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Enrichment of cookies with glutathione by inactive yeast cells (Saccharomyces cerevisiae): Physicochemical and functional properties
Accepted Manuscript Enrichment of cookies with glutathione by inactive yeast cells (Saccharomyces cerevisiae): Physicochemical and functional properties
Serpil Öztürk, İnci Cerit, Selime Mutlu, Omca Demirkol PII:
S0733-5210(17)30508-8
DOI:
10.1016/j.jcs.2017.06.019
Reference:
YJCRS 2392
To appear in:
Journal of Cereal Science
Received Date:
18 October 2016
Revised Date:
30 May 2017
Accepted Date:
29 June 2017
Please cite this article as: Serpil Öztürk, İnci Cerit, Selime Mutlu, Omca Demirkol, Enrichment of cookies with glutathione by inactive yeast cells (Saccharomyces cerevisiae): Physicochemical and functional properties, Journal of Cereal Science (2017), doi: 10.1016/j.jcs.2017.06.019
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Enrichment of cookies with glutathione by inactive yeast cells (Saccharomyces
2
cerevisiae): Physicochemical and functional properties
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Serpil Öztürk, İnci Cerit, Selime Mutlu, Omca Demirkol*
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Department of Food Engineering, Sakarya University, Esentepe, Sakarya 54187, Turkey
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* Corresponding author: Omca Demirkol
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Address: Sakarya University, Faculty of Engineering,
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Department of Food Engineering,
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54187 Esentepe, Sakarya, Turkey
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Phone : + 90 264 295 5921
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Fax
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E-mail: [email protected]
: + 90 264 295 5601
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e-mails
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[email protected]
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[email protected]
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[email protected]
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Abstract
25
Cookie is one of the favorite cereal products which could be formulated by using various
26
ingredients for enrichment. The aim of this study was to determine the effects of the inactivate
27
yeast cells addition to cookie formulation in terms of glutathione (GSH) content, antioxidant
28
activities, physicochemical and sensorial properties. Also, the cookies were produced with
29
GSH (pure, 98%) addition to compare the effects of inactive yeast GSH with pure GSH on
30
revealed properties. According to results, The GSH contents and the antioxidant activities
31
increased in dough and cookie samples with inactive yeast addition compared to control and
32
pure GSH added dough and cookie samples. The increases in GSH contents and antioxidant
33
activities were observed after baking in all samples. The pure GSH addition increased the
34
moisture content, spread ratio and L* value while decreased the hardness and b* values of the
35
cookies. The contrary results were obtained by inactive yeast addition in terms of the
36
physicochemical properties where the protein content of the cookies increased with inactive
37
yeast addition by approximately 25%. The loss of GSH content decreased while the
38
antioxidant activities and nutritional value increased in cookies by inactivate yeast addition.
39 40
Keywords: Glutathione; Yeast; Cookie; Antioxidant activity
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Abbreviations
42
AACC, The American Association of Cereal Chemists
43
ABTS, 2,20-azino-bis/3-ethyl-benzothiazoline-6-sulfonic acid
44
C, control cookie
45
CUPRAC, cupric ion reducing antioxidant capacity
46
CY, cookie with inactive yeast
47
D, control dough
48
DETAPAC, diethylenetriaminepentaacetic acid
49
DG, dough with pure GSH
50
DPPH, 1,1-diphenyl-2-picryl-hydrazyl
51
DY, dough with inactive yeast
52
HMF, Hydroxymethylfurfural
53
GSH, glutathione
54
GSSG, oxidized glutathione
55
NPM, N-(1-pyrenylmaleimide)
56
SBB, serine borate buffer
57
α-TOHs, α-tocopherols
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1. Introduction
59
Glutathione (GSH), a member of thiols, plays important role as an antioxidants and it is
60
defined as a mercaptan. The GSH has a tripeptide structure (γ-glutamylcysteinylglycine) and
61
is found in animals, plants and microorganisms in milimolar concentrations. Antioxidant
62
property of GSH has been studied as one of the most important topic in biological functions.
63
It is important in detoxification of radicals such as hydroxyl radical (OH•), hydrogen peroxide
64
(H2O2) and superoxide anion (O2•). Although GSH is found as reduced form in the cell, it is
65
being oxidized (GSSG) during in antioxidant reactions. The GSH also plays an important role
66
in transportation of amino acids, synthesis of proteins and DNA (Kans et al., 1988; Orlowski
67
and Meister, 1970; Suthanthiran et al., 1990). In the literature, there are studies about GSH
68
existence in some fresh fruits and vegetables, but it is also known that GSH may get lost in
69
processes such as disinfection, heat treatment and drying (Demirkol et al., 2008, 2004;
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Gümüşay et al., 2015). Nowadays, GSH is being used as pharmaceutical component and has a
71
potential in food and cosmetic industry. Still, it has not been widely used in the food industry
72
because of its cost.
73
Yeast usage in fermentation processes like alcoholic beverage production and bread
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leavening made them to be known by humans for almost thousands of years. The current
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scientific knowledge and technology allow the isolation, construction and industrial
76
production of yeast strains which have high biomass yield on carbon source (Bekatorou et al.,
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2006). The acceptability as food or feed of a particular microorganism depends on its
78
nutritional value and safety, including nucleic acid content, presence of toxins and residual
79
undesirable compounds. S. cerevisiae consists of 30–33% of dry materials, 6.5–9.3% of
80
nitrogen, 40.6–58.0% of proteins, 35.0–45.0% of carbohydrates (high β-glucan), 4.0–6.0% of
81
lipids, 5.0–7.5% of minerals like Ca, P, K, Mg, Cu, Fe, Zn, Mn, Cr and various amounts of
82
vitamins such as B1, B2, B3, B5, B6, B7, B9 (Wood, 2013), and high amounts of GSH
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depending on its type and growth conditions (Ángeles Pozo-Bayón et al., 2009; Li et al.,
84
2004; Ravindra, 2000). Yeasts contain low amounts of nucleic acids but essential amino acids
85
mainly lysine with higher amount than bacteria or algae. Thus, yeasts are considered to be one
86
of the most important alternative single cell nutrition sources (Bekatorou et al., 2006; Gupta et
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al., 2013).
88
Inactive form of yeast is produced as nutritional yeast and commercially available in many
89
countries. Yeast cells are inactivated by heat treatment and the process is completed with
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drying. In recent years, using of inactive yeast cells as a part of wine production process has
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become common because of the improving effects of GSH in the sensory properties and
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fermentation. Moreover, it is also considered that the GSH in inactive yeast cells is effective
93
in maintaining the wine color (Ángeles Pozo-Bayón et al., 2009; Andújar-Ortiz et al., 2014).
94
Another application of inactive yeasts in food industry is in removing of toxic substances such
95
as ochratoxin and patulin which have been produced by microorganisms (Piotrowska et al.,
96
2013; Yue et al., 2011).
97
The trends in consumption of foods with high nutritional quality promote the researchers to
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enrich the cookies likewise all other food products. The aim of this study was to compare the
99
changes in the GSH content, antioxidant activities and quality properties of cookies enriched
100
with inactive yeast cells and pure GSH.
101 102
2. Material and methods
103 104
2.1. Materials
105
Acetonitrile, methanol, hydrochloric acid, sodium hydroxide were provided from Merck
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(Darmstadt, Germany). Acetic acid, phosphoric acid and borate were purchased from Fisher
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(St. Louis, MO). GSH, N-(1- Pyrenyl) maleimide (NPM), 2,2-diphenyl- 1-picrylhydrazyl
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(DPPH), Trolox [(±)-6- hydroxy-106 2,5,7,8-tetramethylchroman-2-carboxylic acid],
109
Neocuproine, copper (II) chloride, ammonium acetate, Tris-HCl, serine, diethylenetriamine
110
pentaacetic acid (DETAPAC), 2,20-azino-bis/3-ethyl-benzothiazoline-6-sulfonic acid (ABTS)
111
and potassium persulfate were supplied from Sigma (St. Louis, MO). Instant dry yeast was
112
provided from local markets in Sakarya, Turkey. A hundred gram of instant dry yeast used in
113
the study had a proximate composition of 3.5 g of moisture, 6 g of fat, 39 g of carbonhaydrate
114
and 44 g of protein.
115 116
2.2. Preparation of the cookies
117
Cookies were prepared according to AACC Approved Method No: 10-54 (AACC, 2000)
118
with slight modifications. Formulation of cookies is given in Table 1. Instant dry yeast was
119
heat treated in oven at 120°C for 30 minutes to produce inactive yeast cells. In preliminary
120
study, the inactive yeast cells (2.88 mg GSH/ g yeast) were added to cookie formulation at 5,
121
10, 20, and 30% levels (in flour basis) and the cookies were analyzed for sensorial and
122
physical quality properties and GSH contents (Mutlu et al., 2016). Sensorial properties of the
123
cookies were examined in terms of taste, odor and tenderness. According to the results
124
(Fig.1), 10% level was found as the upper limit in cookies without deterioration in terms of
125
mainly taste. A lower level (5%) was also acceptable in terms of sensorial properties; however
126
the aim of the study was to increase the glutathione content in cookies. Therefore, the upper
127
level, 10%, was chosen to obtain cookies with higher level of glutathione.
128
Hence, the study has continued with the addition of 10% (in flour basis) of inactive yeast
129
(2.88 mg GSH/ g yeast) and 23 mg/recipe of pure GSH which was equaled to the amount of
130
GSH in 10% yeast. Four cookies were baked at a time with a rotary oven (Simsek
131
Laborteknik, Ankara Turkey) at 205°C for 11 min. The baked cookies were cooled at room
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temperature for 2 hours and analyzed for physical quality parameters, and then packed with
133
plastic bags to store at -20C for further analyses.
134
2.3. Preparation of the extracts for determination of the GSH content
135
The dough and cookie extracts were prepared according to the procedure described by
136
Demirkol et al. (2004). The samples were weighted into test tubes and serine borate buffer
137
(SBB) were added (0.1 g/mL) to prevent potential oxidation of biothiols by atmospheric
138
oxygen. The SBB buffer contained 100 mM Tris-HCl, 10 mM borate, 5 mM serine, and 1
139
mM DETAPAC, with the final pH adjusted to 7.0 by a concentrated NaOH solution. The
140
samples were homogenized with tissue tearor (Wiggen Hauser, model D-130 handheld
141
disperser, Germany) in ice bath for 2 min, with 30 second intervals, and then centrifuged at
142
13,130 g for 10 min at 4°C. The supernatants were used to determine the GSH content.
143 144
2.4. Determination of the GSH content by the HPLC method
145
The GSH contents of the extracts were measured by using the chromatographic method to
146
analyze γ-glutamyl cycle intermediates that was developed by Winters et al. (1995), and
147
modified by Demirkol et al. (2004). The supernatants (100 µL) were diluted to 250 µL with
148
distilled water and derivatised with 750 µL N-(1-pyrenyl)maleimide (NPM) solution (1 mM
149
in acetonitrile). The solution was stirred and incubated at room temperature for 5 min, so that
150
the NPM was reacted with free sulfhydryl groups to form fluorescent derivatives. Then, 2 N
151
HCl solution (10 µL) was added to stop the reaction. The solution was filtered through 0.45
152
µm nylon filter and injected into a 5 μm C18 column in a reverse phase HPLC system.
153
The HPLC system (Hitachi, Tokyo, Japan) was comprised of a model L-2130 pump, L-
154
2300 oven, a L-2200 auto sampler, and a Hitachi Chromaster 5440 fluorescence detector
155
which was operated at an excitation wavelength of 330 nm and an emission wavelength of
156
376 nm, and a reversed-phase Reliasil ODS-1 C18 column (5 μm, 250 × 4.6 mm) (Orochem,
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Naperville, USA). The mobile phase consisted of acetonitrile: water solution (70:30) which
158
was adjusted to approximately pH of 2.5 by addition of 1 mL of acetic acid and 1 mL of o-
159
phosphoric acid per liter. The calibration curve was plotted by using 0 to 2500 nM GSH
160
solutions (r2= 0.996).
161 162
2.5. Preparation of the extracts for DPPH and CUPRAC assays
163
The dough and cookie extracts were prepared according to the method described by
164
Capanoglu et al. (2008) to determine DPPH (1,1-diphenyl-2-picryl-hydrazyl) and CUPRAC
165
(cupric ion reducing antioxidant capacity) assays. One gram of a sample was put into test tube
166
and 3 mL of methanol solution (75%, v/v) was added, then the mixture was kept in an
167
ultrasonic water bath for 15 min. The mixture was centrifuged at 13,130 g for 10 min at 4°C,
168
and the supernatant was separated. Three mL of methanol solution was added over the pellet
169
and the procedure was repeated. The collected supernatants were completed to the volume of
170
10 mL with methanol solution.
171 172
2.6. DPPH radical scavenging capacity assay
173
The DPPH assay was applied according to the procedure of Brand-Williams et al. (1995)
174
with some modifications. Three mL of 0.051 mM DPPH in methanol was added to 200 μL of
175
extract and the mixture was incubated at room temperature for 30 min. The DPPH scavenging
176
capacity was evaluated spectrophotometrically (Shimadzu UV-1240, USA) by measuring the
177
decrease in absorbance at 517 nm. Antioxidant capacity was calculated as DPPH radical
178
scavenging capacity (%) = [(A0– A1) / A0] × 100, where A0 was the absorbance of the blank
179
(reacting mixture without the test sample), and A1 was the absorbance of the reacting mixture
180
with the test sample.
181 182
2.7. CUPRAC assay
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The CUPRAC assay was performed according to the method developed by Apak et al.
184
(2004). The sample extract (0.5 mL) and distilled water (0.6 mL) were transferred into a test
185
tube. One mL of 10-2 M copper (II) chloride, 1 mL of 7.5×10-2 M neocuproine, and 1 mL of
186
ammonium acetate buffer solutions were added and the mixture was incubated at room
187
temperature for an hour. The absorbance was determined spectrophotometrically (Shimadzu
188
UV-1240, USA) at 450 nm. The results were expressed as trolox equivalent, milligrams per
189
100 g (r2=0.996).
190 191
2.8. ABTS•+ assay
192
The ABTS (2,20-azino-bis/3-ethyl-benzothiazoline-6-sulfonic acid) assay was applied by
193
QUENCHER method described by Serpen et al. (2008). The stock solution of ABTS•+ was
194
prepared by reacting a 7 mmol/L aqueous solution of ABTS with 2.45 mmol/L K2O8S2. The
195
stock solution was diluted with water/ethanol mixture (50:50, v/v) to obtain the working
196
solution with 0.70-0.80 absorbance value. The dough or cookie sample (10 mg) was mixed
197
with 6 mL of ABTS•+ working solution. The mixture was shaken for 25 min and centrifuged
198
at 9200 g for 2 min at 25°C, and then the absorbance measurement was performed
199
spectrophotometrically (Shimadzu UV-1240, USA) at 734 nm. The results were expressed as
200
trolox equivalent, millimole per gram (r2=0.993).
201 202
2.9. Moisture and protein contents of the cookies
203
The moisture contents (%) of the cookies were measured by gravimetric method in oven at
204
135°C (AACC, 2000). The nitrogen contents were determined by using the Kjeldahl method
205
and were multiplied by a factor of 5.7 to estimate the percent protein content (AACC, 2000).
206 207
2.10. Physical quality parameters of the cookies
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After 2 hours of cooling at room temperature, the cookies were measured for diameter (D)
209
and thickness (T) with a caliper and the spread ratio (D/T) was calculated according to AACC
210
(2000).
211
The hardness values of the cookies were measured by a Texture Analyser (Stable
212
Microsytems, TA-XT plus, UK) equipped with a three-point bending rig. The span between
213
the supports was 4 cm. A load cell of 50 kg was used. The maximum force (Newton) required
214
to break the cookie sample was determined 24 hours after baking.
215
The surface color values of the cookies were determined by using Lovibond Reflectance
216
Colorimeter RT300 (UK), and CIE color values (L*, a*, b*) were measured. The L* value
217
indicates the lightness, 0–100 representing dark to light. The a* value gives the degree of the
218
red-green color. The b* value indicates the degree of the yellow-blue color. The instrument
219
was calibrated by using white and black calibration plates.
220 221
2.11. Sensorial analysis
222
The sensorial properties (taste, odor, tenderness and appearance) of the cookies were
223
screened by panel members by giving the scores ranging between 1 and 5; 5 being the most
224
desirable.
225 226
2.12. Statistical analysis
227
The tests were performed in duplicate and the results were expressed as mean ± standard
228
deviation (SD). The statistical analyses were performed using SPSS (version 11.5, SPSS Inc.,
229
USA). A comparison of the means was confirmed by Duncan’s test at 5% level of
230
significance using one-way analysis of variance (ANOVA).
231 232
3. Results and discussion
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3.1. The GSH content and antioxidant activities of dough and cookie samples
235
The GSH content of the dough and cookie samples are presented in Fig.2. It was found that
236
the GSH contents of dough with inactive yeast (DY) and dough with pure GSH (DG) were
237
higher than those of their respective cookies. The reason of the decreases in GSH content in
238
the cookies was the high baking temperature (205°C). The accelerated degradation of GSH in
239
baking process might be due to the baking heat and the surrounding air oxygen. Gümüşay et
240
al. (2015) reported that the significant losses in GSH content were observed in thermal drying
241
of tomato and ginger samples. In another study, it was shown that there was a decrease in
242
GSH concentration in human milk samples subjected to thermal processing (Silvestre et al.,
243
2008). Both of the dough recipes (DG and DY) had 14 mg GSH/100 g dough (23 mg/recipe),
244
however the levels decreased to 8.16 and 11.42 mg/100 g dough in DG and DY, respectively.
245
This phenomenon might be observed because of the oxidation of GSH during mixing trough
246
breaking of the disulphide bonds in gluten by GSH. The GSH cleaved disulphide bonds and
247
formed protein/GSH mixed disulphides, so the number of GSH decreased at the end of the
248
mixing process and GSH transformed into GSSG (oxidized GSH) (Hüttner and Wieser, 2001;
249
Verheyen et al., 2015). The GSH content of the cookie with pure GSH (CG) (3.90 mg
250
GSH/100 g) was lower than that of the cookie with inactive yeast (CY) (7.37 mg GSH/100 g).
251
Although they had same GSH content at the beginning of the process, CY had almost twice as
252
much GSH as CG after cooking. It is well known that eukaryotic structure of yeast cell wall
253
had excellent potential as capsule material and many advantages with natural properties in
254
microencapsulation technology (Nelson, 2002). Because of the resistance of the cell wall of
255
yeast to high temperature, protection of the intercellular GSH level might be observed
256
(Normand et al., 2005).
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Antioxidant activities of the dough and cookie samples are shown in Table 2. The DPPH
258
scavenging activity, CUPRAC and ABTS•+ assays were applied to determine the antioxidant
259
activity values. The DPPH scavenging activities of the control cookie (C), CG, and CY were
260
found as 21.52%, 15.73%, and 43.66%, respectively. The highest antioxidant activity was
261
belonged to CY, because the yeasts synthesized bioactive compounds which could act as
262
antioxidants like the organic acid and the salt forms of citric acid, coenzyme Q or ubiquinone,
263
glutathione, α-tocopherols (α-TOHs) and other forms of tocopherol (Abbas, 2006). The DPPH
264
scavenging activity of the CG was lower than that of control cookie. The nucleophilic
265
properties of sulphydryl compounds played important role in inhibiting the enzymatic and
266
nonenzymatic browning reactions which was the reason of low DPPH scavenging capacity of
267
CG (Friedman, 1994; Molnar-Perl and Friedman, 1990). DPPH scavenging activities of all
268
cookie samples were higher than those of their respective dough samples. However, there was
269
no significant (p>0.05) difference between the DPPH scavenging activities of DG and DC.
270
Maillard reaction products formed during the baking process could be attributed the increases
271
in antioxidant activities of the cookies (Manzocco et al., 2000).
272
The CUPRAC assay for antioxidant activity analysis is sensitive toward thiol-type
273
antioxidants like glutathione (Apak et al., 2004). Although the CUPRAC value of DG was
274
slightly higher than that of the control dough (D), there was no significant difference (p>0.05)
275
probably due to low amount of GSH in dough samples. The CY had the highest (913.15 mg
276
trolox/100 g) while CG had the lowest CUPRAC value due to the same reasons in DPPH
277
scavenging activity assay. The ABTS•+ values of the cookies were higher than those of the
278
dough samples. However, there was significant difference only between the ABTS•+ values of
279
C and D samples (p<0.05). The CY had the highest ABTS•+ value among all samples due to
280
both bioactive compounds of yeast cells and nonenzymatic browning reaction products.
281
Similar to the CUPRAC assay, ABTS•+ value of DG was slightly higher than that of D
ACCEPTED MANUSCRIPT 282
sample, because thiols were irreversibly oxidized by the ABTS radical cation to higher
283
products such as sulphinic or sulphonic acids (Güngör et al., 2011).
284
Abdel-Samie et al. (2010) added the cumin and ginger as antioxidants to dough and
285
examined the effects on cookie quality. According to results, total phenolic compounds and
286
antioxidant activity of cookies increased by adding of 5% cumin and ginger. In another study,
287
different legume flours were incorporated into cookie and nutritional characteristics were
288
evaluated. Antioxidant activity increased up to 207% in legume flour added cookies (Zucco et
289
al., 2011). In the present study, addition of the inactive yeast cells improved the GSH level
290
and increased the antioxidant activities of the cookies significantly which was remarkable for
291
nutritional value.
292 293
3.2. Physicochemical properties of cookies
294
Physicochemical properties of cookies are presented in Table 3. Water binding capacity of
295
yeast cells are lower than that of wheat flour (Salvador et al., 2006). Replacement of flour
296
(10%) with inactive yeast in cookie formulation caused decrease in the moisture content of
297
cookie (CY) as compared to control cookie. However, there was no significant difference
298
(p>0.05) between the moisture contents of C and CY samples, which indicated that the added
299
inactive yeast level was not enough to make a significant effect but only slight decrease.
300
Adding of pure GSH increased the moisture level of cookie as compared to control one.
301
It was indicated in the literature that, the yeast cell walls had high levels of protein and
302
nitrogen (Bekatorou et al., 2006). Therefore, in the present study, the protein content of the
303
inactive yeast added cookie samples was found high as predicted. A significant difference
304
(p<0.05) was only observed in protein content of CY sample compared to other cookies. The
305
protein level reached to 9.72% by adding of inactive yeast into cookie formulation in CY
306
sample.
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The spread ratio of cookie is one of the quality parameters that affect the consumer
308
requirements. Higher spread ratio is related with higher acceptability. In the present study, the
309
results showed a decrease in spread ratio value in CY sample while increase in CG sample
310
compared to control cookie. Dough viscosity and gluten structure are two of the main
311
parameters that affect spread ratio (Miller and Hoseney, 1997; Pareyt et al., 2008). The GSH
312
is a reducing agent which weakens the structure by cleaving the disulfide bonds in gluten
313
proteins (Kline and Sugihara, 1968; Selomulyo and Zhou, 2007). The pure GSH addition to
314
cookie formulation weakened the gluten structure of cookie dough, therefore the spread ratio
315
of CG was found as higher than that of C sample (p<0.05). Although inactive yeast cells
316
released GSH during mixing, CY had lower spread ratio value than the other samples. It was
317
thought that, even there was a GSH release from damaged inactive yeast cells during mixing;
318
yeast cell wall might provide effective protection towards high amount releases that weakened
319
the gluten structure, so that CY had lower spread ratio value than the other samples.
320
Moreover, the moisture content of CG was higher than that of the other samples. Dough
321
viscosity was positively correlated with water binding capacity and the moisture content. The
322
lower moisture content of CY might also be effective on spreading.
323
The hardness of the cookie is one of the most important quality parameter that specifies the
324
tenderness of the product. The hardness of the cookie sample can be affected by its moisture
325
contents inversely. Pareyt et al. (2008) showed that the hardness values decreased by
326
increasing cookie weight and moisture level in the cookies containing various amounts of
327
gluten. Wang et al. (2002) determined that there were negative correlations between the
328
moisture contents and the hardness values in different bread samples. In the present study, the
329
CG with the highest moisture content had the lowest hardness value, while CY which had the
330
lowest moisture content had the highest hardness value (Table 3). Even there was a significant
331
difference (p<0.05) in hardness values only between CY and other samples, the hardness data
ACCEPTED MANUSCRIPT 332
of CG and C samples were still different (p>0.05). Relatively higher hardness of CY was
333
assumed that the added yeast cells also had an impact on cookie structure due to their granule
334
form.
335
The color of the cookie is the first encounter in which consumers criticized the final
336
product and it is one of the main quality parameters beside texture and flavor. Final color of
337
bakery products is the result of formation of browning compounds from non-enzymatic
338
chemical browning reactions such as Maillard and caramelization. The Maillard reaction that
339
occurs during cooking process affects nutritional issues as much as sensorial properties. The
340
reactions between the reducing sugars and amino acids, proteins, and/or other nitrogen-
341
containing compounds with enough available water during baking produces harmful
342
compounds like HMF and acrylamide, in addition to healthy compounds like antioxidants.
343
Thiol precursor of the GSH is interpreted to be a compound to has inhibitory effect towards
344
Maillard reaction during heat treatment processes (Billaud et al., 2004; Friedman, 1994). In
345
the present study, the cookies showed significant difference (p<0.05) in L* values, while no
346
significant difference (p>0.05) in b* values (Table 3). Only CY had significantly (p<0.05)
347
higher a* value than the other cookies. The L* value increased in CG while decreased in CY
348
compared to C sample. It was thought that, decreasing of L* and increasing of a* values in
349
CY sample were not only because of browning reactions, but also the cause of the burning of
350
added inactive yeast cells. Inhibitory effects of the GSH on Maillard reaction might be the
351
reason of the high L* and low a* values of CG sample. Homogeneity of whiteness in CG as
352
compared to C can be seen in Fig. 3.
353 354
4. Conclusion
355
In the present study, the cookies were enriched with inactive yeast cells and pure GSH, and
356
the changes in the GSH content, antioxidant activities and quality properties were examined.
ACCEPTED MANUSCRIPT 357
It was showed that the industrial dry yeast could be converted into inactive yeast cells by
358
applying suitable heat treatment. The cell wall of yeast was thought to be a protecting barrier
359
during cookie baking that prevented the loss of intercellular GSH and maintained the
360
antioxidant activity. The results indicated that the pure GSH addition both inhibited the
361
Maillard reaction, and improved the physicochemical properties of the dough and cookie
362
samples. However, it decreased the antioxidant activity of cookies along with GSH content.
363
The antioxidant activity and GSH content of inactive yeast cell enriched cookies were almost
364
two times higher than those of the control and pure GSH added ones. Even though the
365
physical properties of inactive yeast added cookie were inferior than or similar to control
366
cookie, its nutritive value increased. Nutritional value of cookie containing inactive yeast cells
367
improved significantly in terms of its protein content and antioxidant activities.
368
In enrichment of cookies with natural ingredients and extracts in order to increase the
369
quality and functionality, S. cerevisiae, today’s the only species fully acceptable as food for
370
humans, is one of these ingredients suits this definition and can be used. Limited numbers of
371
studies on GSH content and antioxidant activity of inactive yeast shows that more researches
372
needed to be conducted.
373 374 375
Acknowledgement Authors would like to thank Dr. Arzu Cağrı Mehmetoğlu for her valuable contributions.
376 377
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Winters, R.A., Zukowski, J., Ercal, N., Matthews, R.H., Spitz, D.R., 1995. Analysis of
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Glutathione, Glutathione Disulfide, Cysteine, Homocysteine, and Other Biological
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Thiols by High-Performance Liquid Chromatography Following Derivatization by N-(1-
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Pyrenyl)maleimide. Anal. Biochem. 227, 14–21
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Wood, A., 2013. Give Yeast a Chance. http://www.alive.com/food/give-yeast-a-chance/
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Yue, T., Dong, Q., Guo, C., Worobo, R.W., 2011. Reducing Patulin Contamination in Apple
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Juice by Using Inactive Yeast. J. Food Prot. 74, 149–153
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Zucco, F., Borsuk, Y., Arntfield, S.D., 2011. Physical and nutritional evaluation of wheat
485
cookies supplemented with pulse flours of different particle sizes. LWT - Food Sci.
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Technol. 44, 2070–2076 Figure Captions
488 489
Fig. 1. Sensory scores of inactive yeast added cookies
490 491
Fig. 2. The GSH content of the cookie and dough samples
492
For each column, values followed by the same letter (a-b) are not significantly different
493
(p>0.05) for each treatment
494
C: Control, G: Glutathione, Y: Yeast, ND: Not Detected
495 496
Fig. 3. Cookies (a) control; (b) added with pure GSH (23 mg/recipe); (c) added with inactive
497
yeast (10% w/w flour)
ACCEPTED MANUSCRIPT 6 a a a
a
Sensory scores
5
b b
a b
b
a a b
b
b
4
a
b c
c 3
c
c
2 1 0 Taste
Odor Control
Fig. 1.
5%
Tenderness 10%
20%
30%
Appearance
ACCEPTED MANUSCRIPT
mg GSH/100 g
14
a
12 10
a
8 6
b
4 2
b
ND
0 C
G Cookie
Fig. 2.
Y Dough
ACCEPTED MANUSCRIPT
(a) Fig. 3.
(b)
(c)
ACCEPTED MANUSCRIPT Table 1 Formulation of cookies Ingredients
Weight (g)
All-purpose shortening
25.0
Fine granulated sucrose
25.6
Brownulated granulated sugar
8.0
Nonfat dry milk
0.8
Salt
1.0
Sodium bicarbonate
1.0
High-fructose corn syrup (HFCS)
1.2
Deionized water
17.5
Flour* (or blend)
80.0
*13% moisture basis
13
Table 2 Antioxidant activities of the dough and cookie samples1 DPPH scavenging activity
CUPRAC
ABTS
(%)
(mg troloks/100 g)
(mM trolox/ g)
Conc. Samples2
Conc. Dough
(mg dm/ ml)
Cookie
Dough
Cookie
Dough
Cookie
554.25±10.55b
105.97±5.03b* 128.78±1.93b
(mg dm/ ml)
C
9.38
12.45±0.80c*
8.21
21.52±1.88b
272.10±6.03b*
G
9.32
14.09±0.29b
8.24
15.73±1.22c
287.01±11.16b* 467.48±2.28c
114.29±3.91b
120.67±3.46b
Y
9.40
24.72±0.57a*
8.21
43.66±1.15a
497.14±15.51a*
142.36±2.70a
151.95±3.65a
1For
each column, values followed by the same letter (a-c) are not significantly different (p>0.05); * shows the significant difference between
dough and cookie samples 2C:
13
913.15±36.47a
Control, G: Glutathione, Y: Yeast, Conc.: Concentration of extract, dm: dry matter
Table 3 Physicochemical properties of the cookies1 Sample2
Moisture
Protein
(%)
(%)
Spread ratio
Hardness (N)
Color L*
a*
b*
C
6.19±0.05b
7.25±0.52b 6.24±0.16b
51.69±2.81b 73.64±0.28b 9.32±0.40b
27.45±0.16a
CG
6.84±0.06a
7.19±0.23b 6.71±0.11a
46.60±2.02b 74.39±0.25a 8.64±0.29b
28.26±0.37a
CY
6.04±0.09b
9.72±0.23a 6.06±0.11b
61.90±2.70a 68.84±0.01c 10.70±0.38a 27.65±0.45a
1For 2C:
13
each column, values followed by the same letter (a-c) are not significantly different (p>0.05)
Control; CG: Cookie with pure GSH; CY: Cookie with yeast
ACCEPTED MANUSCRIPT Highlights
Cookies were enriched with inactive yeast cells and pure glutathione (GSH)
The GSH content, antioxidant activity and quality parameters were determined
The GSH content and antioxidant activity increased with yeast addition
Antioxidant activity was higher in cookie than in dough
Yeast addition improved the quality and nutritional properties of the cookies
Serpil Öztürk, İnci Cerit, Selime Mutlu, Omca Demirkol PII:
S0733-5210(17)30508-8
DOI:
10.1016/j.jcs.2017.06.019
Reference:
YJCRS 2392
To appear in:
Journal of Cereal Science
Received Date:
18 October 2016
Revised Date:
30 May 2017
Accepted Date:
29 June 2017
Please cite this article as: Serpil Öztürk, İnci Cerit, Selime Mutlu, Omca Demirkol, Enrichment of cookies with glutathione by inactive yeast cells (Saccharomyces cerevisiae): Physicochemical and functional properties, Journal of Cereal Science (2017), doi: 10.1016/j.jcs.2017.06.019
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ACCEPTED MANUSCRIPT 1
Enrichment of cookies with glutathione by inactive yeast cells (Saccharomyces
2
cerevisiae): Physicochemical and functional properties
3 4
Serpil Öztürk, İnci Cerit, Selime Mutlu, Omca Demirkol*
5 6
Department of Food Engineering, Sakarya University, Esentepe, Sakarya 54187, Turkey
7 8 9 10 11
* Corresponding author: Omca Demirkol
12
Address: Sakarya University, Faculty of Engineering,
13
Department of Food Engineering,
14
54187 Esentepe, Sakarya, Turkey
15
Phone : + 90 264 295 5921
16
Fax
17
E-mail: [email protected]
: + 90 264 295 5601
18 19
e-mails
20
[email protected]
21
[email protected]
22
[email protected]
ACCEPTED MANUSCRIPT 24
Abstract
25
Cookie is one of the favorite cereal products which could be formulated by using various
26
ingredients for enrichment. The aim of this study was to determine the effects of the inactivate
27
yeast cells addition to cookie formulation in terms of glutathione (GSH) content, antioxidant
28
activities, physicochemical and sensorial properties. Also, the cookies were produced with
29
GSH (pure, 98%) addition to compare the effects of inactive yeast GSH with pure GSH on
30
revealed properties. According to results, The GSH contents and the antioxidant activities
31
increased in dough and cookie samples with inactive yeast addition compared to control and
32
pure GSH added dough and cookie samples. The increases in GSH contents and antioxidant
33
activities were observed after baking in all samples. The pure GSH addition increased the
34
moisture content, spread ratio and L* value while decreased the hardness and b* values of the
35
cookies. The contrary results were obtained by inactive yeast addition in terms of the
36
physicochemical properties where the protein content of the cookies increased with inactive
37
yeast addition by approximately 25%. The loss of GSH content decreased while the
38
antioxidant activities and nutritional value increased in cookies by inactivate yeast addition.
39 40
Keywords: Glutathione; Yeast; Cookie; Antioxidant activity
ACCEPTED MANUSCRIPT 41
Abbreviations
42
AACC, The American Association of Cereal Chemists
43
ABTS, 2,20-azino-bis/3-ethyl-benzothiazoline-6-sulfonic acid
44
C, control cookie
45
CUPRAC, cupric ion reducing antioxidant capacity
46
CY, cookie with inactive yeast
47
D, control dough
48
DETAPAC, diethylenetriaminepentaacetic acid
49
DG, dough with pure GSH
50
DPPH, 1,1-diphenyl-2-picryl-hydrazyl
51
DY, dough with inactive yeast
52
HMF, Hydroxymethylfurfural
53
GSH, glutathione
54
GSSG, oxidized glutathione
55
NPM, N-(1-pyrenylmaleimide)
56
SBB, serine borate buffer
57
α-TOHs, α-tocopherols
ACCEPTED MANUSCRIPT 58
1. Introduction
59
Glutathione (GSH), a member of thiols, plays important role as an antioxidants and it is
60
defined as a mercaptan. The GSH has a tripeptide structure (γ-glutamylcysteinylglycine) and
61
is found in animals, plants and microorganisms in milimolar concentrations. Antioxidant
62
property of GSH has been studied as one of the most important topic in biological functions.
63
It is important in detoxification of radicals such as hydroxyl radical (OH•), hydrogen peroxide
64
(H2O2) and superoxide anion (O2•). Although GSH is found as reduced form in the cell, it is
65
being oxidized (GSSG) during in antioxidant reactions. The GSH also plays an important role
66
in transportation of amino acids, synthesis of proteins and DNA (Kans et al., 1988; Orlowski
67
and Meister, 1970; Suthanthiran et al., 1990). In the literature, there are studies about GSH
68
existence in some fresh fruits and vegetables, but it is also known that GSH may get lost in
69
processes such as disinfection, heat treatment and drying (Demirkol et al., 2008, 2004;
70
Gümüşay et al., 2015). Nowadays, GSH is being used as pharmaceutical component and has a
71
potential in food and cosmetic industry. Still, it has not been widely used in the food industry
72
because of its cost.
73
Yeast usage in fermentation processes like alcoholic beverage production and bread
74
leavening made them to be known by humans for almost thousands of years. The current
75
scientific knowledge and technology allow the isolation, construction and industrial
76
production of yeast strains which have high biomass yield on carbon source (Bekatorou et al.,
77
2006). The acceptability as food or feed of a particular microorganism depends on its
78
nutritional value and safety, including nucleic acid content, presence of toxins and residual
79
undesirable compounds. S. cerevisiae consists of 30–33% of dry materials, 6.5–9.3% of
80
nitrogen, 40.6–58.0% of proteins, 35.0–45.0% of carbohydrates (high β-glucan), 4.0–6.0% of
81
lipids, 5.0–7.5% of minerals like Ca, P, K, Mg, Cu, Fe, Zn, Mn, Cr and various amounts of
82
vitamins such as B1, B2, B3, B5, B6, B7, B9 (Wood, 2013), and high amounts of GSH
ACCEPTED MANUSCRIPT 83
depending on its type and growth conditions (Ángeles Pozo-Bayón et al., 2009; Li et al.,
84
2004; Ravindra, 2000). Yeasts contain low amounts of nucleic acids but essential amino acids
85
mainly lysine with higher amount than bacteria or algae. Thus, yeasts are considered to be one
86
of the most important alternative single cell nutrition sources (Bekatorou et al., 2006; Gupta et
87
al., 2013).
88
Inactive form of yeast is produced as nutritional yeast and commercially available in many
89
countries. Yeast cells are inactivated by heat treatment and the process is completed with
90
drying. In recent years, using of inactive yeast cells as a part of wine production process has
91
become common because of the improving effects of GSH in the sensory properties and
92
fermentation. Moreover, it is also considered that the GSH in inactive yeast cells is effective
93
in maintaining the wine color (Ángeles Pozo-Bayón et al., 2009; Andújar-Ortiz et al., 2014).
94
Another application of inactive yeasts in food industry is in removing of toxic substances such
95
as ochratoxin and patulin which have been produced by microorganisms (Piotrowska et al.,
96
2013; Yue et al., 2011).
97
The trends in consumption of foods with high nutritional quality promote the researchers to
98
enrich the cookies likewise all other food products. The aim of this study was to compare the
99
changes in the GSH content, antioxidant activities and quality properties of cookies enriched
100
with inactive yeast cells and pure GSH.
101 102
2. Material and methods
103 104
2.1. Materials
105
Acetonitrile, methanol, hydrochloric acid, sodium hydroxide were provided from Merck
106
(Darmstadt, Germany). Acetic acid, phosphoric acid and borate were purchased from Fisher
107
(St. Louis, MO). GSH, N-(1- Pyrenyl) maleimide (NPM), 2,2-diphenyl- 1-picrylhydrazyl
ACCEPTED MANUSCRIPT 108
(DPPH), Trolox [(±)-6- hydroxy-106 2,5,7,8-tetramethylchroman-2-carboxylic acid],
109
Neocuproine, copper (II) chloride, ammonium acetate, Tris-HCl, serine, diethylenetriamine
110
pentaacetic acid (DETAPAC), 2,20-azino-bis/3-ethyl-benzothiazoline-6-sulfonic acid (ABTS)
111
and potassium persulfate were supplied from Sigma (St. Louis, MO). Instant dry yeast was
112
provided from local markets in Sakarya, Turkey. A hundred gram of instant dry yeast used in
113
the study had a proximate composition of 3.5 g of moisture, 6 g of fat, 39 g of carbonhaydrate
114
and 44 g of protein.
115 116
2.2. Preparation of the cookies
117
Cookies were prepared according to AACC Approved Method No: 10-54 (AACC, 2000)
118
with slight modifications. Formulation of cookies is given in Table 1. Instant dry yeast was
119
heat treated in oven at 120°C for 30 minutes to produce inactive yeast cells. In preliminary
120
study, the inactive yeast cells (2.88 mg GSH/ g yeast) were added to cookie formulation at 5,
121
10, 20, and 30% levels (in flour basis) and the cookies were analyzed for sensorial and
122
physical quality properties and GSH contents (Mutlu et al., 2016). Sensorial properties of the
123
cookies were examined in terms of taste, odor and tenderness. According to the results
124
(Fig.1), 10% level was found as the upper limit in cookies without deterioration in terms of
125
mainly taste. A lower level (5%) was also acceptable in terms of sensorial properties; however
126
the aim of the study was to increase the glutathione content in cookies. Therefore, the upper
127
level, 10%, was chosen to obtain cookies with higher level of glutathione.
128
Hence, the study has continued with the addition of 10% (in flour basis) of inactive yeast
129
(2.88 mg GSH/ g yeast) and 23 mg/recipe of pure GSH which was equaled to the amount of
130
GSH in 10% yeast. Four cookies were baked at a time with a rotary oven (Simsek
131
Laborteknik, Ankara Turkey) at 205°C for 11 min. The baked cookies were cooled at room
ACCEPTED MANUSCRIPT 132
temperature for 2 hours and analyzed for physical quality parameters, and then packed with
133
plastic bags to store at -20C for further analyses.
134
2.3. Preparation of the extracts for determination of the GSH content
135
The dough and cookie extracts were prepared according to the procedure described by
136
Demirkol et al. (2004). The samples were weighted into test tubes and serine borate buffer
137
(SBB) were added (0.1 g/mL) to prevent potential oxidation of biothiols by atmospheric
138
oxygen. The SBB buffer contained 100 mM Tris-HCl, 10 mM borate, 5 mM serine, and 1
139
mM DETAPAC, with the final pH adjusted to 7.0 by a concentrated NaOH solution. The
140
samples were homogenized with tissue tearor (Wiggen Hauser, model D-130 handheld
141
disperser, Germany) in ice bath for 2 min, with 30 second intervals, and then centrifuged at
142
13,130 g for 10 min at 4°C. The supernatants were used to determine the GSH content.
143 144
2.4. Determination of the GSH content by the HPLC method
145
The GSH contents of the extracts were measured by using the chromatographic method to
146
analyze γ-glutamyl cycle intermediates that was developed by Winters et al. (1995), and
147
modified by Demirkol et al. (2004). The supernatants (100 µL) were diluted to 250 µL with
148
distilled water and derivatised with 750 µL N-(1-pyrenyl)maleimide (NPM) solution (1 mM
149
in acetonitrile). The solution was stirred and incubated at room temperature for 5 min, so that
150
the NPM was reacted with free sulfhydryl groups to form fluorescent derivatives. Then, 2 N
151
HCl solution (10 µL) was added to stop the reaction. The solution was filtered through 0.45
152
µm nylon filter and injected into a 5 μm C18 column in a reverse phase HPLC system.
153
The HPLC system (Hitachi, Tokyo, Japan) was comprised of a model L-2130 pump, L-
154
2300 oven, a L-2200 auto sampler, and a Hitachi Chromaster 5440 fluorescence detector
155
which was operated at an excitation wavelength of 330 nm and an emission wavelength of
156
376 nm, and a reversed-phase Reliasil ODS-1 C18 column (5 μm, 250 × 4.6 mm) (Orochem,
ACCEPTED MANUSCRIPT 157
Naperville, USA). The mobile phase consisted of acetonitrile: water solution (70:30) which
158
was adjusted to approximately pH of 2.5 by addition of 1 mL of acetic acid and 1 mL of o-
159
phosphoric acid per liter. The calibration curve was plotted by using 0 to 2500 nM GSH
160
solutions (r2= 0.996).
161 162
2.5. Preparation of the extracts for DPPH and CUPRAC assays
163
The dough and cookie extracts were prepared according to the method described by
164
Capanoglu et al. (2008) to determine DPPH (1,1-diphenyl-2-picryl-hydrazyl) and CUPRAC
165
(cupric ion reducing antioxidant capacity) assays. One gram of a sample was put into test tube
166
and 3 mL of methanol solution (75%, v/v) was added, then the mixture was kept in an
167
ultrasonic water bath for 15 min. The mixture was centrifuged at 13,130 g for 10 min at 4°C,
168
and the supernatant was separated. Three mL of methanol solution was added over the pellet
169
and the procedure was repeated. The collected supernatants were completed to the volume of
170
10 mL with methanol solution.
171 172
2.6. DPPH radical scavenging capacity assay
173
The DPPH assay was applied according to the procedure of Brand-Williams et al. (1995)
174
with some modifications. Three mL of 0.051 mM DPPH in methanol was added to 200 μL of
175
extract and the mixture was incubated at room temperature for 30 min. The DPPH scavenging
176
capacity was evaluated spectrophotometrically (Shimadzu UV-1240, USA) by measuring the
177
decrease in absorbance at 517 nm. Antioxidant capacity was calculated as DPPH radical
178
scavenging capacity (%) = [(A0– A1) / A0] × 100, where A0 was the absorbance of the blank
179
(reacting mixture without the test sample), and A1 was the absorbance of the reacting mixture
180
with the test sample.
181 182
2.7. CUPRAC assay
ACCEPTED MANUSCRIPT 183
The CUPRAC assay was performed according to the method developed by Apak et al.
184
(2004). The sample extract (0.5 mL) and distilled water (0.6 mL) were transferred into a test
185
tube. One mL of 10-2 M copper (II) chloride, 1 mL of 7.5×10-2 M neocuproine, and 1 mL of
186
ammonium acetate buffer solutions were added and the mixture was incubated at room
187
temperature for an hour. The absorbance was determined spectrophotometrically (Shimadzu
188
UV-1240, USA) at 450 nm. The results were expressed as trolox equivalent, milligrams per
189
100 g (r2=0.996).
190 191
2.8. ABTS•+ assay
192
The ABTS (2,20-azino-bis/3-ethyl-benzothiazoline-6-sulfonic acid) assay was applied by
193
QUENCHER method described by Serpen et al. (2008). The stock solution of ABTS•+ was
194
prepared by reacting a 7 mmol/L aqueous solution of ABTS with 2.45 mmol/L K2O8S2. The
195
stock solution was diluted with water/ethanol mixture (50:50, v/v) to obtain the working
196
solution with 0.70-0.80 absorbance value. The dough or cookie sample (10 mg) was mixed
197
with 6 mL of ABTS•+ working solution. The mixture was shaken for 25 min and centrifuged
198
at 9200 g for 2 min at 25°C, and then the absorbance measurement was performed
199
spectrophotometrically (Shimadzu UV-1240, USA) at 734 nm. The results were expressed as
200
trolox equivalent, millimole per gram (r2=0.993).
201 202
2.9. Moisture and protein contents of the cookies
203
The moisture contents (%) of the cookies were measured by gravimetric method in oven at
204
135°C (AACC, 2000). The nitrogen contents were determined by using the Kjeldahl method
205
and were multiplied by a factor of 5.7 to estimate the percent protein content (AACC, 2000).
206 207
2.10. Physical quality parameters of the cookies
ACCEPTED MANUSCRIPT 208
After 2 hours of cooling at room temperature, the cookies were measured for diameter (D)
209
and thickness (T) with a caliper and the spread ratio (D/T) was calculated according to AACC
210
(2000).
211
The hardness values of the cookies were measured by a Texture Analyser (Stable
212
Microsytems, TA-XT plus, UK) equipped with a three-point bending rig. The span between
213
the supports was 4 cm. A load cell of 50 kg was used. The maximum force (Newton) required
214
to break the cookie sample was determined 24 hours after baking.
215
The surface color values of the cookies were determined by using Lovibond Reflectance
216
Colorimeter RT300 (UK), and CIE color values (L*, a*, b*) were measured. The L* value
217
indicates the lightness, 0–100 representing dark to light. The a* value gives the degree of the
218
red-green color. The b* value indicates the degree of the yellow-blue color. The instrument
219
was calibrated by using white and black calibration plates.
220 221
2.11. Sensorial analysis
222
The sensorial properties (taste, odor, tenderness and appearance) of the cookies were
223
screened by panel members by giving the scores ranging between 1 and 5; 5 being the most
224
desirable.
225 226
2.12. Statistical analysis
227
The tests were performed in duplicate and the results were expressed as mean ± standard
228
deviation (SD). The statistical analyses were performed using SPSS (version 11.5, SPSS Inc.,
229
USA). A comparison of the means was confirmed by Duncan’s test at 5% level of
230
significance using one-way analysis of variance (ANOVA).
231 232
3. Results and discussion
ACCEPTED MANUSCRIPT 233 234
3.1. The GSH content and antioxidant activities of dough and cookie samples
235
The GSH content of the dough and cookie samples are presented in Fig.2. It was found that
236
the GSH contents of dough with inactive yeast (DY) and dough with pure GSH (DG) were
237
higher than those of their respective cookies. The reason of the decreases in GSH content in
238
the cookies was the high baking temperature (205°C). The accelerated degradation of GSH in
239
baking process might be due to the baking heat and the surrounding air oxygen. Gümüşay et
240
al. (2015) reported that the significant losses in GSH content were observed in thermal drying
241
of tomato and ginger samples. In another study, it was shown that there was a decrease in
242
GSH concentration in human milk samples subjected to thermal processing (Silvestre et al.,
243
2008). Both of the dough recipes (DG and DY) had 14 mg GSH/100 g dough (23 mg/recipe),
244
however the levels decreased to 8.16 and 11.42 mg/100 g dough in DG and DY, respectively.
245
This phenomenon might be observed because of the oxidation of GSH during mixing trough
246
breaking of the disulphide bonds in gluten by GSH. The GSH cleaved disulphide bonds and
247
formed protein/GSH mixed disulphides, so the number of GSH decreased at the end of the
248
mixing process and GSH transformed into GSSG (oxidized GSH) (Hüttner and Wieser, 2001;
249
Verheyen et al., 2015). The GSH content of the cookie with pure GSH (CG) (3.90 mg
250
GSH/100 g) was lower than that of the cookie with inactive yeast (CY) (7.37 mg GSH/100 g).
251
Although they had same GSH content at the beginning of the process, CY had almost twice as
252
much GSH as CG after cooking. It is well known that eukaryotic structure of yeast cell wall
253
had excellent potential as capsule material and many advantages with natural properties in
254
microencapsulation technology (Nelson, 2002). Because of the resistance of the cell wall of
255
yeast to high temperature, protection of the intercellular GSH level might be observed
256
(Normand et al., 2005).
ACCEPTED MANUSCRIPT 257
Antioxidant activities of the dough and cookie samples are shown in Table 2. The DPPH
258
scavenging activity, CUPRAC and ABTS•+ assays were applied to determine the antioxidant
259
activity values. The DPPH scavenging activities of the control cookie (C), CG, and CY were
260
found as 21.52%, 15.73%, and 43.66%, respectively. The highest antioxidant activity was
261
belonged to CY, because the yeasts synthesized bioactive compounds which could act as
262
antioxidants like the organic acid and the salt forms of citric acid, coenzyme Q or ubiquinone,
263
glutathione, α-tocopherols (α-TOHs) and other forms of tocopherol (Abbas, 2006). The DPPH
264
scavenging activity of the CG was lower than that of control cookie. The nucleophilic
265
properties of sulphydryl compounds played important role in inhibiting the enzymatic and
266
nonenzymatic browning reactions which was the reason of low DPPH scavenging capacity of
267
CG (Friedman, 1994; Molnar-Perl and Friedman, 1990). DPPH scavenging activities of all
268
cookie samples were higher than those of their respective dough samples. However, there was
269
no significant (p>0.05) difference between the DPPH scavenging activities of DG and DC.
270
Maillard reaction products formed during the baking process could be attributed the increases
271
in antioxidant activities of the cookies (Manzocco et al., 2000).
272
The CUPRAC assay for antioxidant activity analysis is sensitive toward thiol-type
273
antioxidants like glutathione (Apak et al., 2004). Although the CUPRAC value of DG was
274
slightly higher than that of the control dough (D), there was no significant difference (p>0.05)
275
probably due to low amount of GSH in dough samples. The CY had the highest (913.15 mg
276
trolox/100 g) while CG had the lowest CUPRAC value due to the same reasons in DPPH
277
scavenging activity assay. The ABTS•+ values of the cookies were higher than those of the
278
dough samples. However, there was significant difference only between the ABTS•+ values of
279
C and D samples (p<0.05). The CY had the highest ABTS•+ value among all samples due to
280
both bioactive compounds of yeast cells and nonenzymatic browning reaction products.
281
Similar to the CUPRAC assay, ABTS•+ value of DG was slightly higher than that of D
ACCEPTED MANUSCRIPT 282
sample, because thiols were irreversibly oxidized by the ABTS radical cation to higher
283
products such as sulphinic or sulphonic acids (Güngör et al., 2011).
284
Abdel-Samie et al. (2010) added the cumin and ginger as antioxidants to dough and
285
examined the effects on cookie quality. According to results, total phenolic compounds and
286
antioxidant activity of cookies increased by adding of 5% cumin and ginger. In another study,
287
different legume flours were incorporated into cookie and nutritional characteristics were
288
evaluated. Antioxidant activity increased up to 207% in legume flour added cookies (Zucco et
289
al., 2011). In the present study, addition of the inactive yeast cells improved the GSH level
290
and increased the antioxidant activities of the cookies significantly which was remarkable for
291
nutritional value.
292 293
3.2. Physicochemical properties of cookies
294
Physicochemical properties of cookies are presented in Table 3. Water binding capacity of
295
yeast cells are lower than that of wheat flour (Salvador et al., 2006). Replacement of flour
296
(10%) with inactive yeast in cookie formulation caused decrease in the moisture content of
297
cookie (CY) as compared to control cookie. However, there was no significant difference
298
(p>0.05) between the moisture contents of C and CY samples, which indicated that the added
299
inactive yeast level was not enough to make a significant effect but only slight decrease.
300
Adding of pure GSH increased the moisture level of cookie as compared to control one.
301
It was indicated in the literature that, the yeast cell walls had high levels of protein and
302
nitrogen (Bekatorou et al., 2006). Therefore, in the present study, the protein content of the
303
inactive yeast added cookie samples was found high as predicted. A significant difference
304
(p<0.05) was only observed in protein content of CY sample compared to other cookies. The
305
protein level reached to 9.72% by adding of inactive yeast into cookie formulation in CY
306
sample.
ACCEPTED MANUSCRIPT 307
The spread ratio of cookie is one of the quality parameters that affect the consumer
308
requirements. Higher spread ratio is related with higher acceptability. In the present study, the
309
results showed a decrease in spread ratio value in CY sample while increase in CG sample
310
compared to control cookie. Dough viscosity and gluten structure are two of the main
311
parameters that affect spread ratio (Miller and Hoseney, 1997; Pareyt et al., 2008). The GSH
312
is a reducing agent which weakens the structure by cleaving the disulfide bonds in gluten
313
proteins (Kline and Sugihara, 1968; Selomulyo and Zhou, 2007). The pure GSH addition to
314
cookie formulation weakened the gluten structure of cookie dough, therefore the spread ratio
315
of CG was found as higher than that of C sample (p<0.05). Although inactive yeast cells
316
released GSH during mixing, CY had lower spread ratio value than the other samples. It was
317
thought that, even there was a GSH release from damaged inactive yeast cells during mixing;
318
yeast cell wall might provide effective protection towards high amount releases that weakened
319
the gluten structure, so that CY had lower spread ratio value than the other samples.
320
Moreover, the moisture content of CG was higher than that of the other samples. Dough
321
viscosity was positively correlated with water binding capacity and the moisture content. The
322
lower moisture content of CY might also be effective on spreading.
323
The hardness of the cookie is one of the most important quality parameter that specifies the
324
tenderness of the product. The hardness of the cookie sample can be affected by its moisture
325
contents inversely. Pareyt et al. (2008) showed that the hardness values decreased by
326
increasing cookie weight and moisture level in the cookies containing various amounts of
327
gluten. Wang et al. (2002) determined that there were negative correlations between the
328
moisture contents and the hardness values in different bread samples. In the present study, the
329
CG with the highest moisture content had the lowest hardness value, while CY which had the
330
lowest moisture content had the highest hardness value (Table 3). Even there was a significant
331
difference (p<0.05) in hardness values only between CY and other samples, the hardness data
ACCEPTED MANUSCRIPT 332
of CG and C samples were still different (p>0.05). Relatively higher hardness of CY was
333
assumed that the added yeast cells also had an impact on cookie structure due to their granule
334
form.
335
The color of the cookie is the first encounter in which consumers criticized the final
336
product and it is one of the main quality parameters beside texture and flavor. Final color of
337
bakery products is the result of formation of browning compounds from non-enzymatic
338
chemical browning reactions such as Maillard and caramelization. The Maillard reaction that
339
occurs during cooking process affects nutritional issues as much as sensorial properties. The
340
reactions between the reducing sugars and amino acids, proteins, and/or other nitrogen-
341
containing compounds with enough available water during baking produces harmful
342
compounds like HMF and acrylamide, in addition to healthy compounds like antioxidants.
343
Thiol precursor of the GSH is interpreted to be a compound to has inhibitory effect towards
344
Maillard reaction during heat treatment processes (Billaud et al., 2004; Friedman, 1994). In
345
the present study, the cookies showed significant difference (p<0.05) in L* values, while no
346
significant difference (p>0.05) in b* values (Table 3). Only CY had significantly (p<0.05)
347
higher a* value than the other cookies. The L* value increased in CG while decreased in CY
348
compared to C sample. It was thought that, decreasing of L* and increasing of a* values in
349
CY sample were not only because of browning reactions, but also the cause of the burning of
350
added inactive yeast cells. Inhibitory effects of the GSH on Maillard reaction might be the
351
reason of the high L* and low a* values of CG sample. Homogeneity of whiteness in CG as
352
compared to C can be seen in Fig. 3.
353 354
4. Conclusion
355
In the present study, the cookies were enriched with inactive yeast cells and pure GSH, and
356
the changes in the GSH content, antioxidant activities and quality properties were examined.
ACCEPTED MANUSCRIPT 357
It was showed that the industrial dry yeast could be converted into inactive yeast cells by
358
applying suitable heat treatment. The cell wall of yeast was thought to be a protecting barrier
359
during cookie baking that prevented the loss of intercellular GSH and maintained the
360
antioxidant activity. The results indicated that the pure GSH addition both inhibited the
361
Maillard reaction, and improved the physicochemical properties of the dough and cookie
362
samples. However, it decreased the antioxidant activity of cookies along with GSH content.
363
The antioxidant activity and GSH content of inactive yeast cell enriched cookies were almost
364
two times higher than those of the control and pure GSH added ones. Even though the
365
physical properties of inactive yeast added cookie were inferior than or similar to control
366
cookie, its nutritive value increased. Nutritional value of cookie containing inactive yeast cells
367
improved significantly in terms of its protein content and antioxidant activities.
368
In enrichment of cookies with natural ingredients and extracts in order to increase the
369
quality and functionality, S. cerevisiae, today’s the only species fully acceptable as food for
370
humans, is one of these ingredients suits this definition and can be used. Limited numbers of
371
studies on GSH content and antioxidant activity of inactive yeast shows that more researches
372
needed to be conducted.
373 374 375
Acknowledgement Authors would like to thank Dr. Arzu Cağrı Mehmetoğlu for her valuable contributions.
376 377
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378
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Technol. 44, 2070–2076 Figure Captions
488 489
Fig. 1. Sensory scores of inactive yeast added cookies
490 491
Fig. 2. The GSH content of the cookie and dough samples
492
For each column, values followed by the same letter (a-b) are not significantly different
493
(p>0.05) for each treatment
494
C: Control, G: Glutathione, Y: Yeast, ND: Not Detected
495 496
Fig. 3. Cookies (a) control; (b) added with pure GSH (23 mg/recipe); (c) added with inactive
497
yeast (10% w/w flour)
ACCEPTED MANUSCRIPT 6 a a a
a
Sensory scores
5
b b
a b
b
a a b
b
b
4
a
b c
c 3
c
c
2 1 0 Taste
Odor Control
Fig. 1.
5%
Tenderness 10%
20%
30%
Appearance
ACCEPTED MANUSCRIPT
mg GSH/100 g
14
a
12 10
a
8 6
b
4 2
b
ND
0 C
G Cookie
Fig. 2.
Y Dough
ACCEPTED MANUSCRIPT
(a) Fig. 3.
(b)
(c)
ACCEPTED MANUSCRIPT Table 1 Formulation of cookies Ingredients
Weight (g)
All-purpose shortening
25.0
Fine granulated sucrose
25.6
Brownulated granulated sugar
8.0
Nonfat dry milk
0.8
Salt
1.0
Sodium bicarbonate
1.0
High-fructose corn syrup (HFCS)
1.2
Deionized water
17.5
Flour* (or blend)
80.0
*13% moisture basis
13
Table 2 Antioxidant activities of the dough and cookie samples1 DPPH scavenging activity
CUPRAC
ABTS
(%)
(mg troloks/100 g)
(mM trolox/ g)
Conc. Samples2
Conc. Dough
(mg dm/ ml)
Cookie
Dough
Cookie
Dough
Cookie
554.25±10.55b
105.97±5.03b* 128.78±1.93b
(mg dm/ ml)
C
9.38
12.45±0.80c*
8.21
21.52±1.88b
272.10±6.03b*
G
9.32
14.09±0.29b
8.24
15.73±1.22c
287.01±11.16b* 467.48±2.28c
114.29±3.91b
120.67±3.46b
Y
9.40
24.72±0.57a*
8.21
43.66±1.15a
497.14±15.51a*
142.36±2.70a
151.95±3.65a
1For
each column, values followed by the same letter (a-c) are not significantly different (p>0.05); * shows the significant difference between
dough and cookie samples 2C:
13
913.15±36.47a
Control, G: Glutathione, Y: Yeast, Conc.: Concentration of extract, dm: dry matter
Table 3 Physicochemical properties of the cookies1 Sample2
Moisture
Protein
(%)
(%)
Spread ratio
Hardness (N)
Color L*
a*
b*
C
6.19±0.05b
7.25±0.52b 6.24±0.16b
51.69±2.81b 73.64±0.28b 9.32±0.40b
27.45±0.16a
CG
6.84±0.06a
7.19±0.23b 6.71±0.11a
46.60±2.02b 74.39±0.25a 8.64±0.29b
28.26±0.37a
CY
6.04±0.09b
9.72±0.23a 6.06±0.11b
61.90±2.70a 68.84±0.01c 10.70±0.38a 27.65±0.45a
1For 2C:
13
each column, values followed by the same letter (a-c) are not significantly different (p>0.05)
Control; CG: Cookie with pure GSH; CY: Cookie with yeast
ACCEPTED MANUSCRIPT Highlights
Cookies were enriched with inactive yeast cells and pure glutathione (GSH)
The GSH content, antioxidant activity and quality parameters were determined
The GSH content and antioxidant activity increased with yeast addition
Antioxidant activity was higher in cookie than in dough
Yeast addition improved the quality and nutritional properties of the cookies