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Molecules 2018, 23(2), 357; doi:10.3390/molecules23020357

Article
Development of Volatile Compounds during Hydrolysis of Porcine Hemoglobin with Papain
Kathrine Holmgaard Bak 1,*Orcid, Mikael Agerlin Petersen 1Orcid, René Lametsch 1, Erik T. Hansen 2 and Jorge Ruiz-Carrascal 1Orcid
1
Department of Food Science, University of Copenhagen, Rolighedsvej 26, 1958 Frederiksberg C, Denmark
2
Danish Crown Ingredients, Flæsketorvet 41, 1711 Copenhagen V, Denmark
*
Correspondence: khb@food.ku.dk; Tel.: +45-35-334191
Received: 9 January 2018 / Accepted: 1 February 2018 / Published: 8 February 2018

Abstract

:
There is a growing market for the use of hydrolysates from animal side-streams for production of high-protein supplements. However, there can be issues with development of off-flavors, either due to the raw material in question or due to the hydrolysis process itself. This study examined the development of volatile compounds during hydrolysis of hemoglobin. Briefly, porcine hemoglobin was hydrolyzed by 0.5% papain for up to 5 h, and the development of volatile compounds was analyzed via gas chromatography-mass spectrometry. The results showed that there was significant development of a number of volatile compounds with time, e.g., certain Maillard reaction and lipid oxidation products, which are likely candidates for the aroma development during hydrolysis. Furthermore, it was shown that development of a number of the volatiles was due to the hydrolysis process, as these compounds were not found in a control without enzyme.
Keywords:
hydrolysis; enzyme; enzymatic hydrolysis; hemoglobin; papain; aroma; volatile compounds

1. Introduction

Porcine blood is a significant by-product from the slaughter industry, reported to make up 6–7% of the lean meat content of the carcass [1]. Whereas blood was previously often used in dishes such as blood sausage, it is now rarely used in household cooking and, instead, used for other purposes, mainly animal feed [1,2] and fertilizer [2,3,4]. Increasing the use of by-products from animal slaughter can significantly improve both profit for the meat industry and the sustainability of animal production [2].
At the slaughterhouse, blood is usually divided into its two fractions, plasma and the cellular fraction, the latter mainly consisting of hemoglobin [5]. The plasma fraction is currently being used in food products for human consumption, e.g., as an emulsifier [5,6]. This is rarely the case for hemoglobin, partly due to the color, but also due to the strong flavor, which in many products may be considered an off-flavor [7,8], and due to the prooxidant effect of heme [9]. However, due to the high protein content [7] and high lysine content in particular [1,10], there is a large potential for the use of hemoglobin in different types of food products, which are low in lysine.
One way of adding the hemoglobin-derived protein to food products is by adding them in the form of hydrolysates, which may provide additional health benefits [11,12,13,14,15]. Additionally, by hydrolyzing hemoglobin, the heme group can easily be removed, reducing the intense, metallic taste as well as the prooxidant activity [16]. However, hemoglobin hydrolysates, on top of showing bitter taste, which is characteristic for protein hydrolysates in general, show off-flavors that make the product unacceptable for many food applications.
In order to optimize the flavor of hemoglobin hydrolysates, it is crucial to establish whether such off-flavors are already formed during the acquisition and storage of the cellular fraction, or if they are formed during the hydrolysis process. A first approach to tentatively address the origin of this off-flavor is to identify the volatile compounds present in the raw material as well as those formed during the hydrolysis process. Previous studies on different types of hydrolysate have shown that both the raw material and the conditions during the hydrolysis may influence the profile of volatile compounds formed [17,18,19]. In these studies, compounds from lipid oxidation and from Maillard reactions have been found. While some of those compounds may actually contribute positively to the final aroma profile of the hydrolysates, others might constitute off-flavors, inhibiting the use of the hydrolysate in food applications.
Hence, the aim of this work was to determine the development of volatile compounds during hydrolysis of hemoglobin in order to address to what extent these compounds are a result of the hydrolysis itself or already present in the raw material. If possible, candidates for off-flavor-development during hydrolysis of hemoglobin will be identified.
The results showed that during hydrolysis of hemoglobin, there was a significant development of a number of volatile compounds over time, e.g., certain Maillard reaction and lipid oxidation products, which are likely candidates contributing to the aroma of the final product. Furthermore, it was shown that the development of a number of the volatiles was due to the hydrolysis process itself, as these compounds were not found in a control undergoing the same processing conditions but without enzyme.

2. Results and Discussion

Table 1 shows the yield, defined as percentage of the total dry matter content of the hydrolysate recovered in the protein fraction, at each time point during hydrolysis. Not surprisingly, yield appeared to increase with time of hydrolysis, but particularly in the last two hours of hydrolysis, whereas no change in yield was apparent between 1 h and 3 h.
When producing a protein hydrolysate, as high a yield as possible is generally desirable. But of course, the development and disappearance of both desirable and undesirable volatile compounds during the course of hydrolysis should be considered.
Table 2 shows the names and relative concentrations of the volatile compounds detected via gas chromatography-mass spectrometry (GC-MS). Among the 67 volatile compounds that were detected, 23 were found to change significantly in concentration during hydrolysis, and four exhibited an increase by a factor 4 or more. It is likely that some of these compounds could change the flavor of the hydrolysate, though it should be kept in mind that perceived aroma depends on factors such as concentration, threshold, and matrix [20].
The Strecker aldehydes 3-methylbutanal and benzeneacetaldehyde increased by factors 4.5 and 8 and have dry/green and hyacinth odors. 3-Methylbutanoic acid and hexanoic acid (cheesy and rancid odor descriptors) increased 15 and 42 times, respectively (all odor descriptors from [21]). Some of the mentioned compounds contribute with unpleasant/unwanted odors, but even more pleasant odors may be perceived as off-flavors, depending on the context in which the hydrolysate is used.
Figure 1a shows the score plot from the Principal Component Analysis (PCA) on volatile compounds during hydrolysis of porcine hemoglobin. As expected, there is a clear effect of time, especially a clear separation between 0 h and 5 h on PC1, which explains 41% of the variance. The loading plot in Figure 1b shows how the occurrence of different volatile compounds changes during hydrolysis, since compounds to the left of the loading plot are present mainly at 0 h, whereas compounds to the right of the loading plot are present mainly after 5 h of hydrolysis.
Compounds that were found to increase with time of hydrolysis but were not found in the controls without enzyme (results not shown) include: 3-methyl-2-pentanone, benzoic acid, tetradecane, and 3-methylbutanenitrile. These compounds must therefore be a result of the enzymatic hydrolysis.
The most abundant compound was by far benzaldehyde, which also increased in concentration with time (Figure 1b, Table 2). Benzaldehyde and its derivatives have been described as compounds arising from Maillard reactions [22], although other routes of formation have been suggested, such as the oxidation of different toluenes and other hydrocarbons [23].
Compounds derived from the Strecker degradation of amino acids, such as 2- and 3-methylbutanal showed a clearly increasing trend throughout hydrolysis. This makes sense, since the substrates for the formation of such aldehydes, leucine and isoleucine respectively, together with different reducing sugars, are present in blood [8]. Moreover, carbonyl compounds from the oxidation of lipids [24] or proteins [25] have been demonstrated to promote the oxidative degradation of amino acids and yield the corresponding Strecker aldehydes.
Some of the compounds with a higher load on PC1 were methyl ketones (3-methyl-2-pentanone and 3-methyl-2-butanone) and 𝛼-diketones (e.g., 2,3-pentanedione and 2,3-butanedione), meaning that these compounds showed a trend towards increasing their levels throughout hydrolysis time. Both methyl-ketones and 𝛼-diketones have been described as arising as a consequence of microbial metabolism in different food products [26]. This could actually have been possible during hemoglobin hydrolysis, since there is no previous sterilization, and the incubation temperature (55 °C) would allow for the growing of some microorganisms.
Typical compounds from fatty acid autoxidation, such as straight chain aldehydes and alk-1-ols were found. Not all of these compounds showed the same trend. Hexanal, the most extensively used marker for lipid oxidation, showed a clear growing trend during the initial hours of hydrolysis, followed by a decrease to the original level. Other compounds showed only minor, statistically insignificant variation or even a decreasing trend in the case of octanal. The presence of these compounds is most likely a consequence of the oxidation of the phospholipids remaining in the red cell fraction given the conditions during hydrolysis, at 55 °C and with a massive amount of prooxidant heme.
In this study, the effect of hydrolysis time on the development of volatile compounds was analyzed. There are other factors, which could commonly change in an industrial setup, including the raw material and choice of enzyme(s) and its concentration, to name a few. The final produced hydrolysate should be tested in a real product for a realistic evaluation of the effect of hydrolysates on off-flavor and -odor. For instance, Meinert et al. [27] found that addition of hydrolysates of porcine and bovine origin increased the occurrence of off-flavors and -odors in saveloys compared to the control without hydrolysate.

3. Materials and Methods

3.1. Enzymatic Hydrolysis

Hemoglobin was provided by a commercial slaughterhouse and kept frozen in 50 mL tubes at −80 °C prior to use. The appropriate volume of hemoglobin was thawed overnight in the fridge. Four replicas of the hemoglobin were hydrolyzed as follows: Eighty mL of thawed hemoglobin was poured into a blue cap bottle (500 mL) and 160 mL of cold tap water was added. A magnet was added to each bottle for extra friction at the bottom of the bottles. The bottles were placed in a shaking water bath at 55 °C for temperature equilibration. Enzyme stock solution was made (four replica) by weighing out 1.4057 g of the endopeptidase papain (BSC Biochemicals, Hamme, Belgium) and adding 9.378 mL of cold tap water. When the temperature of the hemoglobin had reached 55 °C, 8 mL enzyme stock solution was added to each bottle for a final enzyme concentration of 0.5%.
In a similar way, four control samples without enzyme were produced by simply adding 8 mL of cold tap water instead of the enzyme stock solution.
At time points 0 h, 1 h, 3 h, and 5 h, a 40 mL sample was taken from each blue cap bottle and transferred to a 50 mL tube (for controls, only time points 0 h and 5 h) and immediately placed in a water bath set at 95 °C for 20 min of heat treatment to inactivate the enzyme. After cooling in an ice bath for 5 min, pH was lowered by adding 1400 µL 2 M sulfuric acid in order to precipitate the heme group. The tubes were then centrifuged at 3000 g for 15 min, and the supernatants (protein fraction) were poured into clean 15 mL tubes.
Dry matter content was determined for both the complete sample (taken prior to centrifugation) and the supernatant after overnight drying at 105 °C of a small sub-sample of each sample. The remaining sample was frozen (−20 °C) until further analysis.

3.2. Volatile Compound Analysis

3.2.1. Dynamic Headspace Sampling

Samples were thawed overnight in the fridge (−4 °C). For each time point, 20 mL of each replica was transferred to a 100 mL gas washing flask and closed with a purge head. The samples were equilibrated to 37 °C in a circulating water bath and then purged with nitrogen (150 mL/min) for 60 min under magnetic stirring (200 rpm). The volatile compounds were collected on traps containing 200 mg of Tenax-TA with mesh size 60/80 (Markes International, Llantrisant, UK). Water was removed from the traps by dry-purging with nitrogen (100 mL/min) for 20 min.

3.2.2. Gas Chromatography-Mass Spectrometry

The trapped volatiles were desorbed using an automatic thermal desorption unit (TurboMatrix 350, Perkin Elmer, Shelton, CT, USA). Primary desorption was carried out by heating the trap to 250 °C with a flow (50 mL/min) of carrier gas (H2) for 15.0 min. The stripped volatiles were trapped in a Tenax TA cold trap (30 mg held at 5 °C), which was subsequently heated at 300 °C for 4 min (secondary desorption, outlet split 1:10). This allowed for rapid transfer of volatiles to a gas chromatograph-mass spectrometer (GC-MS, 7890A GC-system interfaced with a 5975C VL MSD with Triple-Axis detector from Agilent Technologies, Palo Alto, CA, USA) through a heated (225 °C) transfer line. Separation of volatiles was carried out on a ZB-Wax capillary column 30 m long × 0.25 mm internal diameter, 0.50 µm film thickness. The column pressure was held constant at 2.3 psi, resulting in an initial flow rate of 1.4 mL/min using hydrogen as the carrier gas. The column temperature program was: 10 min at 30 °C, from 30 °C to 240 °C at 8 °C/min, and finally, 5 min at 240 °C. The mass spectrometer was operating in the electron ionization mode at 70 eV. Mass-to-charge ratios between 15 and 300 were scanned. Peak areas and mass spectra were extracted from the chromatograms using the PARAFAC2 based software PARADISe [28] and mass spectra were identified using the NIST05 database. Volatile compound identification was confirmed by comparison with retention indices (RI) of authentic reference compounds or retention indices reported in literature.

3.3. Data Analysis

3.3.1. GC-MS

Principal component analysis was performed on autoscaled peak areas using the software Latentix 2.12 (Latentix Aps, Frederiksberg, Denmark). Analysis of variance was performed using JMP 13.0.0 (SAS Institute Inc., Cary, NC, USA) and least square means were compared by Student’s t-test (p ≤ 0.05) (JMP 13.0.0, SAS Institute Inc.).

3.3.2. Yield

Yield was calculated for each time point as the percentage of total dry matter content of the hydrolysate recovered in the supernatant (protein fraction) after centrifugation.

4. Conclusions

The obtained results showed that during hydrolysis of hemoglobin, there was a significant development of 23 volatile compounds over time, e.g., certain Maillard reaction and lipid oxidation products, which are likely candidates contributing to the aroma of the final product. Furthermore, it was shown that the development of a number of the volatiles was due to the hydrolysis process itself, as these compounds were not found in a control having undergone the same processing conditions, only without enzyme. The final produced hydrolysate should be tested in a real product for a realistic evaluation of the effect of the hydrolysate on off-flavor and -odor.

Acknowledgments

This project was funded by the Norma og Frode S. Jacobsens Foundation and by Danish Crown Ingredients.

Author Contributions

J.R.-C., K.H.B., E.T.H. and R.L. conceived and designed the experiments; K.H.B. performed the experiments; M.A.P. analyzed the data; J.R.-C., K.H.B., M.A.P. and R.L. wrote the paper.

Conflicts of Interest

This project was partially funded by Danish Crown Ingredients, who participated in the design of the experiment and agreed to publish the results.

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  • Sample Availability: Samples of the compounds are not available from the authors.
Figure 1. (a) Principal Component Analysis (PCA) score plot (minus outliers) for the effect of time on enzymatic hydrolysis of porcine hemoglobin with papain; (b) PCA loading plot showing the occurrence of volatile compounds during hydrolysis. Codes are equivalent to those found in Table 2.
Figure 1. (a) Principal Component Analysis (PCA) score plot (minus outliers) for the effect of time on enzymatic hydrolysis of porcine hemoglobin with papain; (b) PCA loading plot showing the occurrence of volatile compounds during hydrolysis. Codes are equivalent to those found in Table 2.
Molecules 23 00357 g001
Table 1. Yield for each time point of the hydrolysis.
Table 1. Yield for each time point of the hydrolysis.
Time (h)Yield (%)SD (%)
059.8±0.2
165.1±0.2
364.9±6.1
573.7±1.2
Table 2. Relative concentrations as peak area/1000 of volatile compounds detected during hydrolysis.
Table 2. Relative concentrations as peak area/1000 of volatile compounds detected during hydrolysis.
Code 1Compound0 h 21 h3 h5 hRetention Index
Exp.Auth. Std.Literature
alc12-Methyl-1-propanol3334415311001100
alc2Butanol1213152811651165
alc32-Methylbutanol3453681471231 1158–1244
alc43-Methylbutanol13812811646312321230
alc53-Methyl-3-buten-1-ol39 c47 b44 b, c52 a1268 1221–1277
alc61-Pentanol3740403312741274
alc71-Hexanol28 b9 b30 b295 a13721372
alc81-Heptanol5543391714711471
alc91-Octanol6354463215731573
alc10Z-10-Pentadecen-1-ol333111151676
alc11Phenylethyl alcohol11619019361936
alc12Dodecanol25361151992 1919–1984
ald12-Methylpropanal296 a226 b324 a206 b749 770–834
ald22-Methylbutanal192 c242 b260 b353 a907913
ald33-Methylbutanal914 d1406 c2029 b4109 a917917
ald4Pentanal133 b197 a184 a153 ab983983
ald5Hexanal791 b1427 a1508 a844 b10831088
ald6Heptanal1251391388711921192
ald7Octanal209 a234a191 a107 b13031311
ald8Nonanal265 b337ab453 a440 a14061403
ald9(E)-2-Octenal111518141441 1393–1467
ald10Decanal59 b55 b83 b160 a15101511
ald11Benzaldehyde21,821 d38,502 c46,420 b53,809 a15301539
ald12(E)-2-Nonenal15c21 b, c32 a27 ab15491551
ald13Benzeneacetaldehyde27c51 c112 b222 a1650 1592–1684
ald14(E)-2-Decenal222622221660 1592–1682
est1Ethyl acetate744986102864 850–914
est2Hexyl acetate01222312881293
est3Ethyl octanoate1115214461445
est4Ethyl decanoate333216511651
fur12-Pentylfuran51 d111 b137 a89c1243 1193–1258
fur21-(2,4-Dimethyl-furan-3-yl)-ethanone17251091371586
fur32-Furanmethanol217231671 1613–1698
fur45-Ethyldihydro-2(3H)-furanone83655921719
ket12-Butanone9799811051923887906
ket23-Methyl-2-butanone985c1746 b1803 b2122 a929 918–989
ket33,3-dimethyl-2-butanone889594105948 969–986
ket42,3-Butanedione1665 c2079 b, c2404 b3166 a985985
ket54-Methyl-2-pentanone253760731006 993–1040
ket63-Methyl-2-pentanone512 c731 b751 b889 a10141001
ket72,3-Pentanedione85 c120 b122 b168 a10731073
ket84-Methyl-3-penten-2-one29531551961133 1110–1159
ket92,4-Pentanedione71820541162 1167–1230
ket102-Heptanone4543463211981190
ket114-Methyl-2-heptanone213238401219 1206–1224
ket126-Methyl-5-hepten-2-one91244261350 1296–1368
ket132-Nonanone453429814021382
ket14Acetophenone1421381091331663 1600–1695
lac1Butyrolactone50524301641 1593–1673
lac2delta-Hexalactone1411941816 1751–1830
lac3gamma-Heptalactone118838051825 1755–1817
lac4gamma-Nonalactone1401027122063 1981–2068
lac5gamma-Decalactone36231312176 2090–2185
aci1Acetic acid1967172721459 1401–1485
aci23-Methylbutanoic acid13 b23 b2 b197 a1679 1631–1707
aci3Pentanoic acid190817471755
aci4Hexanoic acid52532101859 1797–1889
aci5Octanoic acid191202078 2011–2100
aci6Benzoic acid26 b46 ab49 ab85 a2415 2380–2457
alk1Tetradecane21 b28 b41 a41 a14051400
alk2Hexadecane1916201716021600
alk3Heptadecane858917001700
alk4Octadecane1614242018001800
ben11,2,4-Trimethylbenzene151517171285 1247–1333
phe1Phenol577350592024 1949–2037
oth13-Methylbutanenitrile63c74 b75 b90 a1121 1090–1144
oth2Thiazole20 c36 b40 b63 a1259 1210–1270
1 alc = alcohol, ald = aldehyde, est = ester, fur = furan, ket = ketone, lac = lactone, aci = organic acid, alk = alkane, ben = benzaldehyde, phe = phenol, oth = other; 2 Within each row, different letters (a–d) indicate a significant difference (p ≤ 0.05) in relative concentration between time points.

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