Food Chemistry 234 (2017) 236–244

Contents lists available at ScienceDirect

Food Chemistry

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

Enzyme-assisted extraction enhancing the umami taste amino acidsrecovery from several cultivated mushrooms

http://dx.doi.org/10.1016/j.foodchem.2017.04.1570308-8146/� 2017 Elsevier Ltd. All rights reserved.

⇑ Corresponding author.E-mail addresses: mahesha@food.ku.dk, mahesha.poojary@unicam.it (M.M.

Poojary), vor@food.ku.dk (V. Orlien), paolo.passamonti@unicam.it (P. Passamonti),ko@food.ku.dk (K. Olsen).

Mahesha M. Poojary a,b, Vibeke Orlien a, Paolo Passamonti b, Karsten Olsen a,⇑aDepartment of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 26, Frederiksberg C DK-1958, Denmarkb School of Science & Technology, Chemistry Section, University of Camerino, via S. Agostino, 1, Camerino 62032, Italy

a r t i c l e i n f o a b s t r a c t

Article history:Received 6 December 2016Received in revised form 15 March 2017Accepted 25 April 2017Available online 26 April 2017

Keywords:Enzyme-assisted extractionUmamiMSG-likeMushroomsFlavourzyme�

In this study, enzyme-assisted extraction was performed to extract umami taste and total free aminoacids (FAAs) from the six different mushrooms including shiitake (Lentinus edodes), oyster (Pleurotusostreatus), tea tree (Agrocybe aegerita) and, white, brown and portobello champignons (Agaricus bisporus).b-Glucanase and Flavourzyme� were used as the enzymes for cell wall and proteins hydrolysis, respec-tively. It was found that b-glucanase treatment alone did not enhance the extraction efficiency, howeverin combination, b-glucanase and Flavourzyme� enhanced the extraction efficiency significantly up to20-fold compared to conventional HCl mediated extraction, depending on the mushroom species. Theoptimal conditions for the enzyme treatment were: water as extraction solvent (initial pH = 7), enzymeconcentration of 5% v/w each of b-glucanase and Flavourzyme�, temperature 50 �C and an incubationtime of 1 h. White and brown champignons were found to be the richest source of umami taste FAAs(26.75 ± 1.07 and 25.6 ± 0.9 mg/g DM, respectively).

� 2017 Elsevier Ltd. All rights reserved.

1. Introduction

Umami is recognized as the fifth basic taste after sweet, salty,bitter, and sour, and is described by its pleasant savory or meatyflavors naturally present in many plant and animal based foods.In 1908, Kikunae Ikeda discovered that glutamate is the chemicalbasis of the pleasant taste and he created the term ‘‘umami” forthis distinguishable taste (Ikeda, 1908). The sodium salt of glu-tamic acid (Glu), monosodium glutamate (MSG), is well-knownto exhibit umami-type flavors and is widely used in the food indus-try (Food Standards Australia New Zealand., 2015). The salt ofaspartic acid (Asp), aspartate, also imparts umami taste in foods(Mouritsen, Styrbaek, Johansen, & Mouritsen, 2014). The umamitaste can also be achieved with the 50-ribonucleotides such asinosinate and guanylate (Zhang, Venkitasamy, Pan, & Wang,2013). In humans, the detection or perceived umami taste involvesmultiple receptors present in taste buds which include glutamate-selective G-protein-coupled receptors, mGluR4 and mGluR1, andthe heterodimer T1R1 + T1R3 (Chaudhari, Pereira, & Roper, 2009).

The role of umami in flavor enhancement and appetite regula-tion is well documented (Masic & Yeomans, 2014). Since umami

possesses a savory taste it has the potential to reduce dietarysodium intake (Nakagawa, Kohori, Koike, Katsuragi, & Shoji,2014), thereby assists in regulating hypertension and cardiovascu-lar diseases. Umami ingredients impart meaty flavor, create mouthfullness and makes food richer in taste, thus has a potential inreducing energy and fat intake (Imada, Hao, Torii, & Kimura,2014; Miyaki, Imada, Hao, & Kimura, 2016). In addition, its pleas-ant taste could increase appetite and improve nutrition in mal-nourished and elderly people (Tomoe et al., 2009). Umamiingredients/extracts are considered the ‘‘chef’s choice” within theculinary world. Food industries are searching for natural umami(mono sodium glutamate (MSG)-like) ingredients due to the nega-tive perceptions among consumers regarding ‘‘added MSG” label(Radam, Yacob, Bee, & Selamat, 2010).

Mushrooms are widely used as raw food, functional food, andseasoning particularly due to their complex flavor and are good asource of proteins, carbohydrates, vitamins, minerals and othernutrients (Kalac, 2013). They also possess a wide range of biologi-cal properties including antioxidant, antimicrobial, anticancer andimmuno-modulating activities (Ganeshpurkar, Rai, & Jain, 2010),and are also good sources of umami substances (Phat, Moon, &Lee, 2016). A comprehensive review on umami based ingredientsof edible mushrooms was published recently by Zhang et al.(2013), which clearly suggests that significant amounts of freeGlu and Asp are present in a wide variety of mushrooms, and their

M.M. Poojary et al. / Food Chemistry 234 (2017) 236–244 237

extracts found to elicit umami flavor. Among many other mush-rooms, champignons (Agaricus bisporus, produced in three vari-eties: white champignons, cremini/brown champignons andPortobello), shiitake (Lentinus edodes) and oyster (Pleurotus ostrea-tus) mushrooms are the most widely cultivated edible mushroomsacross the globe (Reis, Barros, Martins, & Ferreira, 2012). The teatree mushroom (Agrocybe aegerita, also called poplar mushroom)is one of the most cultivated mushroom in China (Jiang et al.,2012) and used in several Chinese cuisines. These mushrooms havebeen identified as the richest source of umami substances (Li et al.,2014; Phat et al., 2016; Tsai et al., 2009) and moreover, thesemushrooms have relatively short growth periods and can be culti-vated easily and economically when compared to other ediblemushrooms (Cotter, 2014; Reis et al., 2012).

Conventionally, FAAs are extracted by agitating the mushroomsin dilute hydrochloric acid (Li et al., 2014; Phat et al., 2016; Tsai,Tsai, & Mau, 2008; Tsai et al., 2009; Tsai, Wu, Huang, & Mau,2007). The samples are homogenized before extraction in orderto achieve cell disruption thereby enhance the mass transfer ofanalytes into the solvent. Although, a number of researchers haveused this HCl extraction of FAAs and umami taste amino acids fromvarious mushrooms, there are no reports describing how the pro-cess of extraction can be improved or optimized. Indeed, improvingthe extraction of umami taste from mushrooms will reduce timeand costs of production and may additional increase yield. More-over, utilization of dilute HCl acid as an extraction solvent is notfeasible in food preparations and it complicates downstream pro-cessing. In this respect, enzyme-assisted extraction would be asubstainable alternative method to improve the recovery of umamibased compounds from mushrooms. Enzymes have been proven tobe an environmentally friendly technique for the enhanced releaseof a variety of bioactives and nutraceuticals from various bio-sources (Puri, Sharma, & Barrow, 2012). Enzymes such as cellu-lases, pectinases and hemicellulases are often used to destructthe cell wall components of the plant cell, thereby enhancing therecovery of value-added compounds. Proteases can be used tohydrolyze proteins (Merz et al., 2015a, 2015b) thereby increasingthe yield of FAAs.

In this study, the potential of enzyme-assisted extraction toimprove the umami taste and total FAAs from six different ediblemushrooms was investigated. The industrial b-glucanase (endo-1,3(4)) containing cellulolytic activity was used to hydrolyzemushroom cell wall structures and a peptidase preparation, com-mercially known as Flavourzyme� containing endo- and exopepti-dase activities was used for mushroom protein hydrolysis. Factorsaffecting the enzyme-assisted extraction process, including pH,temperature, and enzyme concentration as well extraction kineticswere investigated.

2. Materials and methods

2.1. Samples

Dried shiitake mushroom, oyster mushroom and white cham-pignon were obtained from OSCAR A/S, Rønnede, Denmark. Driedtea tree mushroom was purchased from a local market in Sichuanprovince, China. Brown champignon and portobello champignonwere purchased in fresh form from a local super market in Copen-hagen, Denmark and dried in a hot air oven at 35 �C for 2 days. Thesamples were ground into powder using a kitchen homogenizer.

2.2. Chemicals and reagents

Amino acid standards mix containing alanine (Ala), sarcosine(Sar), glycine (Gly), valine (Val), a-aminobutyric acid (ABA), b-

aminoisobutyric acid (b-AIB), leucine (Leu), allo-isoleucine (aILE),isoleucine (Ile), threonine (Thr), serine (Ser), proline (Pro), asparticacid (Asp), a-aminoadipic acid (AAA), methionine (Met), 4-hydroxyproline (Hyp), glutamic acid (Glu), phenylalanine (Phe),ornethine (Orn), lysine (Lys), glutamine (Gln), asparagine (Asn),histidine (His), hydroxylysine (Hly), tyrosine (Tyr), tryptophan(Trp), cystine (CAC), norvaline, reagents for solid phase extractionand derivatization of FAAs (included in EZ:Faast GC-FID FAA anal-ysis kit) were purchased from Phenomenex, Værløse, Denmark.Fluorescamine (�98%) and 6-aminocaproic acid (�99%) were pur-chased from Sigma–Aldrich, Munich, Germany.

Enzymes used in the present study, b-glucanase and Flavour-zyme�, were gratefully donated by Novozymes (Bagsvaerd, Den-mark). According to the supplier, b-glucanase (endo-1,3(4)-) wasproduced from Aspergillus aculeatus and has cellulolytic activityand its declared specific enzyme activity was 100 Fungal Beta-Glucanase units (FBG)/g, while Flavourzyme� was produced fromAspergillus oryzae and contained activity of protease and itsdeclared specific enzyme activity was 1000 Leucine Amino Pepti-dase Units (LAPU)/g.

2.3. Enzyme-assisted extraction

Extractions were carried out in batch in 100 mL screw-topSchott bottles placed in a thermostated shaking water bath(±0.1 �C). In a typical experiment, 50 mL of water or buffer solutionwas initially loaded into the flask along with the required volumeof individual enzyme (b-glucanase or Flavourzyme�) or enzymemixture (1:1 v/v mixture of b-glucanase and Flavourzyme�). Thissolution was preheated at required temperature for 15 min. Themushroom sample (1 g) was then loaded into the flask and agitatedfor required duration. On completion of the extraction process,stirring was stopped and the extract was filtered through a What-man� Grade 41 filter paper under vacuum. The filtrate was subse-quently stored at �20 �C until further analysis.

In the present study optimal conditions for the three enzymetreatments (b-glucanase, Flavourzyme� and 1:1 v/v mixture of b-glucanase and Flavourzyme�) was determined by investigatingthe effects of various treatment parameters on the extraction yield,taking shiitake mushroom as a model matrix. In the first set ofexperiments, the effect of pH was determined by varying the pH(4.0, 5.0, 6.0 and uncontrolled) of the extraction medium usingcitrate buffer or Milli-Q water (when Milli-Q water was used, theinitial pH was 7.0 and it was not controlled during the extractionperiod). In this set of experiments, the extraction temperatureand enzyme to substrate ratio was held constant at 50 �C and 5%v/w, respectively. In the second set, the effect of temperaturewas determined by varying the extraction temperature (30, 40,50 and 60 �C), holding the pH at an optimal level and enzyme tosubstrate ratio at 5% v/w. In the final experiment, the optimal pHvalue and optimal temperature were used, while the enzyme con-centration was varied from 0.1 to 5% v/w. In all experiments theextraction time was set to 1 h. Finally, extraction kinetics of thethree enzyme pretreatments were investigated at the optimal con-ditions derived from the previous sets of experiments. In this case,samples (500 mL) were incubated at 50 �C (5% v/w enzymes, pH 7)and sampling for analysis of FAAs were taken at 0.5, 1, 1.5, 2, 3 and5 h intervals. All experiments were carried out in triplicates.Extracts were stored at �20 �C prior to analysis.

2.4. Conventional extraction

Enzyme-assisted extraction developed in this study was com-pared with conventional extraction procedures previouslyreported in the literature (Li et al., 2014; Phat et al., 2016; Tsaiet al., 2008; Tsai et al., 2009; Yang, Lin, & Mau, 2001). Briefly, a

238 M.M. Poojary et al. / Food Chemistry 234 (2017) 236–244

homogenized sample was agitated in 50 mL of 0.1 M HCl for45 min at ambient temperature, followed by filtration through aWhatman Grade 41 filter paper.

2.5. Free amino acids analysis

FAAs analysis was carried out according to the method previ-ously described by Badawy (2012) with minor modifications.100 mL of diluted extract was pipetted into a shell vial along with100 mL of internal standard (norvaline, 20 nmole). The solutionwas aspired gently through a cation exchange sorbent tip (solidphase extraction). The sorbent particles were then washed with200 mL of 33% propanol. Amino acids were then eluted with200 mL of an eluting mix consisting of 0.33 M NaOH, 80% propanoland 20% 3-picoline. Amino acids were then derivatized using 50 mLof derivatizing agent composed of 2:6:2 v/v/v mixture of propyl chloroformate/chloroform/isooctane. The resultant mixture wasthen vortexed for 1 min and 100 mL 90:10 v/v mixture of isooc-tane/chloroform was added. The solution was vortexed for a fur-ther 1 min and 100 mL 1 M HCl was added. The solution wasfurther vortexed for an additional 1 min and allowed to separateinto two phases. The upper layer containing derivatized FAAswas transferred to a GC insert for analysis.

Gas chromatographic analysis was performed using an Agilent6890 gas chromatograph (Agilent, USA) fitted with a flame ioniza-tion detector (FID). Analytes were separated using a ZB–AAA col-umn (10 m length � 0.25 mm ID � 0.25 lm film) and hydrogen asthe carrier gas (flow rate was 1 mL/min). The injector was set at250 �C in a split mode (1:10 ratio). Samples (3 mL) were injectedusing an autosampler. The temperature program was set as fol-lows: 110–320 �C at 32 �C/min. The detector was set at 320 �C. Amultiple point (n = 5) calibration curve with internal standardwas plotted for quantification of amino acids. Results are expressedas mg of FAA per g of sample (dry weight basis), and presented asmean ± standard deviation (SD) (mg/g DM).

2.6. 50-Mononucleotide assay

A detailed experimental protocol for 50-mononucleotide analy-sis is provided in the Supplementary datasheet method M1.1.

2.7. Degree of hydrolysis

A simple, rapid and convenient method was performed todetermine the degree of hydrolysis (DH) based on the determina-tion of primary amino groups using a fluorescence assay as previ-ously described by Kastrup Dalsgaard, Holm Nielsen, and BachLarsen (2007) with additional modifications. In a typical experi-ment, initially, the sample was appropriately diluted in 0.1 Mborate buffer (pH 9.0). A volume of 151 lL of diluted sample wasmixed with 49 mL of the fluorescamine solution (0.2 mg/mL fluo-rescamine in water free acetone) in a well of 96-well plate. Theplate was shaken immediately and the fluorescence was measured5 min after the addition of the fluorescamine in a microplate platereader (Fluoroskan Ascent, Thermo Scientific, USA) using 0.1 Mborate buffer as a blank. The excitation wavelength was set at390 nm and emission at 480 nm. Concentration of the free aminogroups was calculated as 6-aminocaproic acid equivalents usingan external 6-aminocaproic acid standard curve. The degree ofhydrolysis was then calculated according to the following equation(Jamdar et al., 2010):

%DH ¼ As � Ao

Amax � Ao� 100

where As is the total amount of amino groups detected after hydrol-ysis performed at a particular experimental condition, Ao is the total

amount of amino groups present in the original sample, and Amax isthe maximum amount of the amino groups present in the com-pletely hydrolyzed sample.

2.8. Statistical analysis

One-way ANOVA was used to determine significant differencesbetween means using Tukey’s posthoc test (P values < 0.05). Prin-cipal component analysis (PCA) was a multivariate statisticalapproach used to visualize differences in effects of treatmentparameters on the contents of FAA. The XLSTAT (Addinsoft, USA)and Origin Pro 9.1 (OriginLab Corporation, USA) statistical softwarewere used for statistical analyses.

3. Results and discussion

In food products, the MSG-like taste arises from the free Aspand Glu present in the matrix. Presence of these compounds inadequate concentrations improves the overall taste appeal. Prepa-ration of extracts which are rich in umami substances can be usedas cooking stock as well as food ingredients. In this study, three dif-ferent sets of enzymes namely, b-glucanase, Flavourzyme� and 1:1combination of b-glucanase and Flavourzyme� were used toimprove the recovery of MSG-like FAAs from mushrooms. The b-glucanase, a multi component cellulolytic enzyme, has activitiesof cellulase, hemicellulase and xylanase, and is capable ofhydrolyzing endo (1,3)- or (1,4)-linkages in b-d-glucans. Flavour-zyme� is a proteolytic enzyme comprising of aminopeptidases,which is capable of liberating amino acids by hydrolysis of the N-terminal peptide bonds in proteins. Cell-wall degrading enzymeswere employed to hydrolyze the complex polysaccharides in thecell walls (Puri et al., 2012) of mushrooms, which increases cellwall permeability and, consequently improve the extraction yieldsof FAAs. As mentioned, mushrooms are rich in proteins (Kalac,2013) and in order to improve the taste-contributing FAAs con-tents, these proteins can be hydrolyzed using proteases as a sus-tainable and economical approach. In this respect, Flavourzyme�

was used to hydrolyze the proteins and peptides present in allmushrooms. Flavourzyme� is generally used to produce flavor-active amino acids and peptides from proteins (Berends, Appel,Eisele, Rabe, & Fischer, 2014; Merz et al., 2015a). The investigationon Flavourzyme� and the hydrolysis of plant and animal proteins,such as wheat gluten, soy proteins, red hake proteins are describedin the literature (Merz et al., 2015a, 2015b). It has both endo- andexo- peptidase activities (Berends et al., 2014) suitable to yieldgreater amounts of FAAs. The inclusion of cellulolytic enzymes inthe enzymatic hydrolysis of proteins, may allow for easier accessof proteases to proteins (Passos, Yilmaz, Silva, & Coimbra, 2009),which might, in turn, improve the overall yield of FAAs.

A detailed investigation describing the effect of various extrac-tion parameters on the recovery of umami taste FAAs are given inthe following sections.

3.1. Optimization of enzyme-assisted extraction of FAAs

3.1.1. Effect of pHThe influence of pH of themedium on the extraction of MSG-like

and total FAAs fromshiitakemushroomsusingb-glucanase, Flavour-zyme� and combination of b-glucanase and Flavourzyme� is illus-trated in Fig 1A–D. The pH of extraction media was varied between4.0 and 6.0 using a buffer. An experiment was conducted withoutcontrolling the pH of the medium where Milli-Q water served asthe extraction solvent (initial pH = 7.0). In this case, the pHof extrac-tionmedia after the addition of samplewas found to be 5.9 and it didnot change significantly during the course of extraction. As seen in

Fig. 1. Effect of pH on extraction of (A) aspartic acid (B) glutamic acid (C) MSG-like FAAs and (D) total FAAs from shiitake mushroom. C: control (no enzyme); G: b-glucanase;F: Flavourzyme�; G + F: 1:1 mixture of b-glucanase and Flavourzyme�. The enzyme concentration, extraction time, and extraction temperature were set as 5% v/w, 1 h and50 �C, respectively. *pH was not controlled during the reaction. Columns labeled with different letters were significantly different at P < 0.05 (Tukey’s HSD).

M.M. Poojary et al. / Food Chemistry 234 (2017) 236–244 239

Fig. 1A–D the variation in pH did not alone affect the FAAs recoveryfrom control samples (without enzymes). The extractions with b-glucanase did not improve the extraction yield with the tested pHvalues. The inefficiencyofb-glucanasealone inenhancingFAAsyieldmay be explained by the simpler structures and molecular weightsof amino acids, which can easily diffuse out of the cells, whereforecell wall disruption is not essential.

The pH of the media plays a very important role in enzymeactivity as the pH influences the shape and charges of enzymes,thereby affecting its activity (Raju & Madala, 2005). However, thesubstrates are also important as Kao, Tsou, Kao, and Chiang(2011) observed highest Flavourzyme� activity at pH 7.12 duringthe hydrolysis of soy protein, while Shu, Zhang, Chen, Wan, andLi (2015) used pH 6 to hydrolyze goat milk to produce caseinhydrolysate using Flavourzyme� and Berends et al. (2014) usedan even lower pH of 5 for wheat gluten hydrolysis. For the treat-ment with Flavourzyme� alone it is seen (Fig. 1C + D) that pH 6and uncontrolled pH resulted in increased yield of the umami-like FAAs and total FAAs. Combining b-glucanase and Flavour-zyme� an extra effect is observed as a significant improvementin the extraction yield was obtained in the uncontrolled pH assayresulting in the highest extraction of umami-like FAAs and totalFAAs. Water was chosen as the most suitable extraction solventin the subsequent experiments due to the higher extraction effi-ciency. Moreover, water is an ideal extraction solvent for foodindustries as it is economical and reduces costs associated withdownstream processing.

3.1.2. Effect of temperatureThe enzymes used in this study possessed different temperature

activity profiles, therefore different temperature was investigatedto obtain higher recovery. Thus, the total and umami FAAs yields

were investigated at different extraction temperatures (30, 40, 50and 60 �C) at constant enzyme concentration (5% v/w). Increasingtemperature did not improve the extraction yields of the controlassay and the treatment with b-glucanase, while Flavourzyme�

treatment improved the recovery of umami taste (Fig. 2A–C) andtotal FAAs (Fig. 2D). Similar results were obtained by Kao et al.(2011) and Ovissipour et al. (2009) during hydrolysis of soy pro-teins and Persian sturgeon proteins, respectively. The effectivenessof the two enzymes, b-glucanase and Flavourzyme�, together forextraction of total FAAs and the MSG-like amino acids is onlyobserved to a minor degree at 50 �C and probably due to a slightenhancement in hydrolysis of the peptide bonds in proteins. Treat-ment at 60 �C for all assays resulted in significantly decreased yieldof total and MSG-like FAAs, likely due to enzyme inactivation. Ingeneral, enzyme activity increases with temperature, however,their activity decrease at higher temperatures due to denaturationand inactivation (Daniel et al., 2010). Alternatively, a lower yieldcould be due to the loss of FAAs owing to degradation at highertemperatures or association with other macromolecules presentin the extract. Since some amino acids have reactive amino groups,they can react with carbohydrates present in the extract at highertemperatures (Chuyen, 1998), subsequently leading to lower con-centrations of FAAs in the extract. The highest MSG-like and totalFAAs (18.61 ± 1.73 and 70.80 ± 1.35, respectively) were recoveredat 50 �C using a combination of b-glucanase and Flavourzyme�

(Fig. 2C + D). The 50 �C temperature was also used in hydrolysisof date seed flour (Ambigaipalan & Al-Khalifa, 2015), soy proteinisolate (Tsou, Kao, Tseng, & Chiang, 2010), wheat gluten (Berendset al., 2014) using Flavourzyme�.

3.1.3. Effect of enzyme concentrationThe umami taste FAAs (Fig. 3A–C) and total FAAs (Fig. 3D)

contents of the shiitake mushrooms were measured after 1 h

Fig. 2. Effect of temperature on extraction of (A) aspartic acid (B) glutamic acid (C) MSG-like FAAs and (D) total FAAs from shiitake mushroom C:control (no enzyme); G: b-glucanase; F: Flavourzyme�; G + F: 1:1 mixture of b-glucanase and Flavourzyme�. The enzyme concentration, extraction time, and initial pH were set as 5% v/w, 1 h and pH 7(pH was not controlled during the reaction), respectively. Columns labeled with different letters were significantly different at P < 0.05 (Tukey’s HSD).

240 M.M. Poojary et al. / Food Chemistry 234 (2017) 236–244

enzyme-assisted extraction at 50 �C with respect to the differentconcentrations of enzymes used. In general, b-glucanase did notenhance the extraction yield when compared to control sampleseven after using the highest tested enzyme dosage (5% v/w). Com-monly, increasing the enzyme concentration causes an increasingreaction rate, as more enzymemolecules are available to react withthe substrate molecules (Bettelheim, Brown, Campbell, & Farrell,2009). Apparently, this was not the case in the b-glucanase assaydue to the specific action of this enzyme. However, increasingthe Flavourzyme� concentration in the extraction assay signifi-cantly increased the extractability of the FAAs resulting in morethan double yield of Glu and Asp at 5% v/w compared to the con-trol. Combining b-glucanase and Flavourzyme� further enhancedthe total FAAs and MSG-like FAAs yields considerably from 36.7(0.1% v/w, each) to 71.8 mg/g DM (5% v/w, each) and 10.9 (0.1%v/w, each) to 18.4 mg/g DM (5% v/w, each) (Fig. 3), respectively.The improved yield is caused by the higher rate of protein hydroly-sation when the Flavourzyme� concentration increased. Based onthese results the enzyme concentration of 5% v/w was selectedas optimum for extraction of FAA and MSG-like FAAs from mush-rooms. Indeed, it is assumed that at higher enzyme concentrationsextraction yield would increase, but considering the productionsustainability and economy higher concentrations was notassayed.

3.2. Extraction kinetics

In order to optimize the incubation time, the kinetics of releaseof total and MSG-like FAAs from shiitake mushrooms was investi-gated. The effect of the extraction time on the concentration of the

individual FAAs, Asp and Glu, is shown in Supplementary datasheetFig. S1, while Fig. 4 shows the extraction kinetics of the MSG-likeand the total FAAs. The concentration of total FAAs in the controlsamples remained stable throughout the incubation period(Fig. 4B), however, the concentration of MSG-like FAAs decreasedslightly after 2 h of incubation (Fig. 4A). Overall, b-glucanase didnot enhance the extraction yield considerably in line with the pre-vious findings (Section 3.1). A significant increase in the recoveryof umami taste and total FAAs were observed after Flavourzyme�

and combination of b-glucanase and Flavourzyme� treatments,with a � 2-fold increase after 1 h of incubation as compared tothe control groups (Fig. 4). The enzymatic treatment did not causesignificant effects on the yields of total FAAs extraction when theextraction time was further increased up to 5 h. However, a longerincubation time (>1.5 h) caused significant decrease in the recov-ery of umami taste FAAs, likely due to the degradation of Aspand Glu or association of these amino acids with other macro-molecules such as carbohydrates at higher temperatures and pro-longed reaction times.

In order to monitor the progress of hydrolysis, DH was calcu-lated based on the fluorescamine assay. Proteases like Flavour-zyme� will hydrolyze the peptide bonds releasing free aminogroups, which react and can quantitatively be measured by fluores-cence spectroscopy (Kastrup Dalsgaard et al., 2007). The DH valueis then calculated based on the amine concentrations in hydro-lyzed and control samples (Jamdar et al., 2010). As seen in Supple-mentary datasheet Fig. S2, the hydrolysis progressed rapidlyduring the first 1–2 h incubation and then the rate decreased dueto a lower extent of hydrolysis, thereby lowering the DH. Theobservation was similar to typical hydrolysis curves previously

Fig. 3. Effect of enzyme concentration (%v/w) on extraction of (A) aspartic acid (B) glutamic acid (C) MSG-like FAAs and (D) total FAAs from shiitake mushroom. Theextraction temperature, time, and initial pH were set as 50 �C, 1 h and pH 7 (pH was not controlled during the reaction), respectively. Columns labeled with different letterswere significantly different at P < 0.05 (Tukey’s HSD).

Fig. 4. Extraction kinetics of (A) umami taste and (B) total FAAs. The assay (5% v/w enzymes and pH 7 (uncontrolled during the reaction)) was incubated at 50 �C. Symbols inthe line charts labeled with different letters were significantly different at P < 0.05 (Tukey’s HSD).

M.M. Poojary et al. / Food Chemistry 234 (2017) 236–244 241

reported by Bougatef et al. (2009), Jamdar et al. (2010), Klompong,Benjakul, Kantachote, and Shahidi (2007) for the hydrolysis of dif-ferent proteins. At the beginning of incubation, the availability ofexcess protein substrate allowed a rapid hydrolysis leading to ahigher DH value, while the rate subsequently decreased due tothe loss of substrates. A higher DH value was obtained for the sam-ples treated with mixture of b-glucanase and Flavourzyme� when

compared to treatment with Flavourzyme� alone. This indicatesthat b-glucanase assists in extraction of proteins from the cells,which was subsequently hydrolyzed by Flavourzyme�. The DHprofile of the b-glucanase and Flavourzyme� assays is in accor-dance with the higher extraction yields of FAAs obtainedwhen combinations of b-glucanase and Flavourzyme� were used(Figs. 1–4).

242 M.M. Poojary et al. / Food Chemistry 234 (2017) 236–244

3.3. Principal component analysis

PCA was conducted to assess the effects of treatment parame-ters on the FAAs yields. The PCA describing the changes in profilesof individual FAAs affected by different treatments is shown inSupplementary datasheet Fig. S3. For the overview and focus thegroups of taste-contributing amino acid a simplified PCA plot isshown in Fig. 5, which explains 99.86% (PC1 = 97.48% andPC2 = 2.38%) of the total variability. PC1 accounted for the sweet,bitter, umami taste and total FAAs, while PC2 accounted mainlytasteless FAAs. The scores in the bi-plot show a clear discrimina-tion among all extraction methods, based on the distance of thescores in the plot. The umami, bitter and total FAAs were positivelycorrelated with b-glucanase and Flavourzyme� combination treat-ment, distributed in the positive side of PC 1 (lower right quad-rant). Sweet and tasteless FAAs were located in upper rightquadrant of positive PC 2 axis, correlated with Flavourzyme� treat-ment. Amino acids were not clustered at the treatmentscorresponding to the control, conventional extraction andb-glucanase-assisted extraction indicating lower extraction effi-ciency at these conditions, which was consistent with resultsobtained in the optimization experiment (Figs. 1–4).

3.4. Enzyme-assisted extraction of total and MSG-like FAAs from 6different mushrooms

Based on the optimization studies performed on the shiitakemushroom, an incubation temperature of 50 �C, enzyme cocktailof b-glucanase and Flavourzyme� at 5% v/w each, water as extrac-tion solvent (initial pH = 7.0), and incubation time of 1 h wereselected as the optimal conditions to recover total and MSG-likeFAAs from mushrooms. These conditions were extended to extractFAAs from various other commercial mushrooms such as oystermushroom, tea tree mushroom, white champignon, brown cham-pignon and portobello champignon. The FAAs and umami aminoacids extracted from various mushrooms at conventional extrac-tion method as previously described by Li et al. (2014), Phat

Fig. 5. PCA bi-plots comparing the different methods for extraction of taste-contributing and total FAA (n = 3). Loadings: Total-Total FAA; Bitter-FAA contribut-ing to bitter taste; Umami-FAA contributing for sweet taste; Sweet-FAA contribut-ing for sweet taste; Tasteless-tasteless FAA. Scores: Conventional extraction wasperformed at room temperature for 45 min using 0.1 M HCl as extraction solvent.Control treatment (without an enzyme) and enzymatic treatments were performedat 50 �C for 1 h using water as extraction solvent.

et al. (2016), Tsai et al. (2009) and enzyme-assisted extractionmethod developed in the present study is shown in Table 1.

Overall, the enzyme assisted extraction enhanced the extractionyield significantly. The best results were obtained for the oysterand shitake mushrooms, where the two enzyme mixturesenhanced the FAAs yields 7- and 4-fold, respectively, comparedto the conventional extraction (Table 1). In this line, the extractionyields for the groups of taste specific amino acids increased 3- and5-fold for the umami FAAs and up to 20-fold (bitter taste AA foroyster mushroom) compared to those obtained by the conven-tional method. Similar yield enhancements were observed in caseof tea tree mushroom, white champignon, brown champignonand portobello champignon treated with the two-enzyme mixtureof b-glucanase and Flavourzyme�, though the total FAAs andumami FAAs yields increased only around 2-fold when comparedto conventional extraction. The enzyme treatment resulted in thefollowing order of the concentrations of MSG-like amino acids:white champignon (26.75 ± 1.0 mg/g) > brown champignon(25.6 ± 0.90 mg/g DM) > portobello champignon (21.02 ± 0.52 mg/gDM) > shiitake mushroom (18.35 ± 0.12 mg/g DM) > oystermushroom (13.96 ± 1.11 mg/g DM) > tea tree mushroom(10.14 ± 0.12 mg/g DM). The highest total FAAs was detected inbrown champignons (128.28 ± 6.62 mg/g). The presence of FAAsand MSG-like amino acids and their amounts in mushroomsdepend on several factors such as mushroom species, harvestingperiod, storage time, as well as geographic origin (Zhang et al.,2013). To our knowledge, only conventional solvent extractionshave been employed to extract umami amino acids from mush-rooms using 0.1 M HCl as extraction solvent (Li et al., 2014; Phatet al., 2016; Tsai et al., 2008). Yang et al. (2001)) extracted about1.71–1.93 mg/g of MSG-like amino acids from shiitake mush-rooms. The amount of these substances in oyster mushroomswas found to be 2.14 mg/g (Tsai et al., 2009), while it was rangingfrom 10.6 to 13.5 mg/g in champignons, based on maturity stages(Tsai et al., 2007). Li et al. (2014) and Tsai et al. (2008) extracted upto 3.62 mg/g of MSG-like amino acids from tea tree mushrooms.The yield values reported in these studies were similar to thoseobtained by conventional extraction performed in the presentwork; nevertheless, they were considerably lower when comparedto enzyme-assisted extraction developed in the present work. Anumber of recent reports revealed that several other edible mush-rooms are also a promising source for MSG-like substances [re-viewed in detail in Zhang et al. (2013)]. The enzymatic treatmentwas found to significantly affect the extraction yield. Enzyme treat-ment parameters such as pH, temperature, enzyme dosage andtreatment time had significant influence on the recovery of aminoacids. Although the b-glucanase treatment alone did not enhancethe recovery of amino acids, the combination of b-glucanase andFlavourzyme� enhanced the extraction yield significantly. Thiswas indicative that cellulolytic enzyme treatment is not involvedin the extraction of FAAs from the mushroom matrices, however,it aids in the transfer of proteins, which is subsequently hydrolyzedby Flavourzyme� to release FAAs.

3.5. Effect of enzymes on extraction of 50-mononucleotides

Enzyme treatments were found not to improve the extraction50-mononucleotides such as inosinate and guanylate from the var-ious mushrooms. Control experiments (without using an enzyme)showed that a temperature � 70 �C is crucial for extraction ofnucleotides from mushrooms (data not shown). The testedenzymes cannot be used at such high temperature due to loss ofactivity, thus, the enzyme assays were not tested.

Table 1Enzyme-assisted extraction of FAA (mg/g DM) from various cultivated mushrooms.A

Aminoacids

Shiitake mushroom Oyster mushroom Tea tree mushroom

Conventionalextraction

Enzyme-assistedextraction

Conventionalextraction

Enzyme-assistedextraction

Conventionalextraction

Enzyme-assistedextraction

Ala 1.07 ± 0.17 h 3.81 ± 0.13 f 2.30 ± 0.12 g 8.37 ± 0.42 d 3.71 ± 0.21 f 5.75 ± 0.17 eGly 0.29 ± 0.00 e 1.55 ± 0.01 d 0.29 ± 0.00 e 3.32 ± 0.12 b 0.53 ± 0.02 e 1.31 ± 0.03 dVal 0.52 ± 0.02 ef 3.88 ± 0.12 b 0.35 ± 0.01 f 6.21 ± 0.36 a 1.36 ± 0.05 de 3.81 ± 0.18 bLeu 0.39 ± 0.04 g 4.79 ± 0.16 cd 0.3 ± 0.01 g 6.6 ± 0.29 a 1.34 ± 0.06 ef 4.35 ± 0.13 dIle 0.5 ± 0.01 e 3.84 ± 0.05 bc 0.45 ± 0.01 e 5.14 ± 0.44 a 1.05 ± 0.03 de 3.31 ± 0.08 cThr 0.53 ± 0.05 d 3.45 ± 0.23 c 0.33 ± 0.01 d 5.27 ± 0.32 ab 1.00 ± 0.04 d 2.73 ± 0.10 cSer 0.98 ± 0.16 de 4.30 ± 0.01 ab 0.59 ± 0.02 e 4.47 ± 0.39 ab 1.29 ± 0.05 de 2.52 ± 0.14 bcdePro 0.30 ± 0.04 f 0.92 ± 0.03 ef 0.33 ± 0.01 ef 2.60 ± 0.18 de 0.88 ± 0.00 ef 1.5 ± 0.06 efAsn 0.39 ± 0.07 e 2.69 ± 0.28 cd 0.23 ± 0.01 e 3.90 ± 0.36 b 0.80 ± 0.02 e 2.06 ± 0.09 dAsp 0.76 ± 0.08 g 3.51 ± 0.02 d 1.93 ± 0.33 ef 6.07 ± 0.25 b 1.44 ± 0.03 f 3.08 ± 0.19 dMet ND 0.96 ± 0.02 d ND 2.04 ± 0.09 a ND 0.65 ± 0.09 eGlu 2.91 ± 0.98 f 14.85 ± 0.1 c 2.6 ± 0.69 f 7.89 ± 1.17 e 3.50 ± 0.16 f 7.06 ± 0.12 ePhe 0.58 ± 0.00 f 4.66 ± 0.13 c 0.53 ± 0.00 f 6.05 ± 0.23 a 0.56 ± 0.03 f 3.10 ± 0.19 dGln 4.37 ± 0.56 e 7.27 ± 0.07 d 1.94 ± 0.07 f 6.52 ± 0.49 d ND 1.87 ± 0.16 fOrn 1.68 ± 0.19 def 1.73 ± 0.07 cdef ND 2.02 ± 0.66 bcdef 1.35 ± 0.04 f 1.42 ± 0.06 efLys 0.95 ± 0.04 fg 4.82 ± 0.00 ab ND 5.05 ± 0.38 a 1.27 ± 0.02 f 2.72 ± 0.21 deHis 1.79 ± 0.02 d 2.11 ± 0.03 cd ND 3.72 ± 0.22 b 2.47 ± 0.22 c 2.39 ± 0.35 cdTyr 0.72 ± 0.02 ef 1.05 ± 0.00 de 1.16 ± 0.15 d 4.92 ± 0.32 a 0.65 ± 0.17 f 3.02 ± 0.20 aTrp ND 1.63 ± 0.03 d ND 2.88 ± 0.05 b ND 1.74 ± 0.08 cd

Umami 3.67 ± 1.06 f 18.35 ± 0.12 c 4.54 ± 1.03 f 13.96 ± 1.11 d 4.94 ± 0.18 f 10.14 ± 0.12 eSweet 9.6 ± 1.25 d 25.72 ± 0.67 c 6.01 ± 0.25 d 36.49 ± 1.72 b 9.58 ± 0.39 d 19.17 ± 0.60 cBitter 3.78 ± 0.09 ef 21.88 ± 0.29 c 1.64 ± 0.04 f 32.64 ± 1.58 a 6.79 ± 0.39 e 19.36 ± 0.70 cTasteless 1.67 ± 0.06 ef 5.87 ± 0.00 b 1.16 ± 0.15 f 9.97 ± 0.64 a 1.92 ± 0.18 e 5.75 ± 0.28 bTotal 18.72 ± 2.47 f 71.82 ± 0.50 a 13.34 ± 1.47 f 93.06 ± 4.07 c 23.23 ± 1.14 f 54.41 ± 1.53 e

Aminoacids

White champignon Brown champignon Portobello champignon

Conventionalextraction

Enzyme-assistedextraction

Conventionalextraction

Enzyme-assistedextraction

Conventionalextraction

Enzyme-assistedextraction

Ala 7.58 ± 0.02 d 11.59 ± 0.49 c 11.78 ± 0.65 bc 16.17 ± 0.26 a 12.59 ± 0.21 b 16.33 ± 0.52 aGly 1.27 ± 0.04 d 3.55 ± 0.36 b 2.26 ± 0.08 c 5.03 ± 0.21 a 2.35 ± 0.39 c 4.85 ± 0.10 aVal 1.18 ± 0.02 def 5.47 ± 0.67 a 2.04 ± 0.07 cd 5.86 ± 0.45 a 2.34 ± 0.56 c 5.59 ± 0.23 aLeu 1.17 ± 0.04 f 5.42 ± 0.36 bc 1.72 ± 0.05 ef 6.15 ± 0.54 ab 2.02 ± 0.34 e 5.74 ± 0.28 bIle 0.98 ± 0.01 de 4.71 ± 0.64 ab 1.32 ± 0.01 de 4.98 ± 0.61 a 1.45 ± 0.41 d 4.67 ± 0.27 abThr 1.09 ± 0.03 d 4.87 ± 0.72 b 2.59 ± 0.05 c 5.9 ± 0.42 a 2.72 ± 0.42 c 5.52 ± 0.17 abSer 3.01 ± 0.02 bcd 6.36 ± 1.08 a 3.19 ± 0.06 bcd 3.87 ± 2.41 bc 2.81 ± 0.40 bcde 1.88 ± 0.17 cdePro 11.35 ± 0.14 b 14.7 ± 1.91 a 2.02 ± 0.08 ef 4.68 ± 0.57 c 1.77 ± 0.28 ef 4.04 ± 0.27 cdAsn 2.84 ± 0.06 cd 5.53 ± 0.93 a 3.33 ± 0.04 bc 5.04 ± 0.51 a 2.59 ± 0.17 cd 4.01 ± 0.21 bAsp 1.96 ± 0.02 ef 7.14 ± 0.23 a 1.89 ± 0.08 ef 6.14 ± 0.29 b 2.05 ± 0.12 e 4.18 ± 0.17 cMet ND 1.34 ± 0.09 c ND 1.62 ± 0.16 b ND 1.32 ± 0.05 cGlu 14.02 ± 0.27 c 19.61 ± 0.83 a 12.11 ± 0.24 d 19.46 ± 0.71 a 12.16 ± 0.08 d 16.84 ± 0.35 bPhe 1.39 ± 0.04 e 5.27 ± 0.36 bc 1.78 ± 0.09 e 5.91 ± 0.53 ab 1.83 ± 0.29 e 5.35 ± 0.34 abcGln 10.43 ± 0.37 c 16.89 ± 1.11 b 10.43 ± 0.4 c 21.49 ± 1.22 a 10.05 ± 0.00 c 20.53 ± 0.69 aOrn 3.01 ± 0.05 abc 3.13 ± 0.72 ab 2.9 ± 0.28 abcd 2.68 ± 0.92 bcde 4.15 ± 0.28 a 2.90 ± 0.64 abcdLys 1.17 ± 0.03 f 3.99 ± 1.22 abc 1.39 ± 0.04 f 3.9 ± 0.22 bc 1.60 ± 0.05 ef 3.32 ± 0.20 cdHis ND 3.45 ± 0.48 b 2.06 ± 0.24 cd 5.29 ± 0.28 a 2.46 ± 0.09 c 4.81 ± 0.12 aTyr 0.96 ± 0.01 def ND 1.80 ± 0.10 c ND 1.71 ± 0.04 c NDTrp 1.24 ± 0.00 e 2.78 ± 0.15 b 2.05 ± 0.02 c 4.12 ± 0.28 a 1.84 ± 0.08 cd 3.81 ± 0.25 a

Umami 15.98 ± 0.29 d 26.75 ± 1.07 a 14.00 ± 0.32 d 25.6 ± 0.90 a 14.2 ± 0.20 d 21.02 ± 0.52 bSweet 40.58 ± 0.75 b 66.62 ± 6.94 a 38.5 ± 1.64 b 64.85 ± 4.37 a 39.03 ± 2.17 b 60.06 ± 2.51 aBitter 5.97 ± 0.11 e 28.44 ± 2.75 b 10.97 ± 0.48 d 33.93 ± 2.32 a 11.93 ± 1.77 d 31.28 ± 1.31 abTasteless 2.13 ± 0.04 e 3.99 ± 1.22 c 3.19 ± 0.15 d 3.90 ± 0.22 cd 3.31 ± 0.10 cd 3.32 ± 0.20 cdTotal 64.65 ± 1.19 de 125.8 ± 10.28 ab 66.66 ± 2.59 de 128.28 ± 6.62 a 68.48 ± 4.23 d 115.67 ± 3.46 b

A Conventional extraction was performed at room temperature for 45 min using 0.1 M HCl as extraction solvent, without any enzyme treatment. Enzyme-assistedextraction was performed at optimized conditions developed in the present study (extraction was assisted by a mixture of b-glucanase and Flavourzyme� at a concentrationof 5% v/w each; water was used as extraction solvent (with initial pH 7.0); extraction temperature and time were set as 50 �C and 1 h, respectively). FAA are grouped based ontheir taste characteristics: umami (Asp + Glu); sweet (Thr + Ser + Gly + Ala + Pro); bitter (Val + Met + Ile + Leu + Phe + His + Arg + Trp); tasteless (Tyr + Lys). Values are rep-resented as mean ± standard deviation (SD). SD of 0.00 indicates an SD below the significant figures shown; ND-not detected. Means with different letters within a row aresignificantly different (Tukey’s HSD, P < 0.05).

M.M. Poojary et al. / Food Chemistry 234 (2017) 236–244 243

4. Conclusions

It has been shown that enzyme-assisted extraction is a promis-ing method to improve the recovery of MSG-like amino acids frommushrooms. However, the type of enzyme is of crucial importance,as treatment with the cellulolytic enzyme b-glucanase did notenhance the recovery of FAAs, while treatment with protease Fla-vourzyme� enhanced the umami and other taste-contributingFAAs significantly. The optimal recovery of the MSG-like FAAs from

the mushrooms was obtained by extracting with a two-enzymeassay of the cell-wall degrading enzyme b-glucanase and the pro-teolytic enzyme Flavourzyme�. The treatment parameters pH,temperature, enzyme concentration and incubation time signifi-cantly affected the extraction yield but to different extent. Overall,increasing pH, temperature, and concentration resulted inenhanced extraction (though dependent on the actual assay),whereas conducting the incubation more than 1 h caused a lossof FAAs.

244 M.M. Poojary et al. / Food Chemistry 234 (2017) 236–244

Conflict of interest

Authors have declared that no competing interests exist.

Acknowledgements

Authors thank to OSCAR A/S, Denmark, for providing driedmushrooms and Novozymes, Denmark for donating enzymes.Authors are grateful to Marianne N. Lund, Therese Jansson andHongkai Zhu for their assistance in fluorescence analysis. MM Poo-jary thanks to Regione Marche, Italy and University of Camerino forEureka fellowship. MM Poojary also acknowledges assistance pro-vided by Bente Pia Danielsen and Henriette Rifbjerg Erichsen.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.foodchem.2017.04.157.

References

Ambigaipalan, P., & Al-Khalifa, A. S. (2015). Antioxidant and angiotensin Iconverting enzyme (ACE) inhibitory activities of date seed proteinhydrolysates prepared using Alcalase, Flavourzyme and Thermolysin. Journalof Functional Foods, 18, 1125–1137.

Badawy, A. A.-B. (2012). The EZ: Faast family of amino acid analysis kits:Application of the GC-FID kit for rapid determination of plasma tryptophanand other amino acids. Methods in Molecular Biology (Clifton, N.J.), 828, 153–164.

Berends, P., Appel, D., Eisele, T., Rabe, S., & Fischer, L. (2014). Performance ofenzymatic wheat gluten hydrolysis in batch and continuous processes usingFlavourzyme. LWT – Food Science and Technology, 58, 534–540.

Bettelheim, F. A., Brown, W. H., Campbell, M. K., & Farrell, S. O. (2009). Introductionto general, organic and biochemistry (9th ed.). Belmont: Brooks/Cole.

Bougatef, A., Hajji, M., Balti, R., Lassoued, I., Triki-Ellouz, Y., & Nasri, M. (2009).Antioxidant and free radical-scavenging activities of smooth hound (Mustelusmustelus) muscle protein hydrolysates obtained by gastrointestinal proteases.Food Chemistry, 114, 1198–1205.

Chaudhari, N., Pereira, E., & Roper, S. D. (2009). Taste receptors for umami: The casefor multiple receptors. The American Journal of Clinical Nutrition, 90, 738S–742S.

Chuyen, N. V. (1998). Maillard reaction and food processing. In F. Shahidi, C.-T. Ho, &N. van Chuyen (Eds.), Process-induced chemical changes in food (pp. 213–235).US, New York: Springer.

Cotter, T. (2014). Organic mushroom farming and mycoremediation, simple toadvanced and experimental techniques for indoor and outdoor cultivation (1sted.). Vermont: Chelsea Green Publishing.

Daniel, R. M., Peterson, M. E., Danson, M. J., Price, N. C., Kelly, S. M., Monk, C. R., ...Eyring, H. (2010). The molecular basis of the effect of temperature on enzymeactivity. The Biochemical Journal, 425, 353–360.

Food Standards Australia New Zealand. (2015). MSG in food. Retrieved September12, 2016, from http://www.foodstandards.gov.au/consumer/additives/msg/Pages/default.aspx.

Ganeshpurkar, A., Rai, G., & Jain, A. P. (2010). Medicinal mushrooms: Towards a newhorizon. Pharmacognosy Reviews, 4, 127–135.

Ikeda, K. (1908). New seasonings. Journal of Tokyo Chemical Society, 30, 820–836.Imada, T., Hao, S. S., Torii, K., & Kimura, E. (2014). Supplementing chicken broth with

monosodium glutamate reduces energy intake from high fat and sweet snacksin middle-aged healthy women. Appetite, 79, 158–165.

Jamdar, S. N., Rajalakshmi, V., Pednekar, M. D., Juan, F., Yardi, V., & Sharma, A.(2010). Influence of degree of hydrolysis on functional properties, antioxidantactivity and ACE inhibitory activity of peanut protein hydrolysate. FoodChemistry, 121, 178–184.

Jiang, S., Chen, Y., Wang, M., Yin, Y., Pan, Y., Gu, B., ... Sun, H. (2012). A novel lectinfrom Agrocybe aegerita shows high binding selectivity for terminal N-acetylglucosamine. The Biochemical Journal, 443, 369–378.

Kalac, P. (2013). A review of chemical composition and nutritional value of wild-growing and cultivated mushrooms. Journal of the Science of Food andAgriculture, 93, 209–218.

Kao, F. J., Tsou, M. J., Kao, H. C., & Chiang, W. D. (2011). Optimization of enzymatichydrolysis conditions for producing soy protein hydrolysate with maximumlipolysis-stimulating activity. Journal of Food and Drug Analysis, 19, 197–201.

Kastrup Dalsgaard, T., Holm Nielsen, J., & Bach Larsen, L. (2007). Proteolysis of milkproteins lactosylated in model systems. Molecular Nutrition & Food Research, 51,404–414.

Klompong, V., Benjakul, S., Kantachote, D., & Shahidi, F. (2007). Antioxidativeactivity and functional properties of protein hydrolysate of yellow stripetrevally (Selaroides leptolepis) as influenced by the degree of hydrolysis andenzyme type. Food Chemistry, 102, 1317–1327.

Li, W., Gu, Z., Yang, Y., Zhou, S., Liu, Y., & Zhang, J. (2014). Non-volatile tastecomponents of several cultivated mushrooms. Food Chemistry, 143, 427–431.

Masic, U., & Yeomans, M. R. (2014). Umami flavor enhances appetite but alsoincreases satiety. American Journal of Clinical Nutrition, 100, 532–538.

Merz, M., Eisele, T., Berends, P., Appel, D., Rabe, S., Blank, I., ... Fischer, L. (2015a).Flavourzyme, an enzyme preparation with industrial relevance: Automatednine-step purification and partial characterization of eight enzymes. Journal ofAgricultural and Food Chemistry, 63, 5682–5693.

Merz, M., Eisele, T., Claaßen, W., Appel, D., Rabe, S., Stressler, T., & Fischer, L. (2015b).Continuous long-term hydrolysis of wheat gluten using a principally food-gradeenzyme membrane reactor system. Biochemical Engineering Journal, 99,114–123.

Miyaki, T., Imada, T., Hao, S. S., & Kimura, E. (2016). Monosodium L-glutamate insoup reduces subsequent energy intake from high-fat savoury food inoverweight and obese women. The British Journal of Nutrition, 115, 176–184.

Mouritsen, O. G., Styrbaek, K., Johansen, M., & Mouritsen, J. D. (2014). Umamiunlocking the secrets of the fifth taste. New York: Columbia University Press.

Nakagawa, T., Kohori, J., Koike, S., Katsuragi, Y., & Shoji, T. (2014). Sodium aspartateas a specific enhancer of salty taste perception–sodium aspartate is a possiblecandidate to decrease excessive intake of dietary salt. Chemical Senses, 39,781–786.

Ovissipour, M., Abedian, A., Motamedzadegan, A., Rasco, B., Safari, R., & Shahiri, H.(2009). The effect of enzymatic hydrolysis time and temperature on theproperties of protein hydrolysates from Persian sturgeon (Acipenser persicus)viscera. Food Chemistry, 115, 238–242.

Passos, C. P., Yilmaz, S., Silva, C. M., & Coimbra, M. A. (2009). Enhancement of grapeseed oil extraction using a cell wall degrading enzyme cocktail. Food Chemistry,115, 48–53.

Phat, C., Moon, B., & Lee, C. (2016). Evaluation of umami taste in mushroom extractsby chemical analysis, sensory evaluation, and an electronic tongue system. FoodChemistry, 192, 1068–1077.

Puri, M., Sharma, D., & Barrow, C. J. (2012). Enzyme-assisted extraction of bioactivesfrom plants. Trends in Biotechnology, 30, 37–44.

Radam, A., Yacob, M. R., Bee, T. S., & Selamat, J. (2010). Consumers’ perceptions,attitudes and willingness to pay towards food products with ‘‘No Added Msg”labeling. International Journal of Marketing Studies, 2, 65–77.

Raju, S., & Madala, B. (2005). Illustrated medical biochemistry (1st ed.). New Delhi:Jaypee Brothers Medical Publishers (P) Ltd.

Reis, F. S., Barros, L., Martins, A., & Ferreira, I. C. F. R. (2012). Chemical compositionand nutritional value of the most widely appreciated cultivated mushrooms: Aninter-species comparative study. Food and Chemical Toxicology, 50, 191–197.

Shu, G., Zhang, Q., Chen, H., Wan, H., & Li, H. (2015). Effect of five proteasesincluding alcalase, flavourzyme, papain, proteinase k and trypsin onantioxidative activities of casein hydrolysate from goat milk. Acta UniversitatisCibiniensis Series E: Food Technology, 19, 65–74.

Tomoe, M., Inoue, Y., Sanbe, A., Toyama, K., Yamamoto, S., & Komatsu, T. (2009).Clinical trial of glutamate for the improvement of nutrition and health in theelderly. Annals of the New York Academy of Sciences, 1170, 82–86.

Tsai, S. Y., Huang, S. J., Lo, S. H., Wu, T. P., Lian, P. Y., & Mau, J. L. (2009). Flavourcomponents and antioxidant properties of several cultivated mushrooms. FoodChemistry, 113, 578–584.

Tsai, S.-Y., Tsai, H.-L., & Mau, J.-L. (2008). Non-volatile taste components of Agaricusblazei, Agrocybe cylindracea and Boletus edulis. Food Chemistry, 107, 977–983.

Tsai, S. Y., Wu, T. P., Huang, S. J., & Mau, J. L. (2007). Nonvolatile taste components ofAgaricus bisporus harvested at different stages of maturity. Food Chemistry, 103,1457–1464.

Tsou, M.-J., Kao, F.-J., Tseng, C.-K., & Chiang, W.-D. (2010). Enhancing the anti-adipogenic activity of soy protein by limited hydrolysis with Flavourzyme andultrafiltration. Food Chemistry, 122, 243–248.

Yang, J.-H., Lin, H.-C., & Mau, J.-L. (2001). Non-volatile taste components of severalcommercial mushrooms. Food Chemistry, 72, 465–471.

Zhang, Y., Venkitasamy, C., Pan, Z., & Wang, W. (2013). Recent developments onumami ingredients of edible mushrooms – A review. Trends in Food Science andTechnology, 33, 78–92.

本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。

学霸图书馆（www.xuebalib.com）是一个“整合众多图书馆数据库资源，

提供一站式文献检索和下载服务”的24 小时在线不限IP

图书馆。

图书馆致力于便利、促进学习与科研，提供最强文献下载服务。

图书馆导航：

图书馆首页 文献云下载 图书馆入口 外文数据库大全 疑难文献辅助工具