Ultrasound Bath-Assisted Enzymatic Hydrolysis Procedures as

Anal. Chem. 2004, 76, 3541-3547

Ultrasound Bath-Assisted Enzymatic Hydrolysis Procedures as Sample Pretreatment for the Multielement Determination in Mussels by Inductively Coupled Plasma Atomic Emission Spectrometry Carlos Penã-Farfal,† Antonio Moreda-Pin˜eiro,‡ Adela Bermejo-Barrera,‡ Pilar Bermejo-Barrera,*,‡ Hugo Pinochet-Cancino,† and Ida de Gregori-Henrı´quez†

Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Chemistry, University of Santiago de Compostela, Avda, das Ciencias, s/n, 15782 Santiago de Compostela, Spain, and Institute of Chemistry, Faculty of Basic and Mathemathics Sciences, Pontificia Universidad Cato´ lica de Valparaı´so, Avda, Brasil 2950, Valparaı´so, Chile

Ultrasound energy has been applied to speed up enzymatic hydrolysis processes of mussel tissue in order to determine trace and ultratrace elements (As, Al, Cd, Cr, Cu, Fe, Mn, Ni, Pb, Zn). The element releases, by action of three proteases (pepsin, pancreatin, trypsin), lipase, and r-amylase, have been evaluated by inductively coupled plasma atomic emission spectrometry. Different variables such as pH, sonication temperature, ionic strength, hydrolysis time, ultrasound frequency, extracting volume, and enzyme mass were simultaneously studied by applying an experimental design approach (Plackett-Burman design and central composite design). Results showed that the hydrolysis time was statistically nonsignificant (confidence interval of 95%) for most of the elements and enzymes, meaning that the hydrolysis procedure can be finished within a 30-60-min range. These hydrolysis times are far shorter than those obtained when using thermostatic cameras, between 12 and 24 h. Statistically significant factors were the ultrasound frequency (the highest metals releasing at high-ultrasound frequency), pH, sonication temperature, and ionic strength. All metals can be extracted using the same operating conditions (pH of 1.0 and sodium chloride at 1.0% for pepsin; pH of 7.5, temperature at 37 °C, and 0.4 M potassium dihydrogen phosphate/potassium hydrogen phosphate buffer for amylase; pH of 8.0 and 0.5 M potassium dihydrogen phosphate/potassium hydrogen phosphate buffer for pancreatin; pH of 5.0 and 0.5 M potassium dihydrogen phosphate/potassium hydrogen phosphate buffer for lipase; pH of 8.0 and 0.2 M potassium dihydrogen phosphate/potassium hydrogen phosphate buffer for trypsin). Analytical performances, such as limits of detection and quantification, repeatability of the overall procedure, and accuracy, by analyzing DORM-1, DORM-2, and TORT-1 * Corresponding author. Tel.: + 34 600 942346. Fax: + 34 981 595012. E-mail: [email protected]. † Pontificia Universidad Cato´lica de Valparaı´so. ‡ University of Santiago de Compostela. 10.1021/ac049903r CCC: $27.50 Published on Web 05/25/2004

© 2004 American Chemical Society

certified reference materials, were finally assessed for each enzyme. Enzymatic hydrolysis procedures are a group of sample pretreatments that consist of hydrolyzing biomolecules, mainly proteins, by action of enzymes. The enzymatic or enzymic hydrolysis breaks down certain bonds of these biomolecules under certain environmental conditions such as pH, temperature, and ionic strength.1 After enzymatic hydrolysis action, a variable fraction of metal or organometallic species is released from the biological material and the released analytes can be finally measured by either atomic spectrometric or chromatographic techniques, after an adequate separation of the supernatant and the pellet. This sample pretreatment type offers as the main advantage that alteration of organometallic species is less expected. Because of this, enzymatic hydrolysis have been commonly used for metal speciation in biological and environmental samples.2 Moreover, organometallic species degradation is minimized by enzymatic hydrolysis; these processes offer other advantages such as the absence of oxidizing reagents or concentrated mineral acids, commonly needed in wet acid digestion procedures, and a noncorrosive environment as well as a small volume of acid wastes. However, the main drawback of enzymatic hydrolysis is the long time required to complete the hydrolysis process, and hydrolysis times of 12-24 h are typically reported. Thus, conventional thermostatic incubation with pronase E leads to enzymatic hydrolysis times of ∼5-16 h to assess total Se in blood serum3 or total trace elements in mussels.4 Other conventional enzymatic hydrolyses with lipase, cellulase, and protease type XIV exceed several hours too, within 12-24 h.5,6 Therefore, the (1) Holme, D. J.; Peck, H. Analytical Biochemistry, 2nd ed.; Wiley & Sons: New York, 1994. (2) Ebdon, L.; Fisher, A. In Encyclopedia of Analytical Chemistry; Meyers, R. A., Ed.; Wiley & Sons: New York, 2002; pp 3064-3084. (3) Abou-Shakra, F. R.; Rayman M. P.; Ward, N. I.; Hotton, V.; Bastian, G. J. Anal. At. Spectrom. 1997, 12, 429-433. (4) Bermejo-Barrera, P.; Fernańdez-Nocelo, S.; Moreda-Pin ˜eiro, A.; BermejoBarrera, A. J. Anal. At. Spectrom. 1999, 14, 1893-1900. (5) Tan, Y.; Marshall, W. D. Analyst 1997, 122, 13-18.

Analytical Chemistry, Vol. 76, No. 13, July 1, 2004 3541

development of new methods to speed up the enzymatic extraction procedures, guaranteeing quantitative releases or organometallic species stability, is mandatory. The use of ultrasound energy for accelerating or assisting a different process is a current practice, as has been recently reported Luque-Garcıá and Luque de Castro.7 The induced cavitation process occurring in a liquid when ultrasound waves are applied promotes an increase of pressure and temperature because of bubble collapse. The asymmetric cavity collapse when a solid phase is immersed into a liquid phase produces high-speed jets of liquid, which impact on the solid surface. As a consequence, a high analyte transport from the solid particles to the liquid phase (extracting solution) is reached and the extraction or pretreatment times are commonly shortened.8 There are several applications of ultrasound waves to induce different sample pretreatments in order to determine both organic and inorganic analytes. These ultrasound-assisted sample pretreatments can be carried out using both an ultrasound bath or ultrasound probe devices, although the last ones provide a more efficient cavitation process. However, new ultrasound water bathes offer as an advantage the possibility of temperature control, which allows sonication at high temperatures, increasing the extraction yields by a combined action of ultrasound and temperature. Therefore, some quantitative trace metal acid-leaching procedures have been optimized,9 allowing the acid leaching of some trace elements that are not quantitatively released when sonicating at room temperature. In addition, the ultrasound bath units are the best choice when needing a strict control of temperature, such as when carrying out enzymatic hydrolysis. Therefore, although the most reported application of ultrasound waves involves the use of ultrasound probes to leach inorganic compounds or extract organics,7 optimized ultrasound-assisted sample pretreatments for the determination of trace elements in biological material using bath systems have been performed too. These described procedures use diluted acids to leach trace elements from environmental samples such as plants,10 vegetables,11 or mussels12,13 and from clinical materials such as human hair.9 In this work, the novel application of ultrasound energy to accelerate enzymatic hydrolysis procedures was developed. Enzymes such as R-amylase, lipase, pepsin, pancreatin, and trypsin were investigated in order to quantitatively release trace elements from mussel tissue. EXPERIMENTAL SECTION Apparatus. The following equipment was used: Optima 3300 DV inductively coupled plasma atomic emission spectrometer (Perkin-Elmer, Norwalk, CT) equipped with an autosampler AS 91 (Perkin-Elmer); Raypa UCI-150 ultrasonic cleaner bath from (6) Chen, X.; Marshall, W. D. J. Agric. Food Chem. 1999, 47, 3727-3732. (7) Luque-Garcıá, J. L.; Luque de Castro, M. D. Trends Anal. Chem. 2003, 22, 41-47. (8) Majors, R. E. LC-GC 1999, 17 (6S), S8. (9) Bermejo-Barrera, P.; Moreda-Pin ˜eiro, A.; Bermejo-Barrera, A. J. Anal. At. Spectrom 2000, 15, 121-130. (10) Minami, H.; Honjyo, T.; Atsuya, I. Spectrochim. Acta, Part B 1996, 51, 211220. (11) Nascentes, C. C.; Korn, M.; Arruda, M. A. Z. Microchem. J. 2001, 69, 3743. (12) El Azouzi, H.; Cervera, M. L.; de la Guardia, M. J. Anal. At. Spectrom. 1998, 13, 533-538. (13) Bermejo-Barrera, P.; Mun ˜iz-Naveiro, O.; Moreda-Pin ˜eiro, A.; BermejoBarrera, A. Anal. Chim. Acta 2001, 439, 211-227.

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Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

R. Espinar S.L. (Barcelona, Spain), programmable for temperature and time, and with discrete ultrasounds frequencies of 17 and 35 kHz; Centrifuge Centromix (Selecta) and Orion 720A plus pHmeter with a glass-calomel electrode (Orion, Cambridge, U.K.); Samsung domestic microwave oven (Seoul, Korea), programmable for time and microwave power (100-900 W); poly(tetrafluoroethylene) (PTFE) low-pressure bombs with hermetic seals. Chemometrics package was Statgraphics Plus V 5.0 for Windows, 19941999 (Manugistics Inc., Rockville MD). Reagents. All chemicals used were of ultrapure grade and diluted using ultrapure water of resistance 18 MΩ cm-1 obtained from a Milli-Q purification device (Millipore Co., Bedford, MA). Multielement standard solutions were prepared by combining stock standard solutions (1.000 or 10.000 g L-1) supplied by Merck (Poole, Dorset, U.K.). Trypsin was from porcine pancreas and R-amylase from Bacillus subtilis (Merck). Pepsin was from porcine stomach and pancreatin from porcine pancreas (Sigma, St. Lois MO). Lipase type VII was isolated from Candida rugosa (Sigma). AnalaR nitric acid 70.0% and hydrochloric acid 37% were from J.T. Baker B. V. (Deventer, Holland) and sodium hydroxide, potassium dihydrogen phosphate, potassium hydrogen phosphate and sodium chloride from Merck. DORM-1 and DORM-2 (dog-fish muscle) and TORT-1 (lobster hepatopancreas) CRMs were supplied from National Research Council of Canada. To avoid metal contamination, all glassware and plastic ware were washed and kept for 48 h in 10% (v/v) nitric acid and then rinsed several times with ultrapure water before use. Mussel Samples. Fresh mussel samples (Mytilus galloprovincialis) were collected from mussel farms at Rıá de Arousa estuary. Studies were performed using all mussel tissue (muscle and gill) by preparing a mussel pool with all the mussels from each mussel farm. After mechanical blending, homogenization, and a freeze-dry process, the dry mussel samples were pulverized by using a vibrating ball mill and they were kept in polyethylene bottles with hermetic seals. Microwave Acid Digestion. Sample acid digestion was achieved by using a domestic microwave oven and low-pressure PTFE bombs. The digestion process was carried out in two steps using 0.2 g of mussel. The first stage involves the use of 2 mL of concentrated HNO3 and two microwave irradiation cycles at 200 W for 2 min, inserting a cooling stage in an ice bath for 10 min in between. The second step uses 1 mL of 33% m/v H2O2 and two microwave heatings at 300 W for 2 min, also inserting a cooling stage. The acid digests were made up to 10 mL with ultrapure water. Enzymatic Hydrolysis Procedure Using Pepsin, Pancretain, and Trypsin. Around 0.2 g of mussel was weighed into the centrifuge tubes, and 20 mg of enzyme dissolved in 7 mL of PDHP/PHP buffer solution at pH 8 (0.5 M for pancreatin or 0.2 M for trypsin) or in 7 mL of 1.0% (m/v) sodium chloride (pH 1.0 fixed by hydrochloric acid, for pepsin) was added. The mixtures were sonicated at 17 kHz and 37 °C for 30 min, and after centrifugation at 3000 rpm for 15 min, the supernatant was made up to 10 mL. ICP-AES Measurements. Elements were measured by ICPAES (axial configuration) without dilution, using the operating conditions and emission wavelength lines given in Table 1. Preliminary studies on the use of scandium as internal standard revealed

Table 1. ICP-AES Operating Conditions radio frequency power/W gas flows/L min-1 plasma auxiliary nebulizer nebulizer type peristaltic pump speed/mL min-1 stabilization time/s number of replicates detection wavelengths/nm Al As Cd Cu Cr Fe Mn Ni Pb Zn

Table 3. Effects of Changing Values from Low to High Levelsa

1300 15.0 0.5 0.8 cross-flow 1.5 45 4 396.153 197.197 228.802 327.393 267.716 238.204 257.610 231.604 224.688 206.200

Table 2. Experimental Field Definition for the Plackett-Burman (PBD) and Central Composite (CCD) Designsa variable

symbol

low level (-)

high level (+)

enzyme mass/mg reaction volume/mL hydrolysis temperature/°C hydrolysis time/min ultrasound frequency/kHz pHb ionic strength (sodium dihydrogen phosphate concn)/Mc-e dummy factor

W V T t F pH IS

5.0 3.0 25 30 17 6.0 0.05

20.0 7.0 50 60 35 9.0 0.20

D

-1

+1

a The mussel mass was 0.20 g for all experiments. b The low and high levels were 1.0 and 4.0, respectively, when using pepsin. c The ionic strength were related to sodium chloride at 2.0 and 0.0% (m/v) for the high and low levels, respectively, when using pepsin in PBD, and 0.5 and 0.2% (m/v), respectively in CCD. d Sodium dihydrogen phosphate concentration at 0.5 and 0.2 M for the high and low levels, respectively, when using R-amylase in CCD. e Sodium dihydrogen phosphate concentration at 0.5 and 0.1 M for the high and low levels, respectively, when using trypsin, pancreatin, and lipase in CCD.

a lack of accuracy for some elements while the standard addition technique gave accurate results. Therefore, the standard addition graph was used for each enzyme type or acid digest, covering analyte concentration ranges between 0 and 10 mg L-1 (Al, Cu, Fe, Mn, Zn) and between 0 and 2 mg L-1 (As, Cd, Cr, Ni, Pb). RESULTS AND DISCUSSION Enzymatic Hydrolysis Variables under Study. Moreover temperature, pH, and ionic strength, the most reported main variables when using an enzymatic hydrolysis process,1 and other operating conditions that could affect the enzymatic activity such as the enzyme mass, the reaction volume, and the incubation (enzymatic hydrolysis) time were considered in the current study. The variable pH was fixed by using a potassium dihydrogen phosphate/potassium hydrogen phosphate buffer solution for all enzymes, except when using pepsin, for which hydrochloric acid was used to fix the pH. The variable ionic strength is related to the concentration of the buffer system used to fix the pH. Since

P

pH

IS

D

Trypsin ns ns ns ns ns ns ns ns ns ns

ns ns ns ns

+ + + + ns + + + ns

ns + + + + ns + ns ns

ns ns ns ns ns ns ns + ns ns

ns + ns ns ns ns ns + ns ns

Pancreatin ns ns ns ns ns ns ns ns ns ns

ns ns ns ns ns

ns ns ns + ns ns + + -

+ ns + ns + ns ns ns ns

ns ns ns ns ns ns ns ns ns

ns ns ns + ns ns ns ns ns ns

ns ns ns + ns ns ns -

Pepsin + + ns + ns ns ns + ns ns

ns ns ns ns

ns ns -

ns + + ns ns ns ns ns

ns ns ns ns ns ns ns ns ns

ns ns ns ns ns + + ns ns

ns ns + ns + ns + ns ns +

ns ns ns ns ns ns -

Lipase ns ns + + + ns ns ns + ns

+ ns ns ns ns ns + ns ns +

ns + ns ns ns -

ns + + ns + ns ns -

ns ns ns ns ns ns ns ns -

-ns ns ns ns ns -

ns ns + ns + + + ns ns +

ns + + ns + ns ns ns -

R-Amylase + ns + + ns + ns ns + +

ns ns ns ns ns ns ns + ns +

ns ns + ns -

ns ns + ns + ns ns ns

ns ns ns ns ns ns ns ns ns -

W

V

T

Al As Cd Cu Cr Fe Mn Ni Pb Zn

ns ns ns + ns ns ns ns + ns

+ ns + + + + + ns + +

ns ns ns -


ns ns + + + + + + + ns

ns ns + ns ns ns + ns ns +


ns ns ns + + ns ns + + ns

Al As Cd Cu Cr Fe Mn Ni Pb Zn Al As Cd Cu Cr Fe Mn Ni Pb Zn

t

a Positive sign (+) means that the effect of the variable on the response is positive. Negative sign (-) means that the effect of the variable on the response is negative. ns means nonsignificant.

potassium dihydrogen phosphate/potassium hydrogen phosphate was used as buffer solution for all enzymes, except for pepsin, the ionic strength was related to the concentration of potassium dihydrogen phosphate/potassium hydrogen phosphate. However, as an extreme acidic pH was needed when using pepsin, the ionic strength was studied by using different sodium chloride concentrations. In addition, as ultrasound energy is used to speed up the Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

3543

Figure 1. Some estimated response surfaces from the central composite design: trypsin (A-C), pancreatin (D, E), and pepsin (F).

enzymatic process, the variable ultrasound frequency was included. A last variable, called the dummy factor, was also considered in the study. Dummy factors are imaginary variables for which the change from one level to another is not supposed to cause any physical change.14 These variables are commonly used in order to evaluate the possible systematic error or the existence of an important variable that was not been taken into account. The response variables were the percentage of released element according to the equation

recovery )

[]enzymatic hydrolysis []acid digestion

× 100

where [ ]enzymatic hydrolysis is the metal concentration obtained after the enzymatic hydrolysis procedure (1-24 in Tables S1-S5, 1-10 in Tables S6-S9, or 1-16 in Table S10, Supporting Information), and [ ]acid digestion is the metal concentration found after microwave acid digestion. Statistically Significant Variables Affecting the UltrasoundAssisted Enzymatic Hydrolysis. The significance of the variables commented on above was simultaneously evaluated by applying a 28 × 3/32 type III resolution Plackett-Burman design (PBD), for 8 factors, 4 degrees of freedom, 12 runs, and 2 replicates (one PBD for each enzyme type investigated). Table 2 lists the low (-) and high (+) levels of each variable, while the PBD experimental conditions and the values for the response variables when using each enzyme are listed in Tables S1-S5, Supporting Information. The statistical evaluation of results was attained at a 95.0% confidence interval from which a minimum t value of 2.46, 3544

Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

calculated by the Statgraphics routine through an iterative process,15,16 was obtained. Variables which t values higher than (2.46 were considered as statistically significant factors. Table 3 shows a summary of the statistically significant effects of each variable on the trace metals released from mussel tissue when using each enzyme type. It can be seen that the variables pH and ionic strength are significant for most of the elements and enzymes. The significance of these two variables means that although the hydrolysis process has been assisted by ultrasounds, the nature of the metal releasing is attributed to enzymatic activity, being mainly controlled by both pH and ionic strength, and not to other extracting or leaching procedures. The variable temperature appears to be less statistically significant than pH and ionic strength, mainly when using pancreatin, lipase, and R-amylase. These results agree with those previously reported when using conventional enzymatic hydrolysis, which reveled a great influence of the variables pH and ionic strength and partial importance of the variable temperature.17 The variable ultrasound frequency has been found significant for most of the elements and enzymes. The effect of this variable was positive when using lipase and R-amylase, which implies a more efficient metal releasing working at a high ultrasound frequency (35 kHz). The effect of the ultrasound frequency was negative when using pepsin, trypsin, and pancreatin, which means (14) Gardiner, W. P.; Gettinby, G. Experimental design techniques in statistical practice, A practical software-based approach; Horwood Publishing Ltd.: West Sussex, U.K., 1998. (15) Lewis, G. A.; Mathieu, D.; Phan-Tan-Luu, R. Pharmaceutical Experimental Design; Marcel Dekker: New York, 1999. (16) Lenth, R. V. Technometrics 1989, 31, 469-473. (17) Pen ã-Farfal, C.; Moreda-Pin ˜eiro, A.; Bermejo-Barrera, P.; Bermejo-Barrera, A.; Pinochet-Cancino, H.; de Gregori-Hernrı´quez, I. Talanta, in press.

Figure 2. Metal recovery percentages from mussel samples by using the ultrasound-assisted enzymatic procedure, the operating conditions recommended by the manufacturers, and the leaching effect of buffer solutions (blanks).

a decrease on the enzyme activity when exposed to high ultrasound frequencies. Therefore, a low ultrasound frequency of 17 kHz led to high metal recoveries for the proteases. The variable hydrolysis time (t) was found as nonsignificant for most of the elements and enzymes, specially when using proteases (pancreatin, pepsin, trypsin). Otherwise, the effect of this variable is positive when it is statistically significant. Since this variable has been studied within the 30-60-min range, this finding shows that the use of ultrasound energy reduces the enzymatic hydrolysis times to 60 min at maximum, although the time can be decreased to 30 min when using proteases. The great reduction on the enzymatic hydrolysis time when using ultrasound could be because the sonication process disrupts cell membranes. Wolf et al.18 have reported that sonication allows a rapid cytosolic preparation when isolating metallothioneins from biological samples. These authors attributed the fast cytosolic preparation to rapid cell membrane disruptions by the direct action of ultrasound. Therefore, the effect of ultrasound when assisting enzymatic hydrolysis must be the same as when preparing cytosolic solutions. After the fast cell membrane disruptions by ultrasound, the cytosolic content is attacked by enzymes, allowing a shorted pretreating time. (18) Wolf, C.; Ro ¨sick, U.; Bra¨tter, P. Anal. Bioanal. Chem. 2002, 372, 491-494.

The variable enzyme mass was statistically significant for some cases with negative effect (lipase, R-amylase) or positive effect (pancreatin, pepsin, trypsin). Therefore, the low lipase and R-amylase masses (5 mg) were chosen as optimum, while the high pancreatin, pepsin, and trypsin masses (20 mg) were selected. The reaction volume was positively significant for most of the elements and enzymes, meaning that metal release is higher when the reaction volume is increased to 7 mL. Finally, the dummy factor was not significant for any case, meaning that there are no systematic errors or unknown variables affecting the system under study. Optimization of Significant Variables by Central Composite Designs. From results shown above, variables such as pH, ionic strength, and hydrolysis temperature were the most significant factors for R-amylase and pepsin, and they were considered for a further optimization process. However, since the variable temperature has resulted as less important when using lipase, pancreatin, and trypsin, only pH and ionic strength have been optimized for these enzymes. The variables enzyme mass, reaction volume, hydrolysis time, and ultrasound frequency were fixed at convenient values in accordance with the negative or positive effect for each enzyme (Table 3). The set hydrolysis temperature/pH/ ionic strength for R-amylase was simultaneously optimized by applying an orthogonal 23 + star central composite design (CCD) Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

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Table 4. Analysis of Certified Reference Materials after Ultrasound-Assisted Enzymatic Hydrolysis Procedures DORM-1

a

DORM-2

certified value/µg g-1

found value/µg g-1

As Cd Cu Cr Mn Ni Pb

17.7 ( 2.1 0.086 ( 0.012 5.22 ( 0.33 3.60 ( 0.40 1.32 ( 0.26 1.20 ( 0.30 0.40 ( 0.12

18.7 ( 0.7 a 4.92 ( 0.16 2.67 ( 0.21 1.28 ( 0.09 1.8 ( 0.8 a

As Cd Cr Mn Ni Pb

17.7 ( 2.1 0.086 ( 0.012 3.60 ( 0.40 1.32 ( 0.26 1.20 ( 0.30 0.40 ( 0.12

Cr Cu Mn Ni Pb Zn

3.60 ( 0.40 5.22 ( 0.33 1.32 ( 0.26 1.20 ( 0.30 0.40 ( 0.12 21.3 ( 1.0


TORT-1 found value/µg g-1


found value/µg g-1

Enzymatic Hydrolysis by Trypsin 18.0 ( 1.1 17.8 ( 0.8 0.043 ( 0.008 a 2.34 ( 0.16 1.87 ( 0.24 34.7 ( 5.5 b 3.66 ( 0.34 2.93 ( 0.07 19.4 ( 3.1 13.1 ( 0.5 0.065 ( 0.007 a

24.6 ( 2.2 26.3 ( 2.1 439 ( 22 2.4 ( 0.6 23.4 ( 1.0 2.3 ( 0.3 10.4 ( 2.0

23.4 ( 1.3 26.8 ( 0.9 b 2.09 ( 0.02 23.1 ( 0.2 2.0 ( 0.1 8.8 ( 0.1

16.5 ( 0.5 a 3.50 ( 0.22 1.04 ( 0.07 1.00 ( 0.08 a

Enzymatic Hydrolysis by Pancreatin 18.0 ( 1.1 18.2 ( 0.7 0.043 ( 0.008 a 34.7 ( 5.5 b 3.66 ( 0.34 2.81 ( 0.18 19.4 ( 3.1 16.5 ( 0.3 0.065 ( 0.007 a

24.6 ( 2.2 26.3 ( 2.1 2.4 ( 0.6 23.4 ( 1.0 2.3 ( 0.3 10.4 ( 2.0

23.0 ( 1.5 27.2 ( 0.4 2.1 ( 0.1 23.7 ( 0.2 2.2 ( 0.1 9.8 ( 0.1

3.33 ( 0.23 5.42 ( 0.09 1.15 ( 0.07 1.36 ( 0.13 a 20.6 ( 0.6

Enzymatic Hydrolysis by Pepsin 34.7 ( 5.5 b 2.34 ( 0.16 2.15 ( 0.10 3.66 ( 0.34 3.39 ( 0.22 19.4 ( 3.1 18.5 ( 0.3 0.065 ( 0.007 a 25.6 ( 2.3 23.3 ( 0.9

2.4 ( 0.6 439 ( 22 23.4 ( 1.0 2.3 ( 0.3 10.4 ( 2.0 177 ( 10

b b 21.9 ( 0.3 2.3 ( 0.1 11.0 ( 0.6 179 ( 10

Ultrasound Bath-Assisted Enzymatic Hydrolysis Procedures as

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