Identification of Bitter Peptides in Aged Cheddar Cheese - Journal of

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Identification of Bitter Peptides in Aged Cheddar Cheese Konstantinia Karametsi, Smaro Kokkinidou, Ian Ronningen, and Devin G. Peterson* Department of Food Science and Nutrition, University of Minnesota, 145 FScN Building, St. Paul, Minnesota 55108, United States ABSTRACT: The compounds responsible for the bitter taste of aged “sharp” Cheddar cheese were characterized. Sensoryguided fractionation techniques using gel permeation chromatography and multi-dimension semi-preparative reversed-phase high-performance liquid chromatography revealed the presence of multiple bitter compounds. The compounds with the highest perceived bitterness intensity were identified by tandem mass spectrometry de novo peptide sequencing as GPVRGPFPIIV, YQEPVLGPVRGPFPI, MPFPKYPVEP, MAPKHKEMPFPKYPVEPF, and APHGKEMPFPKYPVEPF; all originated from βcasein. Subsequent quantitative liquid chromatography−tandem mass spectrometry analysis reported that the concentrations of GPVRGPFPIIV, YQEPVLGPVRGPFPI, and MPFPKYPVEP increased during maturation by 28.7-, 3.1-, and 1.8-fold, respectively. When directly compared to young “mild” Cheddar, APHGKEMPFPKYPVEPF was reported only in the sharp Cheddar cheese, whereas the concentration of MAPKHKEMPFPKYPVEPF did not change. Further taste re-engineering sensory experiments confirmed the importance of the identified peptides to the bitterness of sharp Cheddar. The bitter intensity of the aged “sharp” Cheddar model (mild Cheddar with equivalent concentrations of the five bitter peptides in the sharp sample) was rated as not significantly different from the authentic sharp Cheddar cheese. Among the five peptides, GPVRGPFPIIV was reported to be the main contributor to the bitterness intensity of sharp Cheddar. Furthermore, a difference from control sensory test also confirmed the significance of the bitter taste to the overall perception of aged Cheddar flavor. The sharp Cheddar model was reported to be significantly more similar to aged “sharp” Cheddar in comparison to the young “mild” Cheddar cheese sample. KEYWORDS: Cheddar cheese, aged, bitterness, peptides

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desirable flavor of mature Cheddar,12 excessive bitterness is generally regarded as a defect2,13 and may limit consumer acceptance. The development of bitterness during the maturation of Cheddar cheese is considered to be the result of the accumulation of hydrophobic peptides that consist of 2−23 amino acid residues in the molecular mass range of 500−3000 Da.2,12,13 The isolation and characterization of the compounds that are responsible for bitterness in foodstuffs have been the subject of numerous studies over the years. Gouda has been analyzed by sensory-guided fractionation techniques, and 12 peptides were found to contribute to bitterness8,9 as well as in whey protein and whey protein hydrolysates14,15 and various kinds of cheeses.3−5 Very limited information is available about the taste active compounds in Cheddar cheese. Broadbent et al.13 used two peptides [β-casein (β-CN) (f193−f209) and α(S1)-casein (α(S1)-CN) (f1−f9)] as indicators of excessive bitter development in Cheddar and model systems. However these peptides, or their breakdown products, have not been adequately characterized for their contribution to bitterness perception limiting the application of these data to better understand Cheddar flavor. The goal of this study was therefore to identify bitter peptides in Cheddar cheese and characterize their sensory contribution to improve our understanding of Cheddar flavor.

INTRODUCTION The chemical stimuli that invoke food flavor can be simplistically defined as a combination of aroma, taste, and chemesthetic compounds. Aroma compounds are volatile and perceived by the olfactory system, while taste compounds can be volatile or non-volatile and are detected by the gustatory system. Chemesthetic compounds are irritants of the pain nerves in the oral cavity. Historically, researchers have looked at food flavor as predominately being influenced by volatile aroma compounds, with minor contributions from taste. This assumption has turned out to be an oversimplification. Understanding the flavor compounds that contribute to the desirable flavor properties of foods provides a basis to develop manufacturing strategies to improve the acceptability of healthier options, such as reduced fat or salt. Taste stimuli have been recognized as key aspects of the flavor properties of cheese.1 A number of researchers have characterized the volatile flavor compounds from Cheddar cheese, resulting in an extensive list including a wide variety of compounds, such as acids, alcohols, esters, aldehydes, ketones, sulfur-containing compounds, and phenolics. On the contrary, only limited information is available on the characterization of the taste of most cheese varieties,1−10 and none is characterized sufficiently to permit duplication of its complete flavor by mixtures of pure compounds.11 The degree of ripening of Cheddar cheese is known to affect the development of all aspects of flavor (taste, aroma, and texture). With regard to taste, over time, characteristic bitterness develops that has been associated with hydrophobic peptides. Although some bitterness is considered a normal component of Cheddar cheese taste and contributes to the © 2014 American Chemical Society

Received: Revised: Accepted: Published: 8034

April 30, 2014 July 10, 2014 July 14, 2014 July 30, 2014 dx.doi.org/10.1021/jf5020654 | J. Agric. Food Chem. 2014, 62, 8034−8041

Journal of Agricultural and Food Chemistry

Article

graphic separation was performed using isocratic elution, with a mobile phase comprising of 1:1 (50/50%) water and methanol and a flow rate of 5 mL/min. The effluent was collected into nine fractions, and the corresponding fractions (termed 1−9) obtained from several runs were pooled and liberated from solvent (freeze-dried 2×). The resulting residues obtained were then dissolved in water (10 mL) and evaluated for bitterness intensity by sensory analysis. First-Dimensional (1D) HPLC Fractionation. Bitter fractions 2, 6, and 8 identified after the sensory screening of GPC isolates were selected and underwent further separation using a HPLC system coupled with a semi-preparative diphenyl column (250 × 10 mm, pursuit 5, Varian, Palo Alto, CA). The mobile phase comprised of water with 0.1% formic acid at pH 2.5 (A) and methanol (B) at a flow rate of 3 mL/min. The employed linear gradient was as follows: starting at 10% B in A (0−15 min), increasing to 20% B in A (15−25 min), increasing to 70% for 5 min (25−35 min), increasing to 100%, held for 5 min (35−40 min), and back to initial conditions. Total subfractions collected for fraction 2 were 16 (2.1−16); total subfractions collected for fraction 6 were 15 (6.1−15); and total subfractions collected for fraction 8 were 14 (8.1−14). All subfractions were liberated from solvent (freeze-dried 2×) and dissolved in distilled water (10 mL) for subsequent sensory analysis. Second-Dimensional (2D) HPLC Fractionation. Bitter fractions 2.9, 2.12, and 2.13 were selected (had the highest perceived bitterness intensity from first-dimension HPLC fractionation) and were subjected to additional chromatography using a reverse-phase C18 column (250 × 10 mm, pursuit 5, Varian, Palo Alto, CA). The mobile phase was monitored at 220 nm and consisted of a series of linear gradients of solvent A (water with 0.1% formic acid) and solvent B (methanol) starting at 10% B (0−15 min), increasing to 20% B (15− 25 min), increasing to 100% B (25−35 min), held for 5 min, and back to initial conditions. Total subfractions collected for 2.9 were 14 (2.9.1−2.9.14); total subfractions collected for 2.12 were 14 (2.12.1− 2.12.14); and total subfractions collected for 2.13 were 14 (2.13.1− 2.13.14). All subfractions were liberated from solvent (freeze-dried 2×) and dissolved in distilled water (10 mL) for subsequent sensory analysis. Liquid Chromatography−Mass Spectrometry/Quadrupole Time-of-Flight−Electrospray Ionization (LC−MS/Q-TOF−ESI) (De Novo Peptide Sequencing). Peptide sequences were analyzed using an Acquity ultra-performance liquid chromatography (UPLC) system coupled with a Waters Q-TOF G2 mass spectrometer equipped with an ESI probe, run in positive mode. The UPLC system consisted of a degasser, binary pumping system, autosampler, column heater, and a reverse-phase C18 column (XBridge RP18 column, 2.5 μm, 50 × 2.1 mm). Mass spectrometric ionization conditions were as follows: desolvation temperature, 400 °C; source temperature, 120 °C; and capillary voltage, 3 kV. For scan mode, the range of m/z was 100−2300, and for daughter ion analysis, a collision energy ramp was used, ramping from 40 to 120 eV. Purification of Synthetic Peptides. Purchased synthetic peptides were purified prior to use for sensory analysis. The individual peptides were dissolved in water and purified by preparative HPLC. The preparative HPLC system (Shimadzu, Baltimore, MD) consisted of a binary solvent delivery system (LC-8A), a manual injector, and a variable-wavelength UV−vis detector (SPD-10A) coupled with a preparative reverse-phase C18 column (250 × 21.2 mm, pursuit 5, Varian, Palo Alto, CA). The mobile phase was monitored at 220 nm and consisted of a series of linear gradients of solvent A (water) and solvent B (methanol), starting at 10% B (0−5 min), increasing to 95% B (5−25 min), increasing to 100% B (25−26 min), held for 5 min, and back to initial conditions. The purified peptides were freeze-dried twice to remove all traces of solvent prior to use. Quantification of Peptides in Mild and Sharp Cheddar Cheese. Analysis was performed on mild and sharp Cheddar cheese. The cheese sample (30 g) was combined with water (120 mL), and leucine enkephalin was added (5 mg/kg of cheese) as the internal standard (IS). The mixture was homogenized for 5 min and then centrifuged at 10000g for 20 min. The supernatant was collected, and the pH was adjusted by formic acid addition (pH ≤ 4.6). The sample

The chemical nature of the identified taste compounds will provide insight as to whether the flavor compounds are produced during processing or during aging and will afford a basis to better understand flavor generation (precursors involved) and, thus, facilitate the development of strategies for control in related products for flavor improvement.

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MATERIALS AND METHODS

Samples. Sharp and mild Cheddar cheese samples were obtained from a local grocery store (Archer Farms, Target, Minneapolis, MN) and stored at −20 °C prior to analysis. Sharp Cheddar is representative of an aged cheese of 9−12 months, whereas mild Cheddar is representative of a young cheese of 2−3 months. Chemicals. Trifluoroacetic acid, formic acid, and high-performance liquid chromatography (HPLC)-grade solvents were obtained from Sigma-Aldrich Co. Synthetic peptides YQEPVLGPVRGPFP, GPVRGPFPIIV, MAPKHKEMPFPKYPVEPF, QEPVLGPVREGPFP, APKHKEMPFPKYPVEPF, YQEPVLGPVRGPFPI, EPVLGPVRGPFP, QEPVLGPVRGPFPI, EMPFPKYPVEPF, LGPVRGPFP, PVLGPVRGPFP, and MPFPKYPVEPF were obtained from EZBiolab, Inc. at a purity of 98%. Preparation of the Methanol-Soluble Extract from Cheddar Cheese. A total of 60 g of Cheddar cheese and 240 mL of water were homogenized for 5 min and, subsequently, centrifuged at 4000g for 20 min at 4 °C. The protein pellet as well as the fat layer were collected and re-extracted with 240 mL of methanol (overall analytical scheme is shown in Figure 1). The methanol layer was removed, filtered, and

Figure 1. Sample preparation scheme and analytical techniques employed for identification of bitter peptides from Cheddar cheese. concentrated using a rotary evaporator. The resulting residue was subsequently diluted in water and freeze-dried twice to obtain the methanol-soluble extract (MSE), which was stored at −20 °C prior to further analysis. Gel Permeation Chromatography (GPC) Fractionation. A total of 1 g of MSE was dissolved in 10 mL of nanopure water/ methanol (1:1) and separated by size-extrusion gel permeation chromatography (100 × 2.6 cm). The GPC system consisted of a binary solvent delivery system (Shimadzu, LC-8A), a manual injector (SIL-10vp), a variable-wavelength ultraviolet−visible (UV−vis) detector (Shimadzu, SPD-10A), and a Sephadex G-15. Chromato8035

dx.doi.org/10.1021/jf5020654 | J. Agric. Food Chem. 2014, 62, 8034−8041


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was recentrifuged at 10000g for 20 min, and the supernatant was again collected, pooled, and filtered through a paper filter (Whatman No. 4, Whatman, Ltd., Pittsburgh, PA). The filtrate was then concentrated and desalinized by solid-phase extraction prior to quantification. Briefly, 6 mL of extract was loaded onto a pre-conditioned C-18 SPE cartridge (500 mg of DSC-18 Supelco). The cartridge was then washed with 4 mL of a 98% water, 2% acetonitrile, and 0.1% formic acid solution to remove salt, and the peptides were then eluted using 1 mL of a 40% water, 60% acetonitrile, and 0.1% formic acid solution. Recovery studies were also performed to test the efficiency of the extraction protocol. Target peptides (YQEPVLGPVRGPFPI, GPVRGPFPIIV, MPFPKYPVEPF, PVLGPVRGPFP, MAPKHKEMPFPKYPVEPF, and APHGKEMPFPKYPVEPF) were added in cheese at appropriate concentrations along with water and an IS solution prior to homogenization, and the same extraction procedure was followed. Recovery extractions were performed in triplicate for each sample. For quantification, the mass spectrometer was operated in positiveion mode and the spectra were collected by multiple reaction monitoring (MRM). Six-point calibration curves (0.5−200 mg/L) were constructed from corresponding ion transitions of each peptide. Leucine enkephalin was used as an IS and monitored at an ion transition of 556 → 136. Quantitative analysis was performed in triplicate. MRM analysis was performed with Waters Acquity UPLC (column oven, binary solvent manager, sample manager, and a RPC18 Acquity UPLC HSS T3 1.8 μm, 2.1 × 100 mm column) coupled with Waters triple-quadruple mass spectrometer (Quattro Premier XE) equipped with an ESI probe. Mass spectrometric ionization conditions were as follows: desolvation temperature, 350 °C; source temperature, 120 °C; and capillary voltage, 3 kV. MRM ion transitions were optimized for each peptide and were as follows: YQEPVLGPVRGPFPI, 835.10 → 136.04; GPVRGPFPIIV, 577.12 → 462.04; MPFPKYPVEPF, 677.05 → 263.31; PVLGPVRGPFP, 569.10 → 169.04; MAPKHKEMPFPKYPVEPF, 726.06 → 263.32; and APHGKEMPFPKYPVEPF, 658.72 → 263.32. Precaution Taken for Sensory Analysis of Food Fractions and Taste Compounds. To remove solvent and buffer compound traces from all fractions isolated from Maillard−catechin reaction models and cocoa, prior to sensory testing, volatiles in each fraction were removed by rotary evaporation and, subsequently, freeze-dried twice. Gas chromatography/mass spectrometry (GC/MS), proton nuclear magnetic resonance (1H NMR), and high-performance ion chromatography revealed that fractions treated by the above protocol are free of solvents and buffer compounds and suitable for sensory analysis. Sensory Analysis: Bitterness Intensity of Chromatographic Fractions (GPC, 1D HPLC, and 2D HPLC). Panelists (five healthy adults, with no history of tasting problems) evaluated the bitterness of the fractions. Bitterness was rated on a 15-point line scale. Four references were provided to panelists [0.03, 0.08, and 0.15% (w/v) caffeine in water], corresponding to bitterness intensity ratings of 2, 5, and 10, respectively. Panelists were asked to avoid eating or drinking 2 h before sensory evaluation. Samples were presented in a randomized order at room temperature in coded 1 oz cups. Sensory Analysis: Cheese Samples and Taste Recombination Models. A trained sensory panel consisting of eight panelists (ages 22−34) evaluated the bitterness intensity of mild and sharp commercial Cheddar cheese samples as well as a recombination “sharp Cheddar model” consisting of mild Cheddar with added purified peptides at levels quantified in sharp Cheddar cheese. Cheddar cheese was weighted, cubed, and added to a food processor. The food processor was pulsed until cheese pieces were pea-sized. Water (control samples) or peptide solution was added at 1 mL/29 g of cheese, to make a 30 g sample. Cheese and water were mixed until the sample was homogeneous and smooth. A total of 5 g (±0.2) of this cheese was weighed and rolled into a ball, put into a coded sample cup, and covered immediately. All evaluations were conducted with nose clips. Panelists were trained to evaluate bitter taste intensity using caffeine solutions as references. Two reference levels were provided to panelists [0.05 and 0.08% (w/w) of caffeine in water], corresponding to bitterness intensity ratings of 2 and 5,

respectively. The performance of a panelist was evaluated by analysis of variation (ANOVA, Statistix 9.0, Analytical Software, Tallahassee, FL). The bitterness and astringency intensity of each individual peptide and a peptide mixture were also evaluated at the corresponding concentrations in water solutions. Sensory Analysis: Difference from the Control. Eight experienced sensory panelists, who regularly consumed Cheddar cheese, were asked to evaluate the size of the overall sensory difference between the commercial sharp Cheddar (control) compared to the sharp model and mild Cheddar cheese. The samples were evaluated in duplicate on a 15-point line scale anchored with “no difference” and “extremely different”. The three sample evaluated were (1) the recombination “sharp Cheddar model” consisting of mild Cheddar with added purified peptides at levels quantified in sharp Cheddar cheese, (2) mild Cheddar cheese, and (3) blind control (sharp Cheddar). A Dunnett’s test for multiple comparisons with a control was applied to the sample means. Statistix 9.0 (Analytical Software, Tallahassee, FL) was used for analysis.

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RESULTS AND DISCUSSION A number of extraction protocols have been developed for the analysis of tastants and more specifically for extraction of bitter peptides from cheese matrixes,1,6,8,9 with water being the most common solvent used. In the current study, the initial watersoluble extract obtained from sharp Cheddar cheese was excessively salty, thus masking the bitter taste attributes. Hence, an alternative extraction method using methanol was developed and implemented (Figure 1) to facilitate initial sensory screening and determine the bitterness profile of mild and sharp Cheddar cheese samples. The MSE of Cheddar cheese was freeze-dried twice, redissolved at 10% (w/v), and then evaluated for bitterness by trained panelists. The mean bitterness rating of the MSE was 6 (slightly higher than a 0.08% caffeine solution). The MSE was subsequently separated by GPC (see Figure 2). Nine distinct

Figure 2. GPC chromatogram of Cheddar cheese isolate and the taste intensity chromatogram. Nine fractions were collected (shown in brackets); the fractions with the highest perceived bitter intensity (2, 6, and 8) are illustrated and selected for further investigation.

fractions were obtained and evaluated by a sensory panel. Fractions 2, 6, and 8 were identified as bitter, with average intensity ratings of 6, 2.5, and 1.5 and yields of 8, 12.5, and 15.8%, respectively (Figure 2). On the basis of the intensity of bitterness ratings of these fractions, fraction 2 was selected for further investigation. 8036



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Figure 3. HPLC second-dimension chromatogram of fraction 2 obtained from GPC. A total of 16 subfractions of fraction 2 were collected (shown in parentheses); the fractions with the highest perceived bitter intensity (2.9, 2.12, and 2.13) are illustrated and selected for further investigation.

Figure 4. Third-dimension HPLC chromatogram of fraction 2.12 obtained from HPLC second-dimension chromatogram. A total of 14 subfractions of fraction 2.12 were collected (shown in parentheses); the fractions with the highest perceived bitter intensity (2.12.4, 2.12.6, and 2.12.10) are illustrated and selected for further investigation.

To decrease the complexity of the GPC isolated bitter-tasting fractions and facilitate identification of the underlying bitter compounds, fraction 2 was further separated using a HPLC system coupled with a diphenyl preparative column in 16 subfractions (see Figure 3). Six of the isolates (2.4, 2.9, 2.11, 2.12, 2.13, and 2.14) were identified as bitter. The fractions with the highest bitter intensity were 2.9, 2.12, and 2.13, corresponding to mean ratings of 4.5, 4, and 6. The remaining fractions (2.14, 2.11, and 2.4) were rated at 0.5 or threshold bitterness and, thus, were not selected for further investigation.

The three main bitter subfractions (2.9, 2.12, and 2.13) were subsequently analyzed by LC−MS, which revealed that more than one predominate peptide was present in each of the selected fractions. Consequently, the fractions were further purified by HPLC using a C-18 semi-preparative column. From this second HPLC separation, 16 fractions were obtained from fraction 2.9 (2.9.1−16) and 2 fractions were reported to be bitter (2.9.9 and 2.9.12). Similarly, 13 fractions were obtained from fraction 2.12 (2.12.1−13), and 12 fractions were obtained from 2.13 (2.13.1−12). Of those, 5 fractions (2.12.4, 2.12.5, 8037



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Figure 5. MS/MS ESI+ spectra for de novo sequence determination of bitter peptide GPVRGPFPIIV.

Figure 6. β-CN(91−120) and β-CN(181−209) sequences of bovine β-CN and assignment of bitter peptides (black lines) identified in sharp Cheddar cheese.

MPFPKYPVEP, MAPKHKEMPFPKYPVEPF, and APHGKEMPFPKYPVEPF were bitter and, thus, were selected for further examination and taste recombination experiments to determine their overall sensory impact in Cheddar cheese. The five identified bitter peptides are hydrolysis products derived from the β-CN(91−120) and β-CN(181−209) sequence regions of bovine β-CN. Peptides YQEPVLGPVRGPFPI and GPVRGPFPIIV correspond to the following β-CN sequences, β-CN(193−207) and β-CN(199−209), and they are similar to bitter peptides previously identified in Gouda cheese.8,9 The formation of β-CN(193−209) can be assigned to the hydrolytic action of chymosin that is responsible for the initial hydrolyisis of β-CN, yielding three peptides, β-I, β-II, and β-III, produced by cleavage of bonds 192−193 and/or 189−190, 163−164, and/or 165−166, and/or 167−168 and 139−140, respectively.16−18 In addition to chymosin action, which mainly takes place during the initial stages of cheesemaking, the generation of β-CN(193−209) can be attributed to the hydrolytic action of the cell envelope proteinase (CEP) of Lactobacillus sp. because Leu192−Tyr193 is a known cleavage site of CEP in Cheddar cheese.19 This peptide [β-CN(193−209)] has been

2.12.6, 2.12.9, and 2.12.10) and 4 fractions (2.13.3, 2.13.4, 2.13.8, and 2.13.9) were found to be bitter, respectively. An example chromatogram of fraction 2.12 is presented in Figure 4. The bitter intensity of the collective resultant fractions ranged from 4 to 0.5, with the highest ratings reported for 2.12.4, 2.12.6, 2.12.10, and 2.13.4. These four fractions were subsequently analyzed by de novo peptide sequencing techniques by time-of-flight tandem mass spectrometry (TOF MS/MS). A total of 12 main peptide candidates were identified as YQEPVLGPVRGPFP, GPVRGPFPIIV, MAPKHKEMPFPKYPVEPF, QEPVLGPVREGPFP, APKHKEMPFPKYPVEPF, YQEPVLGPVRGPFPI, EPVLGPVRGPFP, QEPVLGPVRGPFPI, EMPFPKYPVEPF, LGPVRGPFP, PVLGPVRGPFP, and MPFPKYPVEPF. An example of MS/MS ESI+ spectra of peptide GPVRGPFPIIV is shown in Figure 5. The sequence alignment of the above peptides identified in this study revealed that they originate from β-CN, as showed in Figure 6, which is in agreement with previous studies.4,5,12 Initial sensory screening of synthetic purified peptides revealed that GPVRGPFPIIV, YQEPVLGPVRGPFPI, 8038



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Table 1. Mean Percent Recovery of Identified Bitter Peptides When Using a Water Extraction Protocol, for Both Mild and Sharp Cheddar Cheese Samplesa

a

peptide

mild Cheddar (percent recovery ± error)

MPFPKYPVEPF GPVRGPFPIIV MAPKHKEMPFPKYPVEPF APHGKEMPFPKYPVEPF YQEPVLGPVRGPFPI

55.1 ± 7.4 60.1 ± 8.1 63.1 ± 7.0 ND 67.1 ± 9.4

sharp Cheddar (percent recovery ± error) 56.2 49.1 63.6 70.4 70.6

± ± ± ± ±

6.1 9.6 8.0 8.1 9.5

Analysis conducted in triplicate; mean recovery ± standard error.

Table 2. Mean Concentration of Select Bitter Peptides in Mild and Sharp Cheddar Cheesea

a

peptide

mild Cheddar (concentration ± error) (mg/kg)


38.6 ± 2.9 5.1 ± 0.4 37.9 ± 2.7 ND 1.9 ± 0.2

sharp Cheddar (concentration ± error) (mg/kg) 69.8 146.4 33.8 1.7 5.8

± ± ± ± ±

4.3 14.1 2.7 0.1 0.6

Analysis conducted in triplicate; mean recovery ± standard error.

GPVRGPFPIIV, YQEPVLGPVRGPFPI, and MPFPKYPVEP increased during maturation by 28.7-, 3.1-, and 1.8-fold, respectively. Additionally, peptide APHGKEMPFPKYPVEPF was only found in quantifiable concentrations in the sharp Cheddar cheese sample, confirming that it was generated during the maturation process. The concentration of peptide MAPKHKEMPFPKYPVEPF was not statistically different between the two samples, indicating that maturation is not responsible for the generation of this bitter compound (Table 2). To correlate analytical results with sensory impact of identified bitter peptides, taste re-engineering experiments were performed. A model sharp Cheddar was made by spiking appropriate concentrations of the identified peptides in mild Cheddar cheese, and a descriptive analysis study of the bitterness intensity of the samples was conducted. The results confirmed that the perceived bitterness of the sharp Cheddar recombination model was not significantly different [least significant difference (LSD) all-pairwise comparison of 0.46 at 5% significance level] when compared to a commercial sharp Cheddar sample with mean ratings of 1.89 and 1.58, respectively (Table 3). For comparison, the bitterness of the

previously classified as extremely bitter, and studies have shown that fragments within that sequence exert significant bitterness as well. For example, fragment β-CN(202−209) has been reported to have a low bitterness threshold. Our findings support the contribution of the β-CN(193−209) region to perceived bitterness in aged Cheddar cheese. The formation of peptides MAPKHKEMPFPKYPVEPF [β-CN(102−119)] and APHGKEMPFPKYPVEPF [β-CN(103−119)] can likewise be attributed to chymosin action as well as the CEP of Lactobacillus sp. because Ala101−Met102 is a known cleavage site.19 Moreover, peptide MPFPKYPVEPF [β-CN(109−119)] has also been previously identified in Gouda cheese,20 and its formation can be attributed to plasmin, because Lys108−Gly109 has been identified as a cleavage site of this enzyme.21 For quantification, LC−MS/MS−ESI methods were developed for each peptide and ion transitions, and optimized conditions of MRM methods were determined. Six-point calibration curves for each peptide were constructed, exhibiting good linearity within a concentration range of 0.5−200 mg/kg and regression coefficients ranging from 0.960 to 0.999. On the basis of previous reports,6,22 water extracts have typically been used for isolation and identification of taste components from Cheddar cheese. Thus, an aqueous extraction protocol was developed, and its efficiency was evaluated by performing recovery experiments. Results obtained from peptide spiking experiments and subsequent water extraction demonstrated that mean recovery was between 49.1 and 70.6% (±6.1−9.6%) depending upon the peptide (Table 1), suggesting that water extraction is not a quantitative method of extraction and can lead to underestimation of the levels of peptides present in cheese samples if percent recovery is not considered with the quantification method. Such an underestimation can lead to inaccurate construction of recombination models as well as incorrect conclusions regarding the sensory impact of identified taste active compounds. The concentrations of the five bitter peptides were determined for both mild and sharp Cheddar cheese and are shown in Table 2. Overall results correlated well with the observed taste profile differences between mild and sharp Cheddar cheese (Table 2). Sharp Cheddar was rated as significantly higher in bitterness compared to mild Cheddar. That was depicted in the concentration of bitter peptides, as

Table 3. Mean Bitterness Intensity of Sharp and Mild Cheddar Cheese and a Sharp Cheddar Model Cheesea sample

mean rating

sharp Cheddar (6 months) sharp Cheddar model mild Cheddar (3 months)

1.89 a 1.58 a 1.07 b

a

Eight panelists in duplicate. Samples with different letters within a column are significantly different at p < 0.05.

mild Cheddar was reported to be significantly lower than the sharp Cheddar as expected and indicated the importance of aging for the development of this taste attribute. To further evaluate the contribution of the individual peptides to the overall bitterness perception, the bitterness intensity of each peptide as well as a peptide mixture at levels found in sharp Cheddar were determined in water solutions (Table 4). Overall, each of the five peptides was reported to be above the bitter recognition taste threshold values. Peptide 8039



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Table 4. Mean Bitterness Intensity of Each Peptide and the Peptide Mixture Dissolved in Water at Levels Quantified in Sharp Cheddar Cheesea peptide

a

0.5 4.5 0.9 0.3 0.5

± ± ± ± ±

*Telephone: 612-624-3201. Fax: 612-625-5272. E-mail: dgp@ umn.edu.

0.13 0.61 0.20 0.23 0.15

Funding

This project was partially supported by the Dairy Research Institute, USA. Notes

The authors declare no competing financial interest.

Five panelists in duplicate; mean ± standard error.

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GPVRGPFPIIV had the highest contribution to the bitter taste profile of sharp Cheddar cheese, with a mean bitter intensity rating of 4.5. Furthermore, the peptide mixture had a bitterness rating of 5.4 and accounted for 90% of the bitterness intensity of the initial methanol extract of sharp Cheddar. These findings support the relevance of the identified and characterized bitter peptides in the sharp Cheddar sample. This peptide (GPVRGPFPIIV) also provides a more targeted region to monitor bitterness development in Cheddar than the prior used sequences [β-CN (f193−f209) and α(S1)-CN (f1−f9)].13 The role of the identified bitter peptides on the overall flavor profile of the sharp Cheddar cheese was also examined using a difference from the control test (see Table 5). The flavor of the

sample

mean rating

difference from the control

3.0 6.2 9.4

3.3b 6.5b

REFERENCES

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Table 5. Difference from the Control Test Indicating the Overall Sensory Difference between the Commercial Sharp Cheddar (Control) Compared to the Sharp Cheddar Model (Mild Cheddar Spiked with Appropriate Levels of Bitter Peptides as Sharp Cheese) and Mild Cheddar Cheesea sharp Cheddar (blind control) sharp Cheddar model mild Cheddar

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bitterness rating sharp


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a

Eight panelist in duplicate rated the difference from the control using a 15-point line scale. Data were analyzed by ANOVA, followed by Dunnett’s multiple comparisons to the control. bSignificance at p < 0.05.

re-engineered sharp Cheddar cheese model (mild Cheddar cheese spiked with appropriate levels of bitter peptides) was reported to be significantly more similar to commercial sharp Cheddar than it was to mild Cheddar. This observation also indicated the importance of taste and, in this case, bitterness to recognize the characteristic flavor of an aged Cheddar cheese. Interestingly, the peptides identified in the current study, such as GPVRGPFPIIV, YQEPVLGPVRGPFPI, and MPFPKYPVEPF, have been previously reported to have positive health bioactivities. Peptides GPVRGPFPIIV,23,24 MPFPKYPVEPF, 20 and YQEPVLGPVRGPFPI25,26 were found to have antihypertensive actions, with the later also exhibiting antimicrobial activity. This observation is another example of the interplay between bitter taste and beneficial bioactivity. In summary, bitter peptides in Cheddar were identified and related to the flavor attributes of an aged product. Understanding the molecular drivers of flavor quality provides a basis to develop improved processing technology for flavor optimization. 8040



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(18) Visser, S.; Slangen, K. J. On the specificity of chymosin (rennin) in its action on bovine β-casein. Neth. Milk Dairy J. 1977, No. 31, 16− 30. (19) Sighn, T. K.; Fox, P.; Healy, I. Isolation and identification of further peptides in the diafiltration retentate of the water-soluble fraction of Cheddar cheese. J. Dairy Res. 1997, No. 64, 433−443. (20) Saito, T.; et al. Isolation and structural analysis of antihypertensive peptides that exist naturally in Gouda cheese. J. Dairy Sci. 2000, 83 (7), 1434−1440. (21) Gallagher, D. P.; Singh, T. K.; Mulvihill, D. M. Hydrolysis of porcine β-casein by bovine plasmin and bovine chymosin. Z. Lebensm.Unters. -Forsch. A 1999, 208 (2), 83−89. (22) Yang, B. I. N.; Vickers, Z. Taste components of Cheddar cheese: Fractionation and optimization of Cheddar cheese taste in water. J. Sens. Stud. 2004, 19 (6), 546−559. (23) Contreras, M. M.; et al. Novel casein-derived peptides with antihypertensive activity. Int. Dairy J. 2009, 19 (10), 566−573. (24) Hayes, M.; et al. Casein fermentate of Lactobacillus animalis DPC6134 contains a range of novel propeptide angiotensin-converting enzyme inhibitors. Appl. Environ. Microbiol. 2007, 73, 4658−4667. (25) Birkemo, G. A.; et al. Antimicrobial activity of two peptides casecidin 15 and 17, found naturally in bovine colostrum. J. Appl. Microbiol. 2009, 106 (1), 233−240. (26) Torres-Llanez, M. J.; et al. Angiotensin-converting enzyme inhibitory activity in Mexican Fresco cheese. J. Dairy Sci. 2011, 94 (8), 3794−3800.

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Identification of Bitter Peptides in Aged Cheddar Cheese - Journal of

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