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Umami taste amino acids produced by hydrolyzing extracted protein from tomato seed meal
Accepted Manuscript Umami taste amino acids produced by hydrolyzing extracted protein from tomato seed meal Yin Zhang, Zhongli Pan, Chandrasekar Venkitasamy, Haile Ma, Yunliang Li PII:
S0023-6438(15)00075-4
DOI:
10.1016/j.lwt.2015.02.003
Reference:
YFSTL 4440
To appear in:
LWT - Food Science and Technology
Received Date: 8 August 2014 Revised Date:
16 January 2015
Accepted Date: 3 February 2015
Please cite this article as: Zhang, Y., Pan, Z., Venkitasamy, C., Ma, H., Li, Y., Umami taste amino acids produced by hydrolyzing extracted protein from tomato seed meal, LWT - Food Science and Technology (2015), doi: 10.1016/j.lwt.2015.02.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Umami taste amino acids produced by hydrolyzing
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extracted protein from tomato seed meal
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Yin Zhang 1, 2, Zhongli Pan2, 3∗, Chandrasekar Venkitasamy2, Haile Ma4, 5, Yunliang Li4
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Key Laboratory of Meat Processing of Sichuan, Chengdu University, Chengdu 610106, China
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Department of Biological and Agricultural Engineering, University of California, Davis, One Shields
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Avenue, Davis, CA 95616, USA
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Healthy Processed Foods Research Unit, USDA-ARS-WRRC, 800 Buchanan St., Albany, CA 94710,
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212013, China
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Zhenjiang, Jiangsu 212013, China
Jiangsu Provincial Research Center of Bio-process and Separation Engineering of Agri-products,
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School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu
24 25 26 ∗ Corresponding author. Address: Processed Foods Research Unit, USDA–ARS West Regional Research Center, 800 Buchanan Street, Albany, Berkeley, CA 94710, USA. Tel.: +1 510 559 5861; fax: +1 510 559 5851. E-mail addresses: [email protected], [email protected] (Z. Pan).
ACCEPTED MANUSCRIPT Abstract: Enzymatic hydrolysis was performed for extracting protein to prepare umami taste
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amino acids from defatted tomato seed meal (DTSM) which is a by-product of tomato processing.
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Papain was used as an enzyme for the hydrolysis of DTSM. The particle size distribution of
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DTSM, protein concentration and free amino acids of the hydrolysate were analyzed. Response
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surface methodology was used in the design of experiments and analysis of results. The protein
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extraction ratio of greater than 85.64% was obtained when the DTSM with particle size ≤ 0.25
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mm was hydrolyzed by papain for 5 h. The optimum extraction conditions to produce maximum
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concentration of umami taste amino acids were enzyme activity of 18.22 %; pH of 3; hydrolysis
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temperature of 40 °C; and hydrolysis time of 6 h, which resulted in glutamic acid and aspartic acid
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concentrations of 727.6 µg/ mL and 149.9 µg/ mL, respectively.
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Key words: umami taste; amino acids; glutamic acid; aspartic acid; tomato seed meal
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ACCEPTED MANUSCRIPT 1. Introduction
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Palatability plays a major role in food selection, intake, absorption and digestion. Glutamate, one
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of the basic umami taste compounds, is a major ingredient to make food tasty, palatable and
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acceptable (Zhang et al., 2013). Tomato contains higher content of glutamate procurer glutamic
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acid than other vegetables (Yamaguchi & Ninomiya, 2000). This might be the possible reason for
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the popularity of tomato as a major vegetable, processed into sauces and often used as food
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condiment. Nearly one third of the cultivated tomatoes are processed (Cantarelli et al., 1993),
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leading to generation of huge volumes of tomato pomace as a by-product.
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About 3 to 5% (in weight) of fresh tomato is generated as pomace which consists approximately
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60% of seed and 40% of peel (Celmaa et al., 2009). Generally, the tomato pomace is used as
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livestock fodder or soil amendment or disposed directly into landfill, causing environmental
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problems and resource loss (Sogi et al., 2005). Recent investigation indicated that defatted tomato
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seed meal protein has the cholesterol-lowering effect in male golden Syrian hamster (Shao et al.,
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2013), which motivated research in the recovery and utilization of tomato seed protein.
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Alkaline treatment is the traditionally followed procedure for extraction of proteins from tomato
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seed meal and other complex plant matrices, such as industrial by-products (Robak, 2006).
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However, chemical methods could induce side-reactions, such as hydrolysis and extraction of
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non-protein components, and denaturation of protein with obvious effects on their functional
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properties (Kinsella, 1981). In order to overcome those drawbacks, enzymatic methods were
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developed (Tang et al., 2002), and preferred for their milder process conditions, easier control of
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reaction parameters and minimal secondary product formation (Mannheim & Cheryan, 1992). To
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our knowledge, very limited information is available regarding the use of enzymatic hydrolysis to
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extract protein from the byproducts of tomato processing industries, like DTSM, to develop new
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products.
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The glutamic and aspartic acids are present at 18.99g/ 100g and 11.95 g/ 100g, respectively, in red
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tomato seed meal (Brodowski & Geisman, 1980). They are known as umami taste amino acids or
ACCEPTED MANUSCRIPT monosodium glutamate like (MSG-like) amino acids (Zhang, et al., 2013). These facts imply that
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the tomato seed meal could be used to develop products rich in umami flavors for both vegetarians
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and non-vegetarians. However, little research has been done on the development and optimization
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of processes for preparing flavor components from DTSM using papain hydrolysis and this
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research will be the first of its kind. Previous investigations revealed that use of papain resulted in
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a higher degree of hydrolysis of whey and egg protein compared with the other enzymes such as
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pepsin and trypsinase (Akeson & Stahmann, 1964; Pena-Ramos & Xiong, 2001; Zhang et al.,
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2014). Therefore, papain was used for hydrolysis of protein from DTSM in this study.
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The objectives of our research were 1) to investigate the effect of enzymatic hydrolysis on protein
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extraction from DTSM, 2) to examine the effects of temperature, pH, time and enzyme activity on
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the concentrations of umami taste amino acids, and 3) to determine the optimal conditions for
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preparing high concentration of umami taste amino acids with an enzymatic hydrolysis method.
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2. Materials and methods
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2.1. Materials
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Tomato pomace produced by a popular hot break process was collected from the tomato
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processing plant of Morningstar Company located in Williams, CA. The pomace samples were
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stored in the freezer (-8° C) until it was used. Its crude composition was determined as 95.4% dry
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matter, 22.72% acid detergent fiber, 28.40% crude protein, 58.72% total digestible nutrients,
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21.32% crude fat, and 3.74% ash. Papain obtained from Carica papaya with enzyme activity of
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1.937 U/mg (the enzyme unit U refers to amount of protease needed to hydrolysis 1 µmol of
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casein in 1 min) was used for hydrolysis. All other chemicals used for this study were of analytical
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grade and obtained from Fisher Scientific Inc. (Pittsburgh, PA., U.S.A.).
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2.2. Preparation of tomato seeds and defatted tomato seed meal
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The tomato pomace was thawed at 4 °C and then dried at 40 °C in an oven (637G, Colorado
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Springs Utilities Investment Recovery, Colorado Springs, CO, USA) for 24 h to a moisture
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content of 5.0 ± 0.2%. The seeds were separated from the dried pomace samples using an aspirator
ACCEPTED MANUSCRIPT system (FC2K testing husker, YAMANMOTO, CO., Higashine, Japan). Sample of 70 g of seeds
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were ground to powder using a mill (M2 Stein Mill, The Steinlite Corporation, Atchison, KS, USA)
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for 30 s and sieved in a Tyler Sieve Shaker (RO-TAP Testing Sieve Shaker, W.S. Tyler Co.,
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Cleveland, OH, USA) with a 14 mesh opening sieve. The sieved powder was defatted with hexane
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(1 g meal: 10 mL hexane) for 4 h. The supernatant was evaporated to recover and recycle the
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hexane. The solid residues were placed in fume hood to remove residual hexane until there was no
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hexane odor and then packed in a ziplock plastic bag for later use. The residual fat content of the
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DTSM was determined as 9.49 ± 0.13%.
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A sieve shaker (Retsch Sieve Shaker AS 300, Haan, Germany) was used to determine the particle
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size distribution of DTSM. A sample of 330 g DTSM was loaded to the shaker with 14, 18, 20, 40,
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60, 80, 100 mesh sieves and shaken for 5 min at an amplitude of 1.36 mm/”g”. The meal left onto
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each sieve was weighed and the size distribution was calculated.
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Weight of particles retained on sieve (%) = Mx/ Mt*100
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Where, Mx is the mass of meal left onto one sieve or bottom tray, g; Mt is the total weight of
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defatted meal, g.
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Triplicate experiments were performed and the average values are reported.
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2.4. Effect of particle size on protein extraction
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To determine the effect of particle size on protein extraction, each sample with different particle
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sizes was extracted with the ratio of 2 g meal / 20 mL buffer (phosphate buffer, composed of 0.2
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mol/L Na2HPO4 and 0.1 mol/L citric acid). The papain hydrolysis conditions were pH of 5.4,
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temperature of 54 ºC, and [E]/[S] of 67 U/g for 0 h, 1 h, 3 h, and 5 h in a reciprocal water bath
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shaker (Model R 76, New Brunswick Scientific, Edison, N. J., U.S.A.). At the end of hydrolysis,
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the mixture was heated in boiling water for 20 min to inactivate the protease. The mixture was
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centrifuged with a centrifuge (Eppendorf centrifuge 5804R, Eppendorf North America, Inc., USA)
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at 9000 ×g for 10 min, and then the protein content of supernatant was determined with Biuret
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method (Nowotny, 1979). Triplicate experiments were performed and average values were
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Protein extraction ratio of the DTSM was defined as the ratio of the extracted protein to the total
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protein content of the DTSM. The ratio of the extracted protein was calculated as follow:
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Protein extraction ratio (%) = Msp/ Mtp *100.
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Where Msp is the dry mass of protein in the supernatant, g; Mtp is the dry mass of protein in the
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defatted meal, g.
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2.5. Determination of total free amino acids
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The free amino acid compositions were determined according to the methods of Zhu et al. (2008)
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with some revisions. The hydrolysate samples were precipitated with 10% sulfosalicyclic acid for
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2 h and then centrifuged at 11,000 g for 15 min. The pH of supernatant was adjusted to 2.0, and
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the solution was passed through a microfiltration membrane (0.45 µm). After precolumn
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derivatizing with phthalic dicarboxaldehyde (OPA), the filtrate was subjected to a reversed-phase
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high performance liquid chromatography SYKAM Amino Acid Analyzer S 433D (Sykam GmbH;
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Kleinostheim, Germany) with PEEK column (4.6×150 mm, 7µm, 10% crosslink) to determine the
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free amino acid compositions. Triplicate experiments were performed and average values are
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reported.
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2.6. Experimental design of single factor tests
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In order to determine the effective range of each factor, the response surface experimental method
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was used according to previous investigations (Zhang et al., 2012). The effects of enzyme activity,
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hydrolysis temperature, hydrolysis time, and pH on the concentrations of umami taste amino acids
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were determined. To study the effect of enzyme activity, hydrolysis time, hydrolysis temperature,
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pH were set as 3 h, 50 °C, 5.5, respectively, when the [E]/[S] values were varied as 1.73%, 3.47%,
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5.20%, 8.67%, and 13.8%. To study the pH effect, the [E]/[S], hydrolysis time, and hydrolysis
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temperature were set as 5.20%, 3h, and 50 °C, respectively, when the pH values were varied as 4,
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5, 6, 7, and 8. To study hydrolysis temperature effect, the [E]/[S], hydrolysis time, and pH were set
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as 5.20%, 3h, and 5.5, respectively, when the hydrolysis temperatures were changed as 40 °C,
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50 °C, 60 °C, 70° C, and 80 °C. Similarly, to determine the effect of hydrolysis time, the [E]/[S],
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hydrolysis temperature, and pH were set as 5.20%, 50 °C, and 5.5, respectively, when the
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hydrolysis times were varied as 1, 3, 5, 7, and 9 h.
174 2.7. Response surface experimental design
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Based on the results of the single factor tests, a series of experiments were conducted using the
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response surface methodology (RSM) to optimize the hydrolysis conditions. The non-coded
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values of four independent variables by Central Composite (Uniform Precision) Rotatable Design
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are described in Table 1 and used to determine the response pattern and establish a model. The
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four variables were enzyme activity (X1), hydrolysis pH (X2), hydrolysis temperature (X3), and
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hydrolysis time (X4), with 5 levels of each variable. The dependent variables were concentrations
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of glutamine acid (Y1, CGA) and aspartic acid (Y2, CAA).
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183 2.8. Statistical analysis
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All the tests were conducted in triplicate. The results were analyzed by ANOVA at a significance
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level of 5% (H0: P<0.05). The comparison of means was analyzed by Fisher’s LSD tests using the
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SAS statistical package.
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3. Results and discussion
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3.1. Particle size distribution of defatted tomato seed meal
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The particle size distribution of DTSM is shown in Fig. 1. The results indicated that the largest
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portion of DTSM had particle sizes between 0.85 mm ~ 0.43 mm and the second largest portion
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had particle sizes between 0.43 mm ~ 0.25 mm. The cumulative portion of DTSM having particle
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size ≥ 0.43 mm was 70% which showed 70% of DTSM particles were ≥ 0.43 mm in size. Because
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the particle size of substance influences hydrolysis rate and efficiency, and substrate rheological
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properties (Dimock & Morgenroth, 2006; Yeh et al., 2010), it is vital to test the effect of particle
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sizes on the protein extraction of DTSM. The results of particle size distribution provide
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information on appropriate particle size required to optimize the extraction yield and also on the
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need for further grinding of DTSM into fine particles to improve the extraction efficiency.
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ACCEPTED MANUSCRIPT 3.2. Effect of particle size on protein extraction
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The results indicated that hydrolysis of DTSM having smaller particles (less than 0.25 mm) did
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not significantly affect the protein extraction ratio. However, when DTSM samples with larger
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particles (>0.25 mm) were hydrolyzed, the protein extraction ratio decreased with increase in
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particle size and this trend has become more obvious with the increase in hydrolysis time to 3 and
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5 h. Hydrolysis of fine particles of DTSM resulted in a higher protein extraction ratio, which is
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consistent with the results of previous investigations (Yeh, et al., 2010). The main reason for such
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results is that the fine particles have more specific surface area available for hydrolysis compared
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to larger particles (Dimock & Morgenroth, 2006).
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The statistical analyses showed that DTSM with less than 0.25 mm particle size resulted in
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significantly higher (P<0.05) protein extraction ratio than large particles (≥ 0.25mm). Therefore,
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DTSM was ground into powder with particle size of ≤ 0.25 mm for extracting protein and prepare
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umami taste amino acids. The protein extraction ratio obtained for DTSM with particle sizes of
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0.25 mm ~ 0.18 mm, 0.18 mm ~ 0.15 mm, and <0.15 mm were 85.64%, 93.49% and 87.21%,
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respectively, for 5 h of hydrolysis. These protein extraction ratios were nearly 20% higher than the
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previously reported extraction yield of 66.10% obtained by optimized chemical method (Robak,
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2006). Therefore, the enzymatic hydrolysis is a promising alternative method to extract protein
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from DTSM and capable of producing higher protein yields than the chemical extraction method.
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3.3. Effect of hydrolysis conditions on concentration of umami taste amino acids
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3.3.1. Effect of enzyme activity on concentration of umami taste amino acids
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The results shown in Fig. 2A indicated that the concentration of glutamic acid, aspartic acid, and
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the sum of the two acids (MSG-like) increased with the increase in enzyme activity. Similar trend
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was found by Selvakumar et al. (2012) who used enzymatic hydrolysis of bovine hide to recycle
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collagen and its hydrolysate. The concentration of glutamic acid, aspartic acid, and the sum of two
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acids were significantly (P<0.05) higher at the [E]/[S] of 8.67% and 13.88% (Fig.2A) compared to
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other [E]/[S] values studied. The concentrations of glutamic acid obtained by extractions at the
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[E]/[S] activities of 8.67% and 13.88% were 29.70% and 25.90%, respectively, higher than the
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ACCEPTED MANUSCRIPT glutamic acid concentration obtained by extractions at the [E]/[S] activity of 5.20%. The
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concentrations of aspartic acid obtained at the [E]/[S] activities of 8.67% and 13.88% were
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17.10% and 7.20%, respectively, higher than those values obtained for the [E]/[S] of 5.20%.
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Similarly, the concentrations of the sum of glutamic acid and aspartic acid obtained at the [E]/[S]
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activities of 8.67% and 13.88% were 26.90% and 21.70%, respectively, higher than those values
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obtained for the [E]/[S] of 5.20%. Therefore, these results indicated that the [E]/[S] values of
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5.20%~13.88% were the enzyme activity range which could have the response value near the
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optimum.
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3.3.2. Effect of hydrolysis pH on concentration of umami taste amino acids
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The statistical analysis in Fig.2B indicated that low pH values resulted in a significantly higher
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(P<0.05) concentration of glutamic acid. The sum of the glutamic and aspartic acids
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concentrations decreased significantly (P<0.05) as the pH was increased from 4 to 8. Similar
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result was obtained for the hydrolysis of protein from rawhide with papain (Damrongsakkul et al.,
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2008). The concentration of aspartic acid was significantly (P<0.05) higher at pH value of 6 than
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pH values of 7 and 8. There was no significant (P>0.05) difference in the concentration of aspartic
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acid and the sum of the glutamic and aspartic acids for hydrolysis at pH values of 4, 5, and 6.
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These results suggested that papain hydrolysis performed between pH 4 and pH 6 could result in
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the response value near the optimum.
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3.3.3. Effect of temperature on concentration of umami taste amino acids
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Effect of hydrolysis temperature on the concentration of umami taste amino acids is shown in Fig.
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2C. The concentrations of glutamic acid and the sum of the glutamic and aspartic acids increased
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significantly (P<0.05) with the increase in hydrolysis temperature from 40 °C to 60 °C. The
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concentrations of glutamic acid and the sum of the glutamic and aspartic acids decreased
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significantly (P<0.05) at temperature of 70 °C in comparison to 60 °C. No significant (P>0.05)
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differences in the concentrations of glutamic acid and the sum of the glutamic and aspartic acids
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were noticed for the hydrolysis temperatures of 70 °C and 80 °C. The concentration of aspartic
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acid did not change significantly (P>0.05) as the hydrolysis temperature was increased from 40 °C
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to 80 °C. These results suggested that papain hydrolysis performed between temperature 40 °C
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and 60 °C would result in the response value near the optimum.
261 Generally, the concentrations of the glutamic acid and aspartic acid should increase and stabilize
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at higher value as the hydrolysis temperature is increased. But, the results obtained from this study
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were not as expected (Fig. 2C). The possible reason for the results might be that concentrations of
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umami taste amino acids were influenced not only by the hydrolysis temperature but also by the
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carbohydrates released during hydrolysis. Hydrolysis of chicken protein with papain at different
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temperatures showed that the increase in heating temperature led to a concomitant decrease in the
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content of SH groups and a steady increase in the content of S-S bonds (Wang et al., 2009).
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Furthermore, except protein, other major components of the DTSM such as fiber, fat residues, and
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minerals like phosphorus, calcium and magnesium could also play a role during hydrolysis (Rao,
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1991). Significant losses of protein-bound amino acids have been reported in nutritional studies
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which involved heating of diets, and these losses have subsequently been attributed to the
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presence of carbohydrates (Bohak, 1964). Both glutamic and aspartic acids have two carboxylic
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side chains which are chemically active (Dookeran et al., 1996) and could easily react with the
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exposed hydroxyls in fiber, forming weak chemical bonds. All these factors might be the reasons
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for the unexpected changes in concentrations of amino acids during hydrolysis which needs
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further specific investigations.
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3.3.4. Effect of hydrolysis time on concentration of umami taste amino acids
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Effect of hydrolysis time on concentration of umami taste amino acids is shown in Fig.2D. The
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concentrations of glutamic acid and the sum of the glutamic and aspartic acids increased
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significantly (P<0.05) as the hydrolysis time was increased from 1 h to 5 h. The concentrations of
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glutamic acid and the sum of the glutamic and aspartic acids decreased significantly (P<0.05) as
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the hydrolysis time was increased from 5 h to 7 h. The concentration of glutamic acid and the sum
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of the glutamic and aspartic acids increased slightly as the hydrolysis time was further increased to
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9 h. The concentration of aspartic acid increased slightly as the hydrolysis time increased from 1 h
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to 3 h, but decreased significantly (P<0.05) as the hydrolysis time was increased from 3 h to 7 h,
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and the concentration value at 9 h remained the same as that of 7 h. According to the significance
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analysis, it is suggested that papain hydrolysis time between 3 h and 5 h could result in the
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response value near the optimum.
291 Theoretically, the concentrations of the glutamic acid, aspartic acid, and the sum of the two acids
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should increase and then stabilize with the increase in the hydrolysis time, but the results in Fig.
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2D indicated a different trend. Cui et al. (2009) found that heat treatment and heating time showed
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similar effect on the hydrolysis of proteins from chicken breast meat. Similar results were also
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obtained when the viscera of Persian sturgeon (Acipenser persicus) was hydrolyzed by Alcalase
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proteinase (Ovissipour et al., 2009). Therefore, the possible reasons for the effect of hydrolysis
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time on the concentrations of the glutamic acid, aspartic acid, and the sum of them might be
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similar to those of the hydrolysis temperature.
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3.4. Process optimization of hydrolysis of DTSM
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3.4.1. Experimental results and ANOVA of response surface experimental results
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According to the results of the single factor experiments, a response surface experiment was
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designed with 31 runs as shown in Table 1. The ANOVA of the fitting results (Table 2) indicated
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that both enzyme activity (X1) and pH (X2) influenced the concentration of glutamic acid (CGA)
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and also the concentration of aspartic acid (CAA) significantly (P=0.0001<0.05). The P value of
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X3= 0.181 and X4= 0.228, both >0.05 for CGA indicated that CGA was not significantly
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influenced by both hydrolysis temperature and time. Similarly, the P values of X3= 0.924 and X4=
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0.992, both >0.05 for CAA indicated that the CAA was not significantly influenced by both
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hydrolysis temperature and time.
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The ANOVA of the master model (P=0.0001 <0.05) for both CGA and CAA indicated that the
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model obtained for CGA and CAA, correlated the hydrolysis parameters with the CGA and CAA
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values significantly. Combining the ANOVA of the master models with the P values of lack of fit
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in Table 2 (PCGA=0.055 >0.05, PCAA=0.339 >0.05)), and the R2 of CGA and CAA values (0.964
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and 0.857), it can be concluded that the obtained models could be feasibly used for optimizing the
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hydrolysis conditions for preparation of umami taste amino acids from DTSM (Cheng et al.,
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2012).
319 The final models obtained are shown as formula (1) and (2).
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Y1 = -835.17 + 59.49556*X1 + 92.04182*X2 + 15.39241*X3 + 102.7861*X4 + 0.1299946*X1*X1
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-8.525202*X1*X2 – 0.1287298*X1*X3 +0. 6061348*X1*X4 - 2.127723*X2*X2 - 0.251188*X2*X3
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-9.219375*X2*X4 - 0.085827*X3*X3- 0.833688*X3*X4 - 0.742723*X4*X4
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Y2 = -78.7129 + 12.91026*X1 + 8.379215*X2 + 0.991718*X3 + 18.13591*X4 + 0.06466194*X1*X1 –
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1.581797*X1*X2 -0.0 6206797*X1*X3 – 0.1273041*X1*X4 + 0.777946*X2*X2 + 0.083625*X2*X3 -
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4.295*X2*X4 - 0.009008*X3*X3 + 0.061875*X3*X4 + 0.102946*X4*X4
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(2)
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(1)
3.4.2. Verification of the fitting model
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In order to further verify the reliability of the obtained model, the experimentally determined CGA
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and CAA values were compared with the calculated CGA and CAA (data not show) values. Most
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of the calculated values were very close to the determined values, and the largest relative error
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was less than 23%. This suggested that the obtained models are reliable to be used for predicting
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the optimal hydrolysis conditions to prepare protein with highest CGA and CAA.
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3.4.3. Results of process optimization
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The optimal hydrolysis condition was predicted based on the fitting models. The optimization
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results indicated that the optimal hydrolysis conditions for preparing the highest concentrations of
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glutamic acid and aspartic acid were enzyme activity of 18.22 %; pH of 3; hydrolysis temperature
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of 40 °C; hydrolysis time of 6 h and the corresponding CGA and CAA values were 727.6 µg/ mL
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and 149.9 µg/ mL, respectively. The changes of the CGA and CAA values can be visualized in the
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response surfaces (Fig. 3).
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Previous optimization studies of enzymatic hydrolysis process with response surface methodology
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indicated that the key factors influencing the hydrolysis were mainly decided by the purpose. The
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enzyme activity and pH greatly influenced the papain hydrolysis process of jellyfish umbrella
ACCEPTED MANUSCRIPT collagen (Zhuang et al., 2009), whereas, pH and temperature were the most important factors of
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the Alcalase hydrolysis process optimized by response surface methodology (Guerard et al., 2007).
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The pH was found to be the most important factor in the hydrolysis of shrimp waste with papain to
350
recover chitin (Diniz & Martin, 1996). Therefore, pH and enzyme activity should be strictly
351
monitored and controlled for the optimal processing of papain hydrolysis of DTSM in large scale
352
applications.
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353 4. Conclusions
355
The protein extraction ratio of DTSM could reach to more than 85.64% when the DTSM with
356
particle sizes of ≤ 0.25 mm was hydrolyzed by papain for 5 h. The results proved that the
357
enzymatic hydrolysis is a promising method to extract protein from DTSM. The optimization
358
results showed that the optimal hydrolysis condition for preparing the highest concentration of
359
glutamic acid and aspartic acid were enzyme activity of 18.22%; pH of 3; hydrolysis temperature
360
of 40 °C; and hydrolysis time of 6 h, and the corresponding concentration of the glutamic acid and
361
aspartic acid obtained were 727.6 µg/ mL and 149.9 µg/ mL, respectively. The pH and enzyme
362
activity were the most important factors affecting the hydrolysis of DTSM using papain, which
363
need to be monitored for during papain hydrolysis of DTSM in large scale applications.
366 367 368 369 370 371 372 373 374 375
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evaluation. The Journal of nutrition, 83(3), 257-261. Bohak, Z. (1964). Nε-(DL-2-amino-2-carboxyethyl)-L-lysine, a new amino acid formed on alkaline treatment of proteins. Journal of Biological Chemistry, 239(9), 2878-2887. Brodowski, D., & Geisman, J. (1980). Protein content and amino acid composition of protein of seeds
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Persian sturgeon (Acipenser persicus) viscera. Food chemistry, 115(1), 238-242.
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Sogi, D. S., Bhatia, R., Garg, S. K., & Bawa, A. S. (2005). Biological evaluation of tomato waste seed
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defatted rice bran. 1. Physical processing and enzyme treatments. Journal of agricultural and food
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chemistry, 50(25), 7444-7448.
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Wang, J.-s., Wei, Z.-y., Li, L., Bian, K., & Zhao, M.-m. (2009). Characteristics of enzymatic hydrolysis of thermal-treated wheat gluten. Journal of Cereal Science, 50(2), 205-209. Yamaguchi, S., & Ninomiya, K. (2000). Umami and food palatability. The Journal of nutrition, 130(4), 921S-926S.
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Yeh, A.-I., Huang, Y.-C., & Chen, S. H. (2010). Effect of particle size on the rate of enzymatic hydrolysis of cellulose. Carbohydrate Polymers, 79(1), 192-199. Zhang, Y., Venkitasamy, C., Pan, Z., & Wang, W. (2013). Recent developments on umami ingredients of edible mushrooms–A review. Trends in Food Science & Technology, 33(2), 78-92. Zhang, Y., Wang, W., Wang, X., & Zhang, J. (2014). Bone soup: protein nutrition and enzymatic
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hydrolysis process optimized by response surface method. Journal of Food & Nutrition Research,
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53(1), 1-12.
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Zhang, Y., Wang, W., Zhang, J.-m., & Liu, D.-y. (2012). Application of Response Surface Method to
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Optimizing the Hydrolysis Processing of Pig Bone Extract by Papaya Protease. The Food
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Industry(5), 57-59.
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Zhu, L., Chen, J., Tang, X., & Xiong, Y. L. (2008). Reducing, radical scavenging, and chelation
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properties of in vitro digests of alcalase-treated zein hydrolysate. Journal of agricultural and food
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chemistry, 56(8), 2714-2721.
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Zhuang, Y.-l., Zhao, X., & Li, B.-f. (2009). Optimization of antioxidant activity by response surface
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methodology in hydrolysates of jellyfish (Rhopilema esculentum) umbrella collagen. Journal of
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Zhejiang University SCIENCE B, 10(8), 572-579.
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ACCEPTED MANUSCRIPT Table 1 Central composite rotatable experimental design and results RUN
X1: Enzyme
X2: pH
X4: Time
Y1: CGA
Y2: CAA
(°C)
(h)
(µg/mL)
(µg/mL)
1
5.20
4
50
3
196.96±9.96
46.00±1.64
2
5.20
4
50
5
238.89±14.89
53.99±8.42
3
5.20
4
70
3
213.06±12.11
47.79±0.68
4
5.20
4
70
5
232.26±14.02
62.89±5.27
5
5.20
6
50
3
6
5.20
6
50
5
7
5.20
6
70
3
8
5.20
6
70
5
9
13.88
4
50
3
10
13.88
4
50
5
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activity (%)
X3:Temperature
11
13.88
4
70
12
13.88
4
70
13
13.88
6
50
14
13.88
6
15
13.88
6
16
13.88
6
17
0.86
5
18
18.22
5
19
9.54
3
20
9.54
7
21
9.54
22
9.54
23
9.54
24
9.54
25
9.54
26
9.54
27 28
44.79±4.79
152.88±8.79
29.16±1.64
143.61±14.11
42.11±3.26
141.87±7.85
40.34±4.40
375.79±8.50
80.56±2.32
474.68±4.24
96.30±24.79
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154.60±10.06
405.71±22.52
5
398.16±14.10
76.96±2.83
3
204.21±3.12
49.40±4.12
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3
80.16±8.95
5
210.52±7.85
37.62±1.38
70
3
178.65±7.34
43.38±12.31
70
5
180.76±9.11
39.47±1.77
60
4
120.84±6.82
33.91±2.10
60
4
375.42±10.06
69.77±1.01
60
4
312.81±1.83
62.05±5.68
60
4
146.84±18.14
38.11±0.24 43.13±1.53
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50
40
4
218.93±2.10
5
80
4
189.08±13.93
43.60±2.76
5
60
2
235.67±1.46
48.12±1.09
5
60
6
235.06±43.31
46.64±8.00
5
60
4
231.31±20.80
64.69±7.68
5
60
4
270.48±16.52
54.35±4.16
9.54
5
60
4
238.08±7.49
45.12±1.89
9.54
5
60
4
238.74±4.94
49.69±1.58
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5
29
9.54
5
60
4
253.71±7.56
48.71±2.62
30
9.54
5
60
4
238.78±3.35
43.48±1.24
31
9.54
5
60
4
265.30±15.52
51.98±4.51
ACCEPTED MANUSCRIPT Table 2 Fittingness ANOVA of response surface experimental results ANOVA for Y1(CGA)
ANOVA for Y1 (CAA)
Source
DF
MS
F
Pr> F
DF
MS
F
Pr> F
X1
1
89244.23
137.9945
0.0001
1
1811.344
28.62853
0.0001
X2
1
93793.76
145.0292
0.0001
1
2953.933
46.6873
0.0001
X3
1
1263.676
1.953967
0.18124
1
0.59535
X4
1
1016.732
1.572128
0.227909
1
0.00735
X1*X1
1
171.4396
0.26509
0.613684
1
X1*X2
1
21903.26
33.86806
0.0001
1
X1*X3
1
499.4108
0.772217
0.392538
1
X1*X4
1
110.723
0.171206
0.684535
1
X2*X2
1
129.4588
0.200177
0.660576
X2*X3
1
100.9523
0.156098
X2*X4
1
1359.95
X3*X3
1
X3*X4
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Master
0.923929
0.000116
0.991534
42.41874
0.670434
0.424929
754.0516
11.91789
0.003278
116.1006
1.834986
0.194361
4.8841
0.077194
0.784695
1
17.30616
0.273526
0.608143
0.697992
1
11.18903
0.176844
0.679693
2.102831
0.166347
1
295.1524
4.664923
0.046306
2106.452
3.257115
0.089968
1
23.20393
0.366741
0.55328
1
1112.056
1.719523
0.208262
1
6.125625
0.096816
0.759701
X4*X4
1
15.77449
0.024391
0.877847
1
0.303057
0.00479
0.945681
Model
14
212907.1
23.51493
0.0001
14
6042.819
6.821969
0.000239
(Linear)
4
185318.4
71.63744
0.0001
4
4765.879
18.83134
0.0001
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4
2502.344
0.967317
0.452259
4
89.43648
0.353389
0.837891
6
25086.35
6.464989
0.001307
6
1187.503
3.128108
0.031795
16
1012.329
10
714.794
1.44143
0.339159
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(Cross
EP
(Quadratic)
SC
0.00941
Product) Error
16
10347.57
(Lack of fit)
10
8962.632
(Pure Error)
6
1384.94
6
297.5354
Total
30
223254.7
30
7055.148
R2
0.9638
3.882896
0.055242
0.8565
ACCEPTED MANUSCRIPT FIGURE CAPTIONS: Fig.1. A. Particle size distribution of defatted tomato seed meal and B. Effect of particle size on protein extraction of defatted tomato seed meal
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Fig.2 Effect of hydrolysis conditions on concentration of umami taste amino acids Fig.2A Effect of enzyme activity on concentration of umami taste amino acids (hydrolysis time, hydrolysis temperature and pH were set as 3 h, 50 °C and 5.5, respectively), MSG-like: Sum of
the concentrations of glutamic and aspartic acids; Fig.2B Effect of hydrolysis pH on concentration
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of umami taste amino acids (the [E]/[S], hydrolysis time, and hydrolysis temperature were set as 5.20%, 3h and 50 °C, respectively); Fig.2C Effect of temperature on concentration of umami
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taste(the [E]/[S], hydrolysis time and pH were set as 5.20%, 3h and 5.5, respectively); Fig.2D Effect of hydrolysis time on concentration of umami taste amino acids (the [E]/[S], hydrolysis temperature, and pH were set as 5.20%, 50 °C, and 5.5, respectively)
Fig.3 Response surface of the optimization of protein hydrolysis from DTSM
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X1: Enzyme activity (%) , X2: pH, X3: Temperature (°C), X4: Time (h), Y1: CGA, Y2: CAA
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ACCEPTED MANUSCRIPT
A
B
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Fig.1. A. Particle size distribution of defatted tomato seed meal and B. Effect of particle size on
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protein extraction of defatted tomato seed meal
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B
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D
Fig.2 Effect of hydrolysis conditions on concentration of umami taste amino acids
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Fig.2A Effect of enzyme activity on concentration of umami taste amino acids (hydrolysis
time, hydrolysis temperature, and pH were set as 3 h, 50 °C, and 5.5, respectively), MSG-like: Sum of the concentrations of glutamic and aspartic acids; Fig.2B Effect of hydrolysis pH on concentration of umami taste amino acids (the [E]/[S], hydrolysis time, and hydrolysis
temperature were set as 5.20%, 3h, and 50 °C, respectively); Fig.2C Effect of temperature on concentration of umami taste amino acids ( the [E]/[S], hydrolysis time, pH were set as 5.20%, 3h, and 5.5, respectively); Fig.2D Effect of hydrolysis time on concentration of umami taste amino acids (the [E]/[S], hydrolysis temperature, and pH were set as 5.20%, 50 °C, and 5.5, respectively)
A2
A3
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B2
B3
Fig.3 Response surface of the optimization of protein hydrolysis from DTSM
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X1: Enzyme activity (%) , X2: pH, X3: Temperature (°C), X4: Time (h), Y1: CGA, Y2: CAA
ACCEPTED MANUSCRIPT Highlights 1. Enzymatic hydrolysis for extracting defatted tomato seed meal protein 2. Hydrolyzing extracted protein to prepare umami taste amino acids
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3. Optimizing hydrolysis process to obtain maximum amino acid concentration
S0023-6438(15)00075-4
DOI:
10.1016/j.lwt.2015.02.003
Reference:
YFSTL 4440
To appear in:
LWT - Food Science and Technology
Received Date: 8 August 2014 Revised Date:
16 January 2015
Accepted Date: 3 February 2015
Please cite this article as: Zhang, Y., Pan, Z., Venkitasamy, C., Ma, H., Li, Y., Umami taste amino acids produced by hydrolyzing extracted protein from tomato seed meal, LWT - Food Science and Technology (2015), doi: 10.1016/j.lwt.2015.02.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Umami taste amino acids produced by hydrolyzing
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extracted protein from tomato seed meal
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Yin Zhang 1, 2, Zhongli Pan2, 3∗, Chandrasekar Venkitasamy2, Haile Ma4, 5, Yunliang Li4
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Key Laboratory of Meat Processing of Sichuan, Chengdu University, Chengdu 610106, China
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2
Department of Biological and Agricultural Engineering, University of California, Davis, One Shields
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Avenue, Davis, CA 95616, USA
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3
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USA
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Healthy Processed Foods Research Unit, USDA-ARS-WRRC, 800 Buchanan St., Albany, CA 94710,
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4
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212013, China
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Zhenjiang, Jiangsu 212013, China
Jiangsu Provincial Research Center of Bio-process and Separation Engineering of Agri-products,
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School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu
24 25 26 ∗ Corresponding author. Address: Processed Foods Research Unit, USDA–ARS West Regional Research Center, 800 Buchanan Street, Albany, Berkeley, CA 94710, USA. Tel.: +1 510 559 5861; fax: +1 510 559 5851. E-mail addresses: [email protected], [email protected] (Z. Pan).
ACCEPTED MANUSCRIPT Abstract: Enzymatic hydrolysis was performed for extracting protein to prepare umami taste
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amino acids from defatted tomato seed meal (DTSM) which is a by-product of tomato processing.
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Papain was used as an enzyme for the hydrolysis of DTSM. The particle size distribution of
30
DTSM, protein concentration and free amino acids of the hydrolysate were analyzed. Response
31
surface methodology was used in the design of experiments and analysis of results. The protein
32
extraction ratio of greater than 85.64% was obtained when the DTSM with particle size ≤ 0.25
33
mm was hydrolyzed by papain for 5 h. The optimum extraction conditions to produce maximum
34
concentration of umami taste amino acids were enzyme activity of 18.22 %; pH of 3; hydrolysis
35
temperature of 40 °C; and hydrolysis time of 6 h, which resulted in glutamic acid and aspartic acid
36
concentrations of 727.6 µg/ mL and 149.9 µg/ mL, respectively.
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Key words: umami taste; amino acids; glutamic acid; aspartic acid; tomato seed meal
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ACCEPTED MANUSCRIPT 1. Introduction
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Palatability plays a major role in food selection, intake, absorption and digestion. Glutamate, one
58
of the basic umami taste compounds, is a major ingredient to make food tasty, palatable and
59
acceptable (Zhang et al., 2013). Tomato contains higher content of glutamate procurer glutamic
60
acid than other vegetables (Yamaguchi & Ninomiya, 2000). This might be the possible reason for
61
the popularity of tomato as a major vegetable, processed into sauces and often used as food
62
condiment. Nearly one third of the cultivated tomatoes are processed (Cantarelli et al., 1993),
63
leading to generation of huge volumes of tomato pomace as a by-product.
65
About 3 to 5% (in weight) of fresh tomato is generated as pomace which consists approximately
66
60% of seed and 40% of peel (Celmaa et al., 2009). Generally, the tomato pomace is used as
67
livestock fodder or soil amendment or disposed directly into landfill, causing environmental
68
problems and resource loss (Sogi et al., 2005). Recent investigation indicated that defatted tomato
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seed meal protein has the cholesterol-lowering effect in male golden Syrian hamster (Shao et al.,
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2013), which motivated research in the recovery and utilization of tomato seed protein.
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Alkaline treatment is the traditionally followed procedure for extraction of proteins from tomato
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seed meal and other complex plant matrices, such as industrial by-products (Robak, 2006).
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However, chemical methods could induce side-reactions, such as hydrolysis and extraction of
75
non-protein components, and denaturation of protein with obvious effects on their functional
76
properties (Kinsella, 1981). In order to overcome those drawbacks, enzymatic methods were
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developed (Tang et al., 2002), and preferred for their milder process conditions, easier control of
78
reaction parameters and minimal secondary product formation (Mannheim & Cheryan, 1992). To
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our knowledge, very limited information is available regarding the use of enzymatic hydrolysis to
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extract protein from the byproducts of tomato processing industries, like DTSM, to develop new
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products.
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The glutamic and aspartic acids are present at 18.99g/ 100g and 11.95 g/ 100g, respectively, in red
84
tomato seed meal (Brodowski & Geisman, 1980). They are known as umami taste amino acids or
ACCEPTED MANUSCRIPT monosodium glutamate like (MSG-like) amino acids (Zhang, et al., 2013). These facts imply that
86
the tomato seed meal could be used to develop products rich in umami flavors for both vegetarians
87
and non-vegetarians. However, little research has been done on the development and optimization
88
of processes for preparing flavor components from DTSM using papain hydrolysis and this
89
research will be the first of its kind. Previous investigations revealed that use of papain resulted in
90
a higher degree of hydrolysis of whey and egg protein compared with the other enzymes such as
91
pepsin and trypsinase (Akeson & Stahmann, 1964; Pena-Ramos & Xiong, 2001; Zhang et al.,
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2014). Therefore, papain was used for hydrolysis of protein from DTSM in this study.
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The objectives of our research were 1) to investigate the effect of enzymatic hydrolysis on protein
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extraction from DTSM, 2) to examine the effects of temperature, pH, time and enzyme activity on
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the concentrations of umami taste amino acids, and 3) to determine the optimal conditions for
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preparing high concentration of umami taste amino acids with an enzymatic hydrolysis method.
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2. Materials and methods
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2.1. Materials
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Tomato pomace produced by a popular hot break process was collected from the tomato
102
processing plant of Morningstar Company located in Williams, CA. The pomace samples were
103
stored in the freezer (-8° C) until it was used. Its crude composition was determined as 95.4% dry
104
matter, 22.72% acid detergent fiber, 28.40% crude protein, 58.72% total digestible nutrients,
105
21.32% crude fat, and 3.74% ash. Papain obtained from Carica papaya with enzyme activity of
106
1.937 U/mg (the enzyme unit U refers to amount of protease needed to hydrolysis 1 µmol of
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casein in 1 min) was used for hydrolysis. All other chemicals used for this study were of analytical
108
grade and obtained from Fisher Scientific Inc. (Pittsburgh, PA., U.S.A.).
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2.2. Preparation of tomato seeds and defatted tomato seed meal
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The tomato pomace was thawed at 4 °C and then dried at 40 °C in an oven (637G, Colorado
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Springs Utilities Investment Recovery, Colorado Springs, CO, USA) for 24 h to a moisture
113
content of 5.0 ± 0.2%. The seeds were separated from the dried pomace samples using an aspirator
ACCEPTED MANUSCRIPT system (FC2K testing husker, YAMANMOTO, CO., Higashine, Japan). Sample of 70 g of seeds
115
were ground to powder using a mill (M2 Stein Mill, The Steinlite Corporation, Atchison, KS, USA)
116
for 30 s and sieved in a Tyler Sieve Shaker (RO-TAP Testing Sieve Shaker, W.S. Tyler Co.,
117
Cleveland, OH, USA) with a 14 mesh opening sieve. The sieved powder was defatted with hexane
118
(1 g meal: 10 mL hexane) for 4 h. The supernatant was evaporated to recover and recycle the
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hexane. The solid residues were placed in fume hood to remove residual hexane until there was no
120
hexane odor and then packed in a ziplock plastic bag for later use. The residual fat content of the
121
DTSM was determined as 9.49 ± 0.13%.
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124
A sieve shaker (Retsch Sieve Shaker AS 300, Haan, Germany) was used to determine the particle
125
size distribution of DTSM. A sample of 330 g DTSM was loaded to the shaker with 14, 18, 20, 40,
126
60, 80, 100 mesh sieves and shaken for 5 min at an amplitude of 1.36 mm/”g”. The meal left onto
127
each sieve was weighed and the size distribution was calculated.
128
Weight of particles retained on sieve (%) = Mx/ Mt*100
129
Where, Mx is the mass of meal left onto one sieve or bottom tray, g; Mt is the total weight of
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defatted meal, g.
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Triplicate experiments were performed and the average values are reported.
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2.4. Effect of particle size on protein extraction
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To determine the effect of particle size on protein extraction, each sample with different particle
135
sizes was extracted with the ratio of 2 g meal / 20 mL buffer (phosphate buffer, composed of 0.2
136
mol/L Na2HPO4 and 0.1 mol/L citric acid). The papain hydrolysis conditions were pH of 5.4,
137
temperature of 54 ºC, and [E]/[S] of 67 U/g for 0 h, 1 h, 3 h, and 5 h in a reciprocal water bath
138
shaker (Model R 76, New Brunswick Scientific, Edison, N. J., U.S.A.). At the end of hydrolysis,
139
the mixture was heated in boiling water for 20 min to inactivate the protease. The mixture was
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centrifuged with a centrifuge (Eppendorf centrifuge 5804R, Eppendorf North America, Inc., USA)
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at 9000 ×g for 10 min, and then the protein content of supernatant was determined with Biuret
142
method (Nowotny, 1979). Triplicate experiments were performed and average values were
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Protein extraction ratio of the DTSM was defined as the ratio of the extracted protein to the total
145
protein content of the DTSM. The ratio of the extracted protein was calculated as follow:
146
Protein extraction ratio (%) = Msp/ Mtp *100.
147
Where Msp is the dry mass of protein in the supernatant, g; Mtp is the dry mass of protein in the
148
defatted meal, g.
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2.5. Determination of total free amino acids
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The free amino acid compositions were determined according to the methods of Zhu et al. (2008)
152
with some revisions. The hydrolysate samples were precipitated with 10% sulfosalicyclic acid for
153
2 h and then centrifuged at 11,000 g for 15 min. The pH of supernatant was adjusted to 2.0, and
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the solution was passed through a microfiltration membrane (0.45 µm). After precolumn
155
derivatizing with phthalic dicarboxaldehyde (OPA), the filtrate was subjected to a reversed-phase
156
high performance liquid chromatography SYKAM Amino Acid Analyzer S 433D (Sykam GmbH;
157
Kleinostheim, Germany) with PEEK column (4.6×150 mm, 7µm, 10% crosslink) to determine the
158
free amino acid compositions. Triplicate experiments were performed and average values are
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reported.
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2.6. Experimental design of single factor tests
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In order to determine the effective range of each factor, the response surface experimental method
163
was used according to previous investigations (Zhang et al., 2012). The effects of enzyme activity,
164
hydrolysis temperature, hydrolysis time, and pH on the concentrations of umami taste amino acids
165
were determined. To study the effect of enzyme activity, hydrolysis time, hydrolysis temperature,
166
pH were set as 3 h, 50 °C, 5.5, respectively, when the [E]/[S] values were varied as 1.73%, 3.47%,
167
5.20%, 8.67%, and 13.8%. To study the pH effect, the [E]/[S], hydrolysis time, and hydrolysis
168
temperature were set as 5.20%, 3h, and 50 °C, respectively, when the pH values were varied as 4,
169
5, 6, 7, and 8. To study hydrolysis temperature effect, the [E]/[S], hydrolysis time, and pH were set
170
as 5.20%, 3h, and 5.5, respectively, when the hydrolysis temperatures were changed as 40 °C,
171
50 °C, 60 °C, 70° C, and 80 °C. Similarly, to determine the effect of hydrolysis time, the [E]/[S],
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hydrolysis temperature, and pH were set as 5.20%, 50 °C, and 5.5, respectively, when the
173
hydrolysis times were varied as 1, 3, 5, 7, and 9 h.
174 2.7. Response surface experimental design
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Based on the results of the single factor tests, a series of experiments were conducted using the
177
response surface methodology (RSM) to optimize the hydrolysis conditions. The non-coded
178
values of four independent variables by Central Composite (Uniform Precision) Rotatable Design
179
are described in Table 1 and used to determine the response pattern and establish a model. The
180
four variables were enzyme activity (X1), hydrolysis pH (X2), hydrolysis temperature (X3), and
181
hydrolysis time (X4), with 5 levels of each variable. The dependent variables were concentrations
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of glutamine acid (Y1, CGA) and aspartic acid (Y2, CAA).
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183 2.8. Statistical analysis
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All the tests were conducted in triplicate. The results were analyzed by ANOVA at a significance
186
level of 5% (H0: P<0.05). The comparison of means was analyzed by Fisher’s LSD tests using the
187
SAS statistical package.
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3. Results and discussion
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3.1. Particle size distribution of defatted tomato seed meal
191
The particle size distribution of DTSM is shown in Fig. 1. The results indicated that the largest
192
portion of DTSM had particle sizes between 0.85 mm ~ 0.43 mm and the second largest portion
193
had particle sizes between 0.43 mm ~ 0.25 mm. The cumulative portion of DTSM having particle
194
size ≥ 0.43 mm was 70% which showed 70% of DTSM particles were ≥ 0.43 mm in size. Because
195
the particle size of substance influences hydrolysis rate and efficiency, and substrate rheological
196
properties (Dimock & Morgenroth, 2006; Yeh et al., 2010), it is vital to test the effect of particle
197
sizes on the protein extraction of DTSM. The results of particle size distribution provide
198
information on appropriate particle size required to optimize the extraction yield and also on the
199
need for further grinding of DTSM into fine particles to improve the extraction efficiency.
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ACCEPTED MANUSCRIPT 3.2. Effect of particle size on protein extraction
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The results indicated that hydrolysis of DTSM having smaller particles (less than 0.25 mm) did
203
not significantly affect the protein extraction ratio. However, when DTSM samples with larger
204
particles (>0.25 mm) were hydrolyzed, the protein extraction ratio decreased with increase in
205
particle size and this trend has become more obvious with the increase in hydrolysis time to 3 and
206
5 h. Hydrolysis of fine particles of DTSM resulted in a higher protein extraction ratio, which is
207
consistent with the results of previous investigations (Yeh, et al., 2010). The main reason for such
208
results is that the fine particles have more specific surface area available for hydrolysis compared
209
to larger particles (Dimock & Morgenroth, 2006).
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The statistical analyses showed that DTSM with less than 0.25 mm particle size resulted in
212
significantly higher (P<0.05) protein extraction ratio than large particles (≥ 0.25mm). Therefore,
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DTSM was ground into powder with particle size of ≤ 0.25 mm for extracting protein and prepare
214
umami taste amino acids. The protein extraction ratio obtained for DTSM with particle sizes of
215
0.25 mm ~ 0.18 mm, 0.18 mm ~ 0.15 mm, and <0.15 mm were 85.64%, 93.49% and 87.21%,
216
respectively, for 5 h of hydrolysis. These protein extraction ratios were nearly 20% higher than the
217
previously reported extraction yield of 66.10% obtained by optimized chemical method (Robak,
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2006). Therefore, the enzymatic hydrolysis is a promising alternative method to extract protein
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from DTSM and capable of producing higher protein yields than the chemical extraction method.
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3.3. Effect of hydrolysis conditions on concentration of umami taste amino acids
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3.3.1. Effect of enzyme activity on concentration of umami taste amino acids
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The results shown in Fig. 2A indicated that the concentration of glutamic acid, aspartic acid, and
224
the sum of the two acids (MSG-like) increased with the increase in enzyme activity. Similar trend
225
was found by Selvakumar et al. (2012) who used enzymatic hydrolysis of bovine hide to recycle
226
collagen and its hydrolysate. The concentration of glutamic acid, aspartic acid, and the sum of two
227
acids were significantly (P<0.05) higher at the [E]/[S] of 8.67% and 13.88% (Fig.2A) compared to
228
other [E]/[S] values studied. The concentrations of glutamic acid obtained by extractions at the
229
[E]/[S] activities of 8.67% and 13.88% were 29.70% and 25.90%, respectively, higher than the
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231
concentrations of aspartic acid obtained at the [E]/[S] activities of 8.67% and 13.88% were
232
17.10% and 7.20%, respectively, higher than those values obtained for the [E]/[S] of 5.20%.
233
Similarly, the concentrations of the sum of glutamic acid and aspartic acid obtained at the [E]/[S]
234
activities of 8.67% and 13.88% were 26.90% and 21.70%, respectively, higher than those values
235
obtained for the [E]/[S] of 5.20%. Therefore, these results indicated that the [E]/[S] values of
236
5.20%~13.88% were the enzyme activity range which could have the response value near the
237
optimum.
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3.3.2. Effect of hydrolysis pH on concentration of umami taste amino acids
240
The statistical analysis in Fig.2B indicated that low pH values resulted in a significantly higher
241
(P<0.05) concentration of glutamic acid. The sum of the glutamic and aspartic acids
242
concentrations decreased significantly (P<0.05) as the pH was increased from 4 to 8. Similar
243
result was obtained for the hydrolysis of protein from rawhide with papain (Damrongsakkul et al.,
244
2008). The concentration of aspartic acid was significantly (P<0.05) higher at pH value of 6 than
245
pH values of 7 and 8. There was no significant (P>0.05) difference in the concentration of aspartic
246
acid and the sum of the glutamic and aspartic acids for hydrolysis at pH values of 4, 5, and 6.
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These results suggested that papain hydrolysis performed between pH 4 and pH 6 could result in
248
the response value near the optimum.
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3.3.3. Effect of temperature on concentration of umami taste amino acids
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Effect of hydrolysis temperature on the concentration of umami taste amino acids is shown in Fig.
252
2C. The concentrations of glutamic acid and the sum of the glutamic and aspartic acids increased
253
significantly (P<0.05) with the increase in hydrolysis temperature from 40 °C to 60 °C. The
254
concentrations of glutamic acid and the sum of the glutamic and aspartic acids decreased
255
significantly (P<0.05) at temperature of 70 °C in comparison to 60 °C. No significant (P>0.05)
256
differences in the concentrations of glutamic acid and the sum of the glutamic and aspartic acids
257
were noticed for the hydrolysis temperatures of 70 °C and 80 °C. The concentration of aspartic
258
acid did not change significantly (P>0.05) as the hydrolysis temperature was increased from 40 °C
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to 80 °C. These results suggested that papain hydrolysis performed between temperature 40 °C
260
and 60 °C would result in the response value near the optimum.
261 Generally, the concentrations of the glutamic acid and aspartic acid should increase and stabilize
263
at higher value as the hydrolysis temperature is increased. But, the results obtained from this study
264
were not as expected (Fig. 2C). The possible reason for the results might be that concentrations of
265
umami taste amino acids were influenced not only by the hydrolysis temperature but also by the
266
carbohydrates released during hydrolysis. Hydrolysis of chicken protein with papain at different
267
temperatures showed that the increase in heating temperature led to a concomitant decrease in the
268
content of SH groups and a steady increase in the content of S-S bonds (Wang et al., 2009).
269
Furthermore, except protein, other major components of the DTSM such as fiber, fat residues, and
270
minerals like phosphorus, calcium and magnesium could also play a role during hydrolysis (Rao,
271
1991). Significant losses of protein-bound amino acids have been reported in nutritional studies
272
which involved heating of diets, and these losses have subsequently been attributed to the
273
presence of carbohydrates (Bohak, 1964). Both glutamic and aspartic acids have two carboxylic
274
side chains which are chemically active (Dookeran et al., 1996) and could easily react with the
275
exposed hydroxyls in fiber, forming weak chemical bonds. All these factors might be the reasons
276
for the unexpected changes in concentrations of amino acids during hydrolysis which needs
277
further specific investigations.
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3.3.4. Effect of hydrolysis time on concentration of umami taste amino acids
280
Effect of hydrolysis time on concentration of umami taste amino acids is shown in Fig.2D. The
281
concentrations of glutamic acid and the sum of the glutamic and aspartic acids increased
282
significantly (P<0.05) as the hydrolysis time was increased from 1 h to 5 h. The concentrations of
283
glutamic acid and the sum of the glutamic and aspartic acids decreased significantly (P<0.05) as
284
the hydrolysis time was increased from 5 h to 7 h. The concentration of glutamic acid and the sum
285
of the glutamic and aspartic acids increased slightly as the hydrolysis time was further increased to
286
9 h. The concentration of aspartic acid increased slightly as the hydrolysis time increased from 1 h
287
to 3 h, but decreased significantly (P<0.05) as the hydrolysis time was increased from 3 h to 7 h,
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and the concentration value at 9 h remained the same as that of 7 h. According to the significance
289
analysis, it is suggested that papain hydrolysis time between 3 h and 5 h could result in the
290
response value near the optimum.
291 Theoretically, the concentrations of the glutamic acid, aspartic acid, and the sum of the two acids
293
should increase and then stabilize with the increase in the hydrolysis time, but the results in Fig.
294
2D indicated a different trend. Cui et al. (2009) found that heat treatment and heating time showed
295
similar effect on the hydrolysis of proteins from chicken breast meat. Similar results were also
296
obtained when the viscera of Persian sturgeon (Acipenser persicus) was hydrolyzed by Alcalase
297
proteinase (Ovissipour et al., 2009). Therefore, the possible reasons for the effect of hydrolysis
298
time on the concentrations of the glutamic acid, aspartic acid, and the sum of them might be
299
similar to those of the hydrolysis temperature.
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300
3.4. Process optimization of hydrolysis of DTSM
302
3.4.1. Experimental results and ANOVA of response surface experimental results
303
According to the results of the single factor experiments, a response surface experiment was
304
designed with 31 runs as shown in Table 1. The ANOVA of the fitting results (Table 2) indicated
305
that both enzyme activity (X1) and pH (X2) influenced the concentration of glutamic acid (CGA)
306
and also the concentration of aspartic acid (CAA) significantly (P=0.0001<0.05). The P value of
307
X3= 0.181 and X4= 0.228, both >0.05 for CGA indicated that CGA was not significantly
308
influenced by both hydrolysis temperature and time. Similarly, the P values of X3= 0.924 and X4=
309
0.992, both >0.05 for CAA indicated that the CAA was not significantly influenced by both
310
hydrolysis temperature and time.
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The ANOVA of the master model (P=0.0001 <0.05) for both CGA and CAA indicated that the
313
model obtained for CGA and CAA, correlated the hydrolysis parameters with the CGA and CAA
314
values significantly. Combining the ANOVA of the master models with the P values of lack of fit
315
in Table 2 (PCGA=0.055 >0.05, PCAA=0.339 >0.05)), and the R2 of CGA and CAA values (0.964
316
and 0.857), it can be concluded that the obtained models could be feasibly used for optimizing the
ACCEPTED MANUSCRIPT 317
hydrolysis conditions for preparation of umami taste amino acids from DTSM (Cheng et al.,
318
2012).
319 The final models obtained are shown as formula (1) and (2).
321
Y1 = -835.17 + 59.49556*X1 + 92.04182*X2 + 15.39241*X3 + 102.7861*X4 + 0.1299946*X1*X1
322
-8.525202*X1*X2 – 0.1287298*X1*X3 +0. 6061348*X1*X4 - 2.127723*X2*X2 - 0.251188*X2*X3
323
-9.219375*X2*X4 - 0.085827*X3*X3- 0.833688*X3*X4 - 0.742723*X4*X4
324
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Y2 = -78.7129 + 12.91026*X1 + 8.379215*X2 + 0.991718*X3 + 18.13591*X4 + 0.06466194*X1*X1 –
326
1.581797*X1*X2 -0.0 6206797*X1*X3 – 0.1273041*X1*X4 + 0.777946*X2*X2 + 0.083625*X2*X3 -
327
4.295*X2*X4 - 0.009008*X3*X3 + 0.061875*X3*X4 + 0.102946*X4*X4
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(2)
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(1)
3.4.2. Verification of the fitting model
330
In order to further verify the reliability of the obtained model, the experimentally determined CGA
331
and CAA values were compared with the calculated CGA and CAA (data not show) values. Most
332
of the calculated values were very close to the determined values, and the largest relative error
333
was less than 23%. This suggested that the obtained models are reliable to be used for predicting
334
the optimal hydrolysis conditions to prepare protein with highest CGA and CAA.
335
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3.4.3. Results of process optimization
337
The optimal hydrolysis condition was predicted based on the fitting models. The optimization
338
results indicated that the optimal hydrolysis conditions for preparing the highest concentrations of
339
glutamic acid and aspartic acid were enzyme activity of 18.22 %; pH of 3; hydrolysis temperature
340
of 40 °C; hydrolysis time of 6 h and the corresponding CGA and CAA values were 727.6 µg/ mL
341
and 149.9 µg/ mL, respectively. The changes of the CGA and CAA values can be visualized in the
342
response surfaces (Fig. 3).
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343 344
Previous optimization studies of enzymatic hydrolysis process with response surface methodology
345
indicated that the key factors influencing the hydrolysis were mainly decided by the purpose. The
346
enzyme activity and pH greatly influenced the papain hydrolysis process of jellyfish umbrella
ACCEPTED MANUSCRIPT collagen (Zhuang et al., 2009), whereas, pH and temperature were the most important factors of
348
the Alcalase hydrolysis process optimized by response surface methodology (Guerard et al., 2007).
349
The pH was found to be the most important factor in the hydrolysis of shrimp waste with papain to
350
recover chitin (Diniz & Martin, 1996). Therefore, pH and enzyme activity should be strictly
351
monitored and controlled for the optimal processing of papain hydrolysis of DTSM in large scale
352
applications.
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353 4. Conclusions
355
The protein extraction ratio of DTSM could reach to more than 85.64% when the DTSM with
356
particle sizes of ≤ 0.25 mm was hydrolyzed by papain for 5 h. The results proved that the
357
enzymatic hydrolysis is a promising method to extract protein from DTSM. The optimization
358
results showed that the optimal hydrolysis condition for preparing the highest concentration of
359
glutamic acid and aspartic acid were enzyme activity of 18.22%; pH of 3; hydrolysis temperature
360
of 40 °C; and hydrolysis time of 6 h, and the corresponding concentration of the glutamic acid and
361
aspartic acid obtained were 727.6 µg/ mL and 149.9 µg/ mL, respectively. The pH and enzyme
362
activity were the most important factors affecting the hydrolysis of DTSM using papain, which
363
need to be monitored for during papain hydrolysis of DTSM in large scale applications.
366 367 368 369 370 371 372 373 374 375
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ACCEPTED MANUSCRIPT Table 1 Central composite rotatable experimental design and results RUN
X1: Enzyme
X2: pH
X4: Time
Y1: CGA
Y2: CAA
(°C)
(h)
(µg/mL)
(µg/mL)
1
5.20
4
50
3
196.96±9.96
46.00±1.64
2
5.20
4
50
5
238.89±14.89
53.99±8.42
3
5.20
4
70
3
213.06±12.11
47.79±0.68
4
5.20
4
70
5
232.26±14.02
62.89±5.27
5
5.20
6
50
3
6
5.20
6
50
5
7
5.20
6
70
3
8
5.20
6
70
5
9
13.88
4
50
3
10
13.88
4
50
5
RI PT
activity (%)
X3:Temperature
11
13.88
4
70
12
13.88
4
70
13
13.88
6
50
14
13.88
6
15
13.88
6
16
13.88
6
17
0.86
5
18
18.22
5
19
9.54
3
20
9.54
7
21
9.54
22
9.54
23
9.54
24
9.54
25
9.54
26
9.54
27 28
44.79±4.79
152.88±8.79
29.16±1.64
143.61±14.11
42.11±3.26
141.87±7.85
40.34±4.40
375.79±8.50
80.56±2.32
474.68±4.24
96.30±24.79
SC
154.60±10.06
405.71±22.52
5
398.16±14.10
76.96±2.83
3
204.21±3.12
49.40±4.12
M AN U
3
80.16±8.95
5
210.52±7.85
37.62±1.38
70
3
178.65±7.34
43.38±12.31
70
5
180.76±9.11
39.47±1.77
60
4
120.84±6.82
33.91±2.10
60
4
375.42±10.06
69.77±1.01
60
4
312.81±1.83
62.05±5.68
60
4
146.84±18.14
38.11±0.24 43.13±1.53
TE D
50
40
4
218.93±2.10
5
80
4
189.08±13.93
43.60±2.76
5
60
2
235.67±1.46
48.12±1.09
5
60
6
235.06±43.31
46.64±8.00
5
60
4
231.31±20.80
64.69±7.68
5
60
4
270.48±16.52
54.35±4.16
9.54
5
60
4
238.08±7.49
45.12±1.89
9.54
5
60
4
238.74±4.94
49.69±1.58
AC C
EP
5
29
9.54
5
60
4
253.71±7.56
48.71±2.62
30
9.54
5
60
4
238.78±3.35
43.48±1.24
31
9.54
5
60
4
265.30±15.52
51.98±4.51
ACCEPTED MANUSCRIPT Table 2 Fittingness ANOVA of response surface experimental results ANOVA for Y1(CGA)
ANOVA for Y1 (CAA)
Source
DF
MS
F
Pr> F
DF
MS
F
Pr> F
X1
1
89244.23
137.9945
0.0001
1
1811.344
28.62853
0.0001
X2
1
93793.76
145.0292
0.0001
1
2953.933
46.6873
0.0001
X3
1
1263.676
1.953967
0.18124
1
0.59535
X4
1
1016.732
1.572128
0.227909
1
0.00735
X1*X1
1
171.4396
0.26509
0.613684
1
X1*X2
1
21903.26
33.86806
0.0001
1
X1*X3
1
499.4108
0.772217
0.392538
1
X1*X4
1
110.723
0.171206
0.684535
1
X2*X2
1
129.4588
0.200177
0.660576
X2*X3
1
100.9523
0.156098
X2*X4
1
1359.95
X3*X3
1
X3*X4
RI PT
Master
0.923929
0.000116
0.991534
42.41874
0.670434
0.424929
754.0516
11.91789
0.003278
116.1006
1.834986
0.194361
4.8841
0.077194
0.784695
1
17.30616
0.273526
0.608143
0.697992
1
11.18903
0.176844
0.679693
2.102831
0.166347
1
295.1524
4.664923
0.046306
2106.452
3.257115
0.089968
1
23.20393
0.366741
0.55328
1
1112.056
1.719523
0.208262
1
6.125625
0.096816
0.759701
X4*X4
1
15.77449
0.024391
0.877847
1
0.303057
0.00479
0.945681
Model
14
212907.1
23.51493
0.0001
14
6042.819
6.821969
0.000239
(Linear)
4
185318.4
71.63744
0.0001
4
4765.879
18.83134
0.0001
M AN U
TE D
4
2502.344
0.967317
0.452259
4
89.43648
0.353389
0.837891
6
25086.35
6.464989
0.001307
6
1187.503
3.128108
0.031795
16
1012.329
10
714.794
1.44143
0.339159
AC C
(Cross
EP
(Quadratic)
SC
0.00941
Product) Error
16
10347.57
(Lack of fit)
10
8962.632
(Pure Error)
6
1384.94
6
297.5354
Total
30
223254.7
30
7055.148
R2
0.9638
3.882896
0.055242
0.8565
ACCEPTED MANUSCRIPT FIGURE CAPTIONS: Fig.1. A. Particle size distribution of defatted tomato seed meal and B. Effect of particle size on protein extraction of defatted tomato seed meal
RI PT
Fig.2 Effect of hydrolysis conditions on concentration of umami taste amino acids Fig.2A Effect of enzyme activity on concentration of umami taste amino acids (hydrolysis time, hydrolysis temperature and pH were set as 3 h, 50 °C and 5.5, respectively), MSG-like: Sum of
the concentrations of glutamic and aspartic acids; Fig.2B Effect of hydrolysis pH on concentration
SC
of umami taste amino acids (the [E]/[S], hydrolysis time, and hydrolysis temperature were set as 5.20%, 3h and 50 °C, respectively); Fig.2C Effect of temperature on concentration of umami
M AN U
taste(the [E]/[S], hydrolysis time and pH were set as 5.20%, 3h and 5.5, respectively); Fig.2D Effect of hydrolysis time on concentration of umami taste amino acids (the [E]/[S], hydrolysis temperature, and pH were set as 5.20%, 50 °C, and 5.5, respectively)
Fig.3 Response surface of the optimization of protein hydrolysis from DTSM
AC C
EP
TE D
X1: Enzyme activity (%) , X2: pH, X3: Temperature (°C), X4: Time (h), Y1: CGA, Y2: CAA
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
A
B
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Fig.1. A. Particle size distribution of defatted tomato seed meal and B. Effect of particle size on
AC C
EP
protein extraction of defatted tomato seed meal
SC
RI PT
ACCEPTED MANUSCRIPT
B
EP
C
TE D
M AN U
A
D
Fig.2 Effect of hydrolysis conditions on concentration of umami taste amino acids
AC C
Fig.2A Effect of enzyme activity on concentration of umami taste amino acids (hydrolysis
time, hydrolysis temperature, and pH were set as 3 h, 50 °C, and 5.5, respectively), MSG-like: Sum of the concentrations of glutamic and aspartic acids; Fig.2B Effect of hydrolysis pH on concentration of umami taste amino acids (the [E]/[S], hydrolysis time, and hydrolysis
temperature were set as 5.20%, 3h, and 50 °C, respectively); Fig.2C Effect of temperature on concentration of umami taste amino acids ( the [E]/[S], hydrolysis time, pH were set as 5.20%, 3h, and 5.5, respectively); Fig.2D Effect of hydrolysis time on concentration of umami taste amino acids (the [E]/[S], hydrolysis temperature, and pH were set as 5.20%, 50 °C, and 5.5, respectively)
A2
A3
M AN U
SC
A1
RI PT
ACCEPTED MANUSCRIPT
B1
B2
B3
Fig.3 Response surface of the optimization of protein hydrolysis from DTSM
AC C
EP
TE D
X1: Enzyme activity (%) , X2: pH, X3: Temperature (°C), X4: Time (h), Y1: CGA, Y2: CAA
ACCEPTED MANUSCRIPT Highlights 1. Enzymatic hydrolysis for extracting defatted tomato seed meal protein 2. Hydrolyzing extracted protein to prepare umami taste amino acids
AC C
EP
TE D
M AN U
SC
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3. Optimizing hydrolysis process to obtain maximum amino acid concentration