrazyl (DPPH) was used to determine if the three cheeses contained peptides that may exhibit antioxidant activity. Antioxidative activity of the cheese peptide extracts was determined by a modied method of Apostolidis, Kwon, and Shetty (2007). Briey, three separate ratios of 60 lM DPPH in ethanol to peptide were prepared: 3 ml:250 ll (Apostolidis et al., 2007), 1 ml:500 ll and 1 ml:1 ml. A control of MQ water and DPPH was set up for each ratio before centrifugation at 9470g (Mikro 20, Hettich Zentrifugen, Tuttlingen, Germany) for 2 min. The absorbance was read at 517 nm. The percentage of inhibition was calculated by:

Acontrol Aextract Acontrol

100

2.6. Statistical analysis The data was analysed using one-way analysis of variance (ANOVA) followed by the Tukeys test to determine signicant differences between treatment groups. The analysis was carried out using SPSS version 17.0 (SPSS Inc., Chicago, USA). 3. Results and discussion 3.1. Extraction and separation of cheese peptides 3.1.1. Average peptide concentrations All three commercial Australian Cheddar cheese peptide extracts had concentrations higher than 10 mg/ml. The commercial cheese B fraction containing peptides larger than 10 kDa was the most concentrated (59.8 mg/ml) and the peptide extract of commercial cheese A had the lowest concentration (10 mg/ml) of peptide. 3.1.2. Separation by RP-HPLC The peptide proles of the three cheese peptide extracts showed similarities. However, commercial cheese A has the highest amount of peptide peaks (196 peaks), followed by B (194 peaks), and then C (182 peaks). 3.2. Antimicrobial activity The peptide extracts were screened for the presence of antimicrobial activity by determining if the growth of three bacteria E. coli, B. cereus and S. aureus were inhibited. The three cheese peptide extracts inhibit E. coli and B. cereus when compared with the positive control containing tryptone water. The commercial cheese A peptide extract (0.245 mg/70 ll) inhibited B. cereus the most overall. E. coli was inhibited the most by commercial cheese C peptides (0.588 mg/70 ll). The results show that B. cereus (Fig. 1B) is inhibited the most by commercial cheese A peptides greater than 10 kDa (1.156 mg/ 70 ll). E. coli is also inhibited the most by commercial cheese A peptides greater than 10 kDa (Fig. 1A); however, the inhibition of S. aureus is low for all three fractionated cheese peptide extracts (Fig. 1C). The results for S. aureus had no signicant differences.

  100 Apositive control Anegative control

  Asample Anegative control

2.4. Determination of antihypertensive activity Antihypertensive activity of the cheese peptide extracts and the fractionated cheese peptides was determined by RP-HPLC using the modied method of Nakamura et al. (1995). This method uses the angiotensin-converting enzyme (ACE) (Sigma-Aldrich, Castle Hill, Australia) and the substrate HippurylHistidylLeucine (HHL) (Sigma-Aldrich, Castle Hill, Australia), which produces hippuric acid. The amount of hippuric acid produced was used to determine antihypertensive activity. 5 mM His-Hip-Leu was dissolved in 50 mM HEPES buffer (containing 0.3 M NaCl pH 8.3). 200 ll HHL solution was added to 50 ll peptide or 50 ll distilled water and incubated for 3 min at 37 C. Then 20 ll 0.1U ACE was added and incubated for 30 min at 37 C. The reaction was stopped by adding 250 ll 1 M HCl and vortexed. The mixture was ltered using 0.2 lm membrane lter with syringe. The mixture was separated using the Alltima amino C18 column with a 0.4 ml/min ow rate and isocratic program using 50% methanol with 0.1% TFA for 22 min by RP-HPLC. Pure huppuric acid was run as a standard using the same program. The height and retention time of the hippuric acid was measured using class-VP 7.3 software (Shimadzu, Melbourne, Australia) and the percentage of inhibition of ACE was determined by the height of hippuric acid peak in the control sample (ACE, HHL), in the sample containing inhibitor (ACE, HHL and peptide) and in the sample without ACE (HHL, peptide) as follows:

S.R. Pritchard et al. / Food Research International 43 (2010) 15451548

1547

A
Percentage of inhibition (%)

30 20 10 0 -10 -20 -30 A5 A10 A10+ B5 B10 B10+ C5 C10 C10+

* * *

Type of cheese peptide extract Percentage of inhibition (%)

50 40 30 20 10 0 -10 A5 A10 A10+ B5 B10 B10+ C5 C10 C10+

The fractionated cheese peptide extracts containing peptides larger than 10 kDa show the greatest inhibition of DPPH. These extracts show similar inhibition with no signicant differences observed (Fig. 2). Other research has demonstrated that Cheddar cheeses enriched with herb or fruit have antioxidant activity (Apostolidis et al., 2007) and that the extent of antioxidant activity of watersoluble Cheddar cheese extracts is dependant on the ripening stage of the cheese (Gupta et al., 2009). In comparison, these results indicate that three Australian Cheddar cheese peptides have antioxidant activity. 3.4. Antihypertensive activity

* *

Type of cheese peptide extract

Percentage of inhibition (%)

10 5 0 -5 -10 -15 -20 -25 -30 A5 A10 A10+ B5 B10 B10+ C5 C10 C10+

Type of cheese peptide extract

Fig. 1. Average percentage of inhibition of bacteria by fractionated cheese peptides. (A) E. coli, (B) B. cereus, (C) S. aureus. Data shown is the mean standard error of the mean (n = 6). Signicantly different when compared to A10+. A: commercial cheese A; B: commercial cheese B; C: commercial cheese C; 5:<5 kDa peptides; 10:<10 kDa peptides; 10+:>10 kDa peptides.

3.3. Antioxidative activity The peptide extracts as well as the fractionated peptide extracts were screened for the presence of antioxidant activity by determining if they inhibit 2,2-diphenyl-1-picrylhydrazyl (DPPH), a free radical. Three different ratios of peptide extract to DPPH were examined to determine if the percentage of inhibition increased with higher concentrations of peptide. The results indicate that the higher the concentration of peptide, the higher the inhibition of DPPH. The peptide extracts data (ratio 1 ml DPPH:1 ml peptide) had higher levels of inhibition than the fractionated peptide extracts (Fig. 2) that have a ratio of 3 ml DPPH to 250 ll peptide. Commercial cheese A peptides (5 mg/ml) by far has the greatest inhibition of DPPH, followed by commercial cheese B peptides (5.875 mg/ml) and commercial cheese C peptides (9.5 mg/ml).

Percentage inhibition of DPPH (%)

There are few studies that have examined the antimicrobial activity of Cheddar cheese. This study has shown that three Australian commercial Cheddar cheese peptides exhibit antimicrobial activities. Similarly, various Italian cheese water-soluble peptides have shown high antimicrobial activity (0.61 mg/ml peptide concentration) against various bacteria including E. coli, Bacillus megaterium, Listeria innocua, and S. aureus (Rizzello et al., 2005).

Antihypertensive activity was determined by measuring the inhibition of the angiotensin-converting enzyme (ACE). Overall, commercial cheese A appears to have the lowest concentration required to inhibit 50% ACE activity (Fig. 3). Several other investigations have revealed potential antihypertensive peptides in cheese (Btikofer, Meyer, Sieber, Walther, & Wechsler, 2008; Gmez-Ruiz, Ramos, & Recio, 2002; Haileselassie et al., 1999; Ong & Shah, 2008; Ryhnen, Pihlanto-Leppl, & Pahkala, 2001; Tonouchi, Suzuki, Uchida, & Oda, 2008). Also, several studies have shown enzyme-modied cheeses contain antihypertensive peptides (Haileselassie et al., 1999; Tonouchi et al., 2008). Fermented cheese with bacteria including Lactobacillus acidophilus LAFTI L10 has also shown ACE-inhibitory activity (Ong & Shah, 2008). Further, 10 different Swiss cheese types were shown to contain the two antihypertensive peptides VPP and IPP in varying concentrations (Btikofer et al., 2008; Meyer, Butikofer, Waither, Wechsler, & Sieber, 2009). Another study that analysed 13 commercial cheeses showed that both vintage and mature Cheddar cheese contains antihypertensive peptides as well as immunomodulatory and opioid peptides (Dionysius et al., 2000). Caseinophosphopeptides have also been isolated from cheese (Lund & Ardo, 2004; Roudot-Algaron, Le Bars, Kerhoas, Einhorn, & Gripon, 1994; Singh, Fox, & Healy, 1997) as well as anti-proliferative and antihypertensive peptides isolated from Gouda cheese (Meisel & Gnther, 1998; Saito, Nakamura, Kitazawa, Kawai, & Itoh, 2000). In conclusion, these preliminary results have shown that all three Cheddar cheese peptide extracts exhibited bioactivity. The peptides extracted from commercial cheese A, which was a vintage cheese, have shown the strongest inhibitory activity in all three bioactive properties tested. Commercial cheese A possibly was matured for the longest time; therefore, more bioactive peptides may have been generated in this time.

18 16 14 12 10 8 6 4 2 0

A5

A10 A10+ B5

B10 B10+ C5

C10 C10+

Type of cheese peptide extract

Fig. 2. Average percentage of inhibition of DPPH by fractionated cheese peptides (ratio: 3 ml DPPH:250 ll peptide). Mean data shown standard error of mean (n = 3). No signicant differences observed. A: commercial cheese A; B: commercial cheese B; C: commercial cheese C; 5:<5 kDa peptides; 10:<10 kDa peptides; 10+:>10 kDa peptides.

1548

S.R. Pritchard et al. / Food Research International 43 (2010) 15451548

0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00

Type of cheese peptide extract

Fig. 3. Average peptide concentration required to inhibit 50% ACE. Data shown is mean standard error of mean (n = 7). Indicates signicantly different (P < 0.05) compared with A10+. A: commercial cheese A; B: commercial cheese B; C: commercial cheese C; 5:<5 kDa peptides; 10:<10 kDa peptides; 10+:>10 kDa peptides.

The fractions exhibiting the strongest activity will be further fractionated and screened for bioactivity using RP-HPLC, and the screening methods above. The peptides with the strongest activity will be sequenced by LCMS and characterised using databases.

References
Apostolidis, E., Kwon, Y. I., & Shetty, K. (2007). Inhibitory potential of herb, fruit, and fungal-enriched cheese against key enzymes linked to type 2 diabetes and hypertension. Innovative Food Science & Emerging Technologies, 8, 4654. Btikofer, U., Meyer, J., Sieber, R., Walther, B., & Wechsler, D. (2008). Occurrence of the angiotensin-converting enzyme-inhibiting tripeptides Val-Pro-Pro and IlePro-Pro in different cheese varieties of Swiss origin. Journal of Dairy Science, 91, 2938. Clare, D. A., & Swaisgood, H. E. (2000). Bioactive milk peptides: A prospectus. Journal of Dairy Science, 83, 11871195. Dionysius, D. A., Marschke, R. J., Wood, A. J., Milne, J., Beattie, T. R., Jiang, H., et al. (2000). Identication of physiologically functional peptides in dairy products. The Australian Journal of Dairy Technology, 55, 103. Gmez-Ruiz, J. ., Ramos, M., & Recio, I. (2002). Angiotensin-converting enzymeinhibitory peptides in Manchego cheeses manufactured with different starter cultures. International Dairy Journal, 12, 697706. Gupta, A., Mann, B., Kumar, R., & Sangwan, R. B. (2009). Antioxidant activity of Cheddar cheeses at different stages of ripening. International Journal of Dairy Technology, 63, 339347.

Haileselassie, S. S., Lee, B. H., & Gibbs, B. F. (1999). Purication and identication of potentially bioactive peptides from enzyme-modied cheese. Journal of Dairy Science, 82, 16121617. Kitts, D. D., & Weiler, K. (2003). Bioactive proteins and peptides from food sources. Applications of bioprocesses used in isolation and recovery. Current Pharmaceutical Design, 9, 13091323. Korhonen, H. J. (2009a). Bioactive milk proteins and peptides: From science to functional applications. The Australian Journal of Dairy Technology, 64, 1625. Korhonen, H. J. (2009b). Milk-derived bioactive peptides: From science to applications. Journal of Functional Foods, 1, 177187. Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of the head bacteriophage T4. Nature, 227, 680685. Lund, M., & Ardo, Y. (2004). Purication and identication of water-soluble phosphopeptides from cheese using Fe(III) afnity chromatography and mass spectrometry. Journal of Agricultural and Food Chemistry, 52, 66166622. Meisel, H., & Gnther, S. (1998). Food proteins as precursors of peptides modulating human cell activity. Nahrung/Food, 42, 175176. Meyer, J., Butikofer, U., Waither, B., Wechsler, D., & Sieber, R. (2009). Hot topic: Changes in angiotensin-converting enzyme inhibition and concentrations of the tripeptides Val-Pro-Pro and Ile-Pro-Pro during ripening of different Swiss cheese varieties. Journal of Dairy Science, 92, 826836. Nakamura, Y., Yamamoto, N., Sakai, K., Okubo, A., Yamazaki, S., & Takano, T. (1995). Purication and characterization of angiotensin I-converting enzyme inhibitors from sour milk. Journal of Dairy Science, 78, 777783. Ong, L., & Shah, N. P. (2008). Release and identication of angiotensin-converting enzyme-inhibitory peptides as inuenced by ripening temperatures and probiotic adjuncts in Cheddar cheeses. LWT Food Science and Technology, 41, 15551566. Rizzello, C. G., Losito, I., Gobbetti, M., Carbonara, T., Debarl, M. D., & Zimbonin, P. G. (2005). Antibacterial activities of peptides from the water-soluble extracts of Italian cheese varieties. Journal of Dairy Science, 88, 23482360. Roudot-Algaron, F., Le Bars, D., Kerhoas, L., Einhorn, J., & Gripon, J. C. (1994). Phosphopeptides from Comte cheese. Nature and origin. Journal of Food Science, 59, 544547. Rutherfurd-Markwick, K. J., & Moughan, P. J. (2005). Bioactive peptides derived from food. Journal of AOAC International, 88, 955966. Ryhnen, E.-L., Pihlanto-Leppl, A., & Pahkala, E. (2001). A new type of ripened, low-fat cheese with bioactive properties. International Dairy Journal, 11, 441447. Saito, T., Nakamura, T., Kitazawa, H., Kawai, Y., & Itoh, T. (2000). Isolation and structural analysis of antihypertensive peptides that naturally exist in Gouda cheese. Journal of Dairy Science, 83, 14341440. Singh, T. K., Fox, P. F., & Healy, A. (1997). Isolation and identication of further peptides in the dialtration retentate of the water-soluble fraction of Cheddar cheese. Journal of Dairy Research, 64, 433443. Tonouchi, H., Suzuki, M., Uchida, M., & Oda, M. (2008). Antihypertensive effect of an angiotensin converting enzyme inhibitory peptide from enzyme modied cheese. Journal of Dairy Research, 75, 284290. Verdini, R. A., Zorrilla, S. E., & Rubiolo, A. C. (2004). Characterisation of a soft cheese proteolysis by RP-HPLC analysis of its nitrogenous fractions. Effect of ripening time and sampling zone. International Dairy Journal, 14, 445454.

IC50 of ACE

B10+

B5

B10

C5

C10

C10+

A5

A10

A10+