LWT - Food Science and Technology 64 (2015) 1028e1035

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LWT - Food Science and Technology

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

Microbiological, functional and rheological properties of low fat

yogurt supplemented with Pleurotus ostreatus aqueous extract
Ana Carolina Pelaes Vital, Priscila Akie Goto, Letícia Naomi Hanai,
Sandra Maria Gomes-da-Costa, Benício Alves de Abreu Filho, Celso Vataru Nakamura,
Paula Toshimi Matumoto-Pintro*

, Av. Colombo, 5790, Maringa
Universidade Estadual de Maringa
, PR 87020-900, Brazil

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

Article history: Pleurotus ostreatus aqueous extract was incorporated to milk aiming to produce a yogurt with different
Received 29 January 2015 functional and rheological characteristics. Viable counts of Streptococcus thermophilus and Lactobacillus
Received in revised form bulgaricus, pH, lactic acid production, changes in rheological and structural properties (syneresis, texture
29 June 2015
profile analyses, color and microstructure), antioxidant capacity and total phenolic were followed
Accepted 1 July 2015
throughout 28 days of cold storage. Addition of POE increased S. thermophilus and L. bulgaricus CFU
Available online 7 July 2015
counts. Low fat yogurts with POE exhibited lower syneresis and firmness, but more adhesiveness,
springiness and cohesiveness than control. Supplemented yogurts were darker, contained more poly-
Keywords:
Polyphenols
phenols and exhibited higher antioxidant activity than controls in cold storage. Overall, the results
Gel formation indicated that POE can be used to manufacture low fat yogurt with functional activity and at the same
Syneresis time modifies rheological properties.
Texture © 2015 Elsevier Ltd. All rights reserved.
Yogurt microstructure

1. Introduction Polyphenols may interact covalently or non-covalently with

Yogurt and fermented milk products are some of the most
popular foodstuffs in the world. Besides the health benefits related
with their nutritional profile and the presence of live microorgan-
isms, rheological properties and texture characteristics play a very
important role in sensory evaluation and in consumer acceptability.
The most typical defects of fermented milk products are low vis-
cosity and reduced firmness or syneresis and liquid consistency
(Domagała, Wszołek, Tamime, & Kupiec-Teahan, 2013). Manufac-
turers attempt to ensure appropriate texture and prevent these
defects by increasing the total solids content of milk, by adding milk
ingredients and stabilizers (Matumoto-Pintro, Rabiey, Robitaille, &
Britten, 2011). In traditional yogurt, the protein gel is mainly sta-
bilized by weak, non-covalent interactions. The introduction of new
covalent bonds leads to gel formation with different structure and
properties (Domagała et al., 2013), and the use of phenolic com-
pounds offers this opportunity, since the ability of polyphenols to
interact covalently with proteins has also been suggested
(O'Connell & Fox, 1999).
* Corresponding author.
E-mail address: ptmpintro@gmail.com (P.T. Matumoto-Pintro).
proteins. The non-covalent interactions have been suggested to be
created by hydrophobic interactions, which may subsequently be
stabilized by hydrogen bonding (Prigent et al., 2003). Caseins show
a tendency to associate with other proteins according to the hy-
drophobic character of the micelle because of the relatively high
charge. Besides, caseins are proline-rich proteins, which in turn
have a strong affinity for the hydroxyl (
OH) group of phenolic
compounds. The proteinepolyphenol interaction is maximal at the
isoelectric point of the protein and the specific functionality of
phenolic compounds in dairy products is based on their ability to
interact with milk proteins (O'Connell & Fox, 2001; Yuksel, Avci, &
Erdem, 2010).
The oyster mushroom (Pleurotus spp.) is one of the most
commercialized edible mushroom genres in the world. Pleurotus
ostreatus is source of bioactive compounds such as phenolic com-
pounds, dietary fiber (non-digestible carbohydrates, as sources of
prebiotics) and has antioxidant activity, anti-inflammatory, anti-
viral, immunomodulatory effects, in addition to lowering choles-
terol (Aida, Shuhaimi, Yazid, & Maaruf, 2009; Papaspyridi,
Katapodis, Gonou-Zagou, Kapsanaki-Gotsi, & Christakopoulos,
2010).
The objective of this study was investigate the effects on

http://dx.doi.org/10.1016/j.lwt.2015.07.003
0023-6438/© 2015 Elsevier Ltd. All rights reserved.
 A.C. Pelaes Vital et al. / LWT - Food Science and Technology 64 (2015) 1028e1035
functionality (interaction between polyphenoleprotein, antioxi-
dant activity and survival of microorganisms) and structural
properties (syneresis, texture, microstructure) of low fat yogurt
supplementation with a natural ingredient, P. ostreatus (ranging
concentration of 0.25e1%) aqueous extract during 28 days of cold
storage.
2. Materials and methods
2.1. Material
FolineCiocalteu reagent, Fast Blue BB, gallic acid, quercetin, 2,20 -
Azinobis-3-ethylbenzotiazoline-6-sulfonic acid (ABTS), phosphate
buffer, sodium carbonate and potassium persulfate were from
Sigma Aldrich. Aluminum chloride, potassium ferricyanide and
ferric chloride were from Vetec. Culture media MRS, M17 and
peptone water were from Himedia. Skim milk powder (Elege ^,
Brazil) and mushroom P. ostreatus were purchased from a local
store. A commercial yogurt culture (BV- Bela Vista, YOG-03, Brazil),
consisting of Lactobacillus delbrueckii subsp. bulgaricus and Strep-
tococcus thermophilus, was used to prepare the yogurt.
2.2. Preparation of mushroom extract
P. ostreatus was dried at 55  C (PO), crushed and made into a fine
powder. Different concentrations of PO (0%, 0.5%, 1.0%, 1.5% and 2%)
were stirred in Millipore water for 30 min at 70  C and stored at 4  C
overnight. The aqueous extract (POE) was filtered (250 mm) and
used in yogurt production. For antioxidant activity and total
phenolic content, the aqueous extract was freeze-dried (FPOE).
2.3. Total phenolic content
2.3.1. Folin-Ciocalteu assay
Total phenolic content (TPC) for FPOE was determined with
modifications (Singleton & Rossi, 1965). FPOE (1:20 w/v) was mixed
with water and centrifuged for 15 min at 10,000 rpm. An aliquot of
supernatant (125 mL) was mixed with Folin-Ciocalteu reagent (1:1
deionized water) (125 mL) and sodium carbonate (28 g/L)
(2250 mL); the samples were incubated in the dark at 25  C for
30 min and absorbance was measured at 725 nm using a spectro-
photometer (Evolution™ 300 e Thermo Scientific). Results were
expressed as mg gallic acid equivalent (GAE)/g of dried weight. The
standard curve of gallic acid ranging from 0 to 300 mg/L.
2.3.2. Fast Blue BB assay
TPC was measured in low fat yogurt (LF-yogurt) and FPOE by the
Fast Blue BB assay (Medina, 2011). LF-yogurt (1:1 w/v) and FPOE
(1:20 w/v) samples were mixed with water, homogenized for
15 min and centrifuged for 10 min at 10,000 rpm. One mL was
added to 0.1 mL of 0.1% Fast Blue BB reagent (prepared in water).
Then, 0.1 mL of 5% NaOH was added and the reaction was allowed
to sit at room temperature for 90 min. The optical density was
measured at 420 nm. Results were expressed as mg GAE/100 g LF-
yogurt and mg GAE/g FPOE. The standard curve of gallic acid
ranging from 0 to 100 mg/L.
2.4. Antioxidant activity
2.4.1. Ferric reducing antioxidant power (FRAP)
The ferric reducing antioxidant power (FRAP) was assessed by
the method described by Zhu, Hackman, Ensunsa, Holt, and Keen
(2002). LF-yogurt (1:1 w/v) and FPOE (1:20 w/v) samples were
mixed with water and an aliquot of sample (250 mL) was mixed
with 50 mM sodium phosphate buffer pH 7.0 (1.25 mL), 1%
1029
potassium ferricyanide (1.25 mL) and incubated at 50  C for 20 min.
After trichloroacetic acid (10%) (1.25 mL) addition, the mixture was
centrifuged at 3000 rpm for 10 min. The upper layer solution
(2.5 mL) was mixed with 0.1% ferric chloride (500 mL) and the
absorbance was measured at 700 nm. Results were expressed as mg
GAE/100 g LF-yogurt and mg GAE/g FPOE. The standard curve of
gallic acid ranging from 0 to 300 mg/L. Increased absorbance of the
reaction mixture indicated increased reducing power.
2.4.2. ABTS assay
The ABTS assay was conducted according to Re et al. (1999) with
modifications. ABTSþ cation was generated through the interac-
tion of 7 mM ABTS (5 mL) with 140 mM potassium persulfate
(88 mL). The cation was incubated in the dark at room temperature
for 16 h. The ABTS activated radical was diluted with ethanol to an
absorbance of 0.70 ± 0.02 and the radical scavenging (%) was
measured at 734 nm. Sample (1:20 w/v water for FPOE and 1:1 w/v
methanol for yogurt) (40 mL) was mixed with ABTSþ solution
(1960 mL) and absorbances were recorded after 6 min. The radical
scavenging activity (%) was obtained by the equation:
Radical scavenging activity of ABTSð%Þ
¼ ð1
ðA samplet¼0 =A samplet Þ  100Þ
A samplet¼0: absorbance of the sample at time zero;
A samplet: Absorbance of the samples over time.
2.5. Yogurt milk standardization with aqueous extract (POE)
Reconstituted skim milk was mixed with aqueous extract (1:1,
v/v) to a final concentration of protein content 4.2%. The final
concentration of PO in yogurt milk was 0% (without POE), 0.25%,
0.50%, 0.75%, and 1%. Heat treatment was applied to standardized
yogurt milk at 90  C for 3 min. The initial pH of yogurt milk was 6.6
(±0.01).
2.6. Yogurt production
A commercial yogurt culture consisting of L. bulgaricus and
S. thermophilus was used to prepare yogurt. For starter culture
preparation, sterilized reconstituted skim milk powder (12%, w/v)
was inoculated with 0.1% of culture and incubated at 41  C until pH
5.3. For each batch of LF-yogurt (Control (without POE), A (0.25%), B
(0.50%), C (0.75%), and D (1%)), supplemented milk was preheated
to 41  C, inoculated at 3% (v/v) with starter culture and distributed
in plastic containers for texture analysis, antioxidant activity, pH,
acidity and color measurement and Falcon tubes for the syneresis
susceptibility. Inoculated milk was incubated at 41  C until pH 4.6.
LF-yogurt fermentation was stopped by cooling to 4  C and stored
at 4  C until analyses of 1, 7, 14, 21 and 28 days.
2.7. pH and titratable acidity determination
The changes in pH during fermentation and storage were
monitored using a glass electrode pH meter (Tecnopon, mPA-210).
For titratable acidity determination, 10 g of LF-yogurt was mixed
with 10 mL of distilled water and titrated with 0.1 M sodium hy-
droxide solution until pH 8.3 ± 0.01. The titratable acidity was
expressed as g lactic acid/100 g of yogurt and calculated using the
following equation:
Titratable acidityðg=100 gÞ ¼ ðV  f  0:9Þ=m
V ¼ volume (mL) of 0.1 M sodium hydroxide solution used in the
titration; m ¼ mass (g) of test sample; 0.9 ¼ conversion factor for
1030 A.C. Pelaes Vital et al. / LWT - Food Science and Technology 64 (2015) 1028e1035
lactic acid; and f ¼ molarity of sodium hydroxide solution
(ISO11869, 1997).
2.8. Microbiological analysis
M17 culture media was used for quantifying the S. thermophilus
and MRS agar for L. bulgaricus. Plates were incubated under
anaerobic conditions at 37  C for 48 h and 37  C for 72 h, respec-
tively (IDF, 1997). After colonies were counted, the results were
treated as log colony-forming units (cfu) per gram yogurt.
2.9. Syneresis susceptibility evaluation
LF-yogurts were prepared in a Falcon tube (25 g) and centri-
fuged at 2200 rpm for 10 min at 4  C (Robitaille et al., 2009).
Syneresis susceptibility was calculated as the weight percentage of
whey released by centrifugation.
2.10. Low fat yogurt texture analysis
Texture profile analyses (TPA) were performed using a Brook-
field texture analyzer-CT III with a TA/1000 cylindrical probe; the
speed of penetration was 1 mm/s, distance target was 5 mm and
trigger was 15 g. For these tests, samples were taken out of the
refrigerator (4  C) just before test operation. Hardness, adhesive-
ness, cohesiveness, and springiness values were obtained.
2.11. Color evaluation
Color was evaluated in supplemented and control yogurt sam-
ples by CIELAB color scale, measuring the L* (100 ¼ white;
0 ¼ black), a* (þ, red;
, green) and b* (þ, yellow;
, blue) pa-
rameters using a Minolta Chroma Meter CR-400 colorimeter with
illuminate D65 as a reference.
2.12. Microstructure
Microstructure was followed the procedure described by
Matumoto-Pintro et al. (2011). Yogurt samples were frozen fixed in
liquid nitrogen and lyophilized. Samples were mounted on
aluminum stubs and coated with a gold layer (Spotter coater, Bal-
tec, SCD 050). Observations were made using a scanning electron
microscope (SEM) (Superscan, Shimadzu SS-550) at 15 kV.
2.13. Statistical analysis
All experiments were repeated four times and each measure-
ment was performed in triplicate. Analysis of variance (ANOVA)
was performed according to a complete factorial design using the
LSD multiple comparisons procedure of Statistical Analysis System
(SAS) 9.1 software package (SAS Institute Inc., Cary, NC, USA). Dif-
ferences were considered significant at p < 0.05 and results
expressed as mean and standard error or standard deviation.
3. Results and discussion
3.1. Total phenolic content and antioxidant activity of P. ostreatus
extract
TPC in FPOE were analyzed using Folin-Ciocalteu and Fast Blue
BB reagent. The amount in extract was 16.55 mg GAE/g dw for Folin
and 10.34 mg GAE/g dw for Fast Blue BB (Table 1). Some authors
analyzed P. ostreatus for TPC and found between 12.1 and 15.7 mg
GAE/g dw using methanol as solvent and 4.27 mg GAE/g dw using
ethanol (Dubost, Ou, & Beelman, 2007; Elmastas, Isildak, Turkekul,
& Temur, 2007; Yang, Fu, & Li, 2012). These variations may be
related to different factors. Phenolic compounds are influenced by
the climate and location, harvest time, as well as processing and
storage conditions, extraction and analytical methods. The
reducing power ability of the ethanolic extract of P. ostreatus
(10 mg/mL concentration) was found to be an absorbance of 1.367,
which was relatively more pronounced than BHT (Jayakumar,
Thomas, & Geraldine, 2009)); in this study, FRAP was 2.40 mg
GAE/g FPOE, equivalent to an absorbance of 1.632 at a concentra-
tion of 50 mg/mL. The ABTSþ scavenging ability (96.90%) also
demonstrated a higher antioxidant activity of FPOE.
3.2. Low fat yogurt fermentation
Phenolic compounds present in the POE are able to interact with
milk proteins; these may affect the functional properties of dairy
products (O'Connell & Fox, 2001). During fermentation pH de-
creases as the lactic acid is produced by the starter culture d
S. thermophilus and L. bulgaricus. The time taken for LF-yogurts to
reach pH 4.6 was affected by the addition of POE (Fig. 1), and the
multiplication of S. thermophilus and L. bulgaricus was slightly
greater in yogurts supplemented (Table 2), specially in LF-yogurts B,
C and D, which could have been the reason for the shorter incu-
bation time needed to reach pH 4.6 for these samples. This may be
explained due to the prebiotic effect of PO, as its contains dietary
fiber represented by non-digestible carbohydrates like chitin, b-
and a-glucans, xylans, mannans and galactans (Aida et al., 2009).
The ability of fiber to accelerate the acidification rate of milk in
yogurt manufacturing has also been reported (McCann, Fabre, &
Day, 2011; Puvanenthiran, Stevovitch-Rykner, McCann, & Day,
2014). Studies have indicated that PO extracts assist probiotic
bacteria growth rates and the symbiotic effect of the extract might
be successful with some Lactobacillus strains (Aida et al., 2009;
Synytsya et al., 2009).
3.3. Viable cell counts during storage
Phenolic compounds are known antimicrobial agents and the
survival of yogurt microbiota in supplemented yogurts has to be
checked (Chouchouli et al., 2013; O'Connell & Fox, 2001). Yogurt
cultures must remain viable above the required level of 7 log CFU
being active at the end of the shelf life during the cold storage
(Chandan & O'Rell, 2006; Najgebauer-Lejko, Sady, Grega, & Walc-
zycka, 2011).
The viable counts of S. thermophilus and L. bulgaricus decreased
at the end of storage; however, the addition of POE causes signifi-
cant changes in the populations of lactic acid bacteria compared to
the control (Table 2). LF-yogurts B (0.50%), C (0.75%) and D (1%) are
significantly different (p < 0.05) compared to the control for both
microorganisms; this effect can also be observed on most days
analyzed in yogurt A (0.25%).
Studies reported that the mixed yogurt culture, L. bulgaricus and
S. thermophilus, appeared unaffected by the addition of prebiotics
during cold storage, although a slight but not significant (p < 0.05)
increase in cell concentration was observed with the addition of
prebiotics (Vasiljevic, Kealy, & Mishra, 2007). Another study
showed that the addition of prebiotic had a substantial positive
effect on the performance of selected Lactobacillus strains (Donkor,
Nilmini, Stolic, Vasiljevic, & Shah, 2007).
During storage, supplemented LF-yogurts showed a significant
difference in the decrease in pH (Table 2) until day 14 and control
until the 21st, taking a longer time to reduce the pH. Yogurt D (1%)
showed the lowest pH values during storage (4.25), followed by the
others supplemented yogurts and then the control and showed a
significant difference (p < 0.05) in relation to the control in all
 A.C. Pelaes Vital et al. / LWT - Food Science and Technology 64 (2015) 1028e1035 1031

Table 1
Total phenolic content (mg GAE/g), ferric ion reducing power (mg GAE/g) and ABTS radical scavenging capacity (%) of freeze-dried mushroom aqueous extract (FPOE).

TPCFa (mgGAE/g) TPCBb (mgGAE/g) FRAPc (mgGAE/g) ABTSd (% 6 min)

FPOE 16.55 ± 0.08 10.34 ± 0.14 2.40 ± 0.01 96.90 ± 0.30

Results are expressed as mean ± standard deviation.

a
TPCF e Total phenolic content by Folin-Ciocalteu.
b
TPCB e Total phenolic content by Fast Blue BB.
c
FRAP e Ferric ion reducing power.
d
ABTS e ABTS radical scavenging capacity (%).

3.4. Changes in the rheological and structural properties

Changes in the rheological and structural properties of LF-

yogurts did not differ significantly, exhibiting the same behavior
during storage; for this reason, only differences between treat-
ments are shown in Figs. 2e4.

Fig. 1. Change in pH during the fermentation time of low fat yogurt supplemented
with Pleurotus ostreatus aqueous extract. Control (without PO), A (0.25%), B (0.5%), C
(0.75%) and D (1% PO). PO e Pleurotus ostreatus.
evaluated times. In terms of lactic acid, as well as pH, supplemented
yogurts showed a significant (p < 0.05) increase during 14 days.
Differences mainly between control and D (1%) were observed over
time.
3.4.1. Syneresis
Gel formation of milk proteins is an important step in yogurt. LF-
yogurts are known to have certain problems such as syneresis and
texture. Syneresis is the shrinkage of the gel, which then leads to
whey separation (Lucey, 2004; McCann et al., 2011; Ramchandran
& Shah, 2010). Syneresis evaluation results are presented in
Fig. 2. Control showed a higher syneresis (p < 0.05) compared to
supplemented yogurts during storage. The lowest syneresis was
observed with yogurt D (1%) followed by the other concentrations
in decreasing order. Syneresis occurs due to the loss of the ability of
the yogurt gel to retain all of the serum phase because of weak-
ening of the gel network. Casein micelles aggregate through iso-
electric precipitation by the action of lactic acid bacteria. The casein
strands can be broken and the size of the aggregates decreases.
Syneresis and the rearrangement of proteins occurs during storage
(Everett & McLeod, 2005; Lucey, 2002).
Yogurt serum separation can be reduced by increasing the total
solids content of milk, subjecting the milk to severe heat

Table 2
Effect of low fat yogurt supplementation with Pleurotus ostreatus aqueous extract on pH, viability of S. thermophilus and L. delbrueckii subsp. bulgaricus (CFU/g) and titratable
acidity (g lactic acid/100 g) during 28 days of storage at 4  C.

Type of yogurt Period of storage (days)

1 7 14 21 28

pH
Control 4.60 ± 0.023aA 4.47 ± 0.017aB 4.34 ± 0.015aC 4.31 ± 0.015aD 4.29 ± 0.005aD
A 4.56 ± 0.005abA 4.43 ± 0.017abB 4.26 ± 0.005bC 4.28 ± 0.005bC 4.28 ± 0.005abC
B 4.58 ± 0.025abA 4.45 ± 0.026abB 4.29 ± 0.030bC 4.28 ± 0.025bC 4.28 ± 0.005abC
C 4.60 ± 0.030abA 4.44 ± 0.020bcB 4.29 ± 0.025bC 4.30 ± 0.010abC 4.28 ± 0.015bC
D 4.55 ± 0.045bA 4.42 ± 0.005cB 4.28 ± 0.010bC 4.25 ± 0.017cC 4.26 ± 0.005cC
Titratable acidity
Control 0.87 ± 0.026cD 0.96 ± 0.009cC 1.04 ± 0.007cB 1.04 ± 0.089bAB 1.09 ± 0.058bA
A 0.91 ± 0.023bcC 1.02 ± 0.027bB 1.14 ± 0.043abA 1.10 ± 0.034bA 1.10 ± 0.050bA
B 0.92 ± 0.026bC 1.02 ± 0.028bB 1.13 ± 0.028bA 1.10 ± 0.024bA 1.09 ± 0.044bA
C 0.94 ± 0.006bC 1.04 ± 0.023bB 1.11 ± 0.035bA 1.10 ± 0.015bA 1.11 ± 0.013bA
D 1.00 ± 0.033aC 1.11 ± 0.019aB 1.19 ± 0.028aA 1.19 ± 0.012aA 1.21 ± 0.023aA
Viability of S. thermophilus (108 cfu/g)
Control 5.30 ± 0.36dB 5.66 ± 0.83cB 6.53 ± 0.45bA 6.56 ± 0.25cA 5.80 ± 0.45dB
A 5.86 ± 0.25cC 6.06 ± 0.20cBC 7.23 ± 0.30aA 6.80 ± 0.26bcAB 6.56 ± 0.25cB
B 7.30 ± 0.30bAB 7.53 ± 0.30bA 7.20 ± 0.20aAB 6.96 ± 0.20bcB 6.76 ± 0.20bcB
C 7.53 ± 0.25abAB 8.03 ± 0.15abA 7.43 ± 0.15aB 7.16 ± 0.45abB 7.30 ± 0.36abB
D 7.86 ± 0.11aB 8.60 ± 0.36aA 7.50 ± 0.30aB 7.60 ± 0.40aB 7.70 ± 0.26aB
Viability of L. bulgaricus (108 cfu/g)
Control 6.46 ± 0.41dAB 6.56 ± 0.32dA 6.13 ± 0.30cBC 6.30 ± 0.10dABC 5.90 ± 0.20cC
A 6.80 ± 0.36cdB 7.20 ± 0.26cA 6.63 ± 0.15bB 6.60 ± 0.17cdB 6.50 ± 0.20bB
B 7.33 ± 0.30bcB 8.26 ± 0.25bA 6.80 ± 0.10bB 6.80 ± 0.20bcB 6.56 ± 0.25bB
C 7.50 ± 0.36abB 8.50 ± 0.20abA 7.20 ± 0.30aBC 7.06 ± 0.20abC 6.83 ± 0.30abC
D 8.00 ± 0.26aB 8.90 ± 0.30aA 7.33 ± 0.15aC 7.36 ± 0.25aC 7.13 ± 0.20aC

Means with different small letters in the same column are significantly different (p < 0.05). Means with different uppercase letters in the same line are significantly different
(p < 0.05). Control e low fat yogurt without PO; A (0.25%); B (0.5%); C (0.75%); D (1% PO); PO - Pleurotus ostreatus; Results are expressed as mean ± standard deviation.
1032 A.C. Pelaes Vital et al. / LWT - Food Science and Technology 64 (2015) 1028e1035

Fig. 2. Effect of Pleurotus ostreatus aqueous extract (POE) addition on the syneresis of
yogurt during storage.

treatments, increasing homogenization pressure or by adding sta-

bilizers (gelatin, pectin, starches, whey protein concentrate, gums)
that interact with the casein network (Everett & McLeod, 2005;
Matumoto-Pintro et al., 2011).
Polyphenols have a significant affinity for proteins that lead to
the formation of soluble complexes, which can grow in size and
even form sediments. Most models suggest that pro-
teinepolyphenol complexes are formed by multiple weak in-
teractions (mainly hydrophobic) between the amino acid side
chains and the polyphenol aromatic rings, indicating that the as-
sociation of polyphenols with proteins is mainly a surface phe-
nomenon; however, sometimes, these interactions could be
complemented by hydrogen bonding, which reinforce and stabilize
the complexes (Charlton et al., 2002; Oliveira et al., 2015).
These polyphenolecasein stable complexes, due to the interac-
tion of proteinepolyphenol, may be the reason for the reduced
syneresis in LF-yogurts supplemented with POE. Stable complexes
with stronger internal bonds may lead to a reduction in the rear-
rangement of proteins during storage, providing more stability to
casein networks, maintaining water in the network and reducing
syneresis. The formation of proteinepolyphenol complexes is
influenced by the nature of the protein, the nature of the poly-
phenol, the temperature of the system and the presence of other
components that can affect the interaction (Prigent et al., 2003).

3.4.2. Texture profile analysis

Firmness is considered as an important parameter for yogurt
texture (Fig. 3). Control had the highest firmness value during
storage. This was most likely due to a higher protein rearrangement
in the control yogurt (Prasanna, Grandison, & Charalampopoulos,
2013). The addition of POE changed the gel firmness significantly
between treatments, and a lower firmness was showed for all
supplemented yogurts. The increased softness of yogurt with POE
could be attributed to increased water in the gel system due to the
decreased syneresis. It has also been observed that the addition of Fig. 3. Changes in gel firmness, springiness, adhesiveness and cohesiveness of low fat
POE contributed to the increase of cohesiveness, adhesiveness and yogurts supplemented with Pleurotus ostreatus aqueous extract (POE).
springiness when compared with the yogurt control (Fig. 3). An
increase in adhesiveness could indicate a tendency of the LF-yogurt
with POE to become associated with the surface of the textur-
ability of a sample after the deformation regains its condition, may
ometer (Magenis et al., 2006); therefore, more work is needed to
also be attributed to the higher amount of water in the gel, making
overcome the forces of attraction between yogurt surface and
it softer and with a less brittle network (Fig. 2), which means that
texturometer probe surface. An increase in springiness, which is the
 A.C. Pelaes Vital et al. / LWT - Food Science and Technology 64 (2015) 1028e1035
the sample has a greater capacity to regain its initial position. This is
in agreement with the results of cohesiveness, as LF-yogurts made
with POE showed higher values of cohesiveness (greater strength of
internal bonds).
3.4.3. Color measurement
The values of L*, a*, b* obtained according to the CIE color scale
were presented in Fig. 4. The treated results indicated that LF-
yogurt supplemented with POE (above 0.5%) was yellower, red
and darker than control. These colors could be attributed to the POE
color (brown).
Fig. 4. Effect of Pleurotus ostreatus aqueous extract (POE) supplementation on low fat
yogurt color: L* (Lightness), a* (redness) and b* (yellowness).
1033
3.4.4. Microstructure
LF-yogurt microstructures were observed by scanning electron
microscopy (Fig. 5). Yogurt microstructures consisted of a three-
dimensional network of aggregates of casein micelles, in which
the globular shape is observable, interspaced by void zones
(Ramírez-Sucre & Ve
lez-Ruiz, 2013). LF-yogurts made with
different concentrations of POE showed particular networks. Con-
trol yogurt exhibited a continuous branched network, with large
void spaces, which may lead to a large structural rearrangement
and contraction of the protein network during storage (Matumoto-
Pintro et al., 2011). A more compact microstructure was observed
with increasing concentrations of POE. Void spaces observed in
control yogurt almost disappeared when POE was added in C
(0.75%) and D (1%). This microstructural arrangement results in less
protein rearrangement and reduced syneresis susceptibility, as
showed in Fig. 2, specially to LF-yogurt D, making LF-yogurt softer
and more stable during storage (Fig. 3).
3.5. Antioxidant potential of low fat yogurt supplemented with P.
ostreatus aqueous extract
For the determination of antioxidant properties of yogurts, two
methods were used which allowed the ability to reduce pro-
oxidant metal ions (FRAP method) and radical scavenging activity
(ABTS method). The concentration of phenolic compounds was
analyzed with the Fast Blue BB method, since there is less inter-
ference from the non-phenolic antioxidants, reducing substances
and proteins which are naturally present in food (Medina, 2011).
The results of the antioxidant FRAP, ABTS and TPCB assays are
presented in Table 3; values were determined for the first and the
last storage day of LF-yogurts.
Supplemented LF-yogurts exhibit significantly higher TPCB and
antioxidant activity than control for both times analyzed. Antioxi-
dant activity of yogurts decrease with storage time. This result is
probably associated with the formation of a complex between
polyphenols and milk proteins (Lamothe, Azimy, Bazinet, Couillard,
& Britten, 2014). Another study also reported an increase in the
antioxidant activity of yogurt supplemented with grape seeds
extract and a decrease in the antiradical capacity during storage
(Chouchouli et al., 2013). Najgebauer-Lejko et al. (2011) also re-
ported a decrease in anti-radical power during the storage of yo-
gurts supplemented with tea.
The level of polyphenols in yogurts with POE on the first day
ranged from 2.18 to 4.36 mg GAE/100 g and did not change
significantly during storage, except for D (1%). This seems to be
related to a greater number of connections between proteins and
polyphenols, since yogurt D showed stronger bonds (Fig. 3), which
might decrease the polyphenol recovery.
4. Conclusion
The multiplication of S. thermophilus and L. bulgaricus was
greater in yogurts supplemented with P. ostreatus aqueous extract
(POE). The utilization of POE in LF-yogurts improves rheological
properties and texture characteristics (lower firmness but higher
cohesiveness, adhesive, springiness and less syneresis). Scanning
electron microscopy showed different structures, which were more
open and less dense for control. The supplemented yogurts with
POE contained more total phenolics and exhibited higher antioxi-
dant activity than control at the beginning and the end of storage
(day 28). The POE may be a convenient yogurt ingredient because it
seems to improve the antioxidant ability and the rheological and
structural properties, instead the darker color.
1034 A.C. Pelaes Vital et al. / LWT - Food Science and Technology 64 (2015) 1028e1035

Fig. 5. Scanning electron microscopy (SEM) images of low fat yogurt supplemented with Pleurotus ostreatus aqueous extract. Control (without PO), A (0.25%), B (0.5%), C (0.75%) and
D (1% PO). PO e Pleurotus ostreatus.

Table 3
Total phenolic content (mg GAE/100 g), ABTS radical scavenging activity (%) and reducing power (mg GAE/100 g) of control and supplemented low fat yogurts at day 1 and day
28.

Analysis Control A B C D

Day 1
TPCBa (mg/100 g) 2.18 ± 0.105dA 2.93 ± 0.337cA 3.35 ± 0.325bcA 3.92 ± 0.162abA 4.36 ± 0.077aA
FRAPb (mg/100 g) 3.40 ± 0.016eA 3.95 ± 0.049dA 4.49 ± 0.071cA 4.86 ± 0.083bA 5.19 ± 0.118aA
ABTSc (% 6 min) 21.55 ± 1.949cA 25.90 ± 0.102bA 26.85 ± 0.821bA 27.2 ± 0.102bA 29.12 ± 1.086aA
Day 28
TPCB (mg/100 g) 2.04 ± 0.123dA 2.48 ± 0.125cA 3.23 ± 0.261bA 3.72 ± 0.046aA 4.00 ± 0.076aB
FRAP (mg/100 g) 3.10 ± 0.0187eB 3.49 ± 0.063dB 3.80 ± 0.049cB 4.15 ± 0.107bB 4.43 ± 0.005aB
ABTS (% 6 min) 20.04 ± 1.876eB 23.11 ± 0.691dB 25.06 ± 0.100cB 26.95 ± 0.395bB 28.37 ± 0.307aA

Means with different small letters in the same line are significantly different (p < 0.05). Means with different uppercase letters in the same column are significantly different
(p < 0.05) Control e low fat yogurt without PO; A (0.25%); B (0.5%); C (0.75%); D (1% PO); PO - Pleurotus ostreatus. Results are expressed as mean ± standard deviation.
a
TPCB e Total phenolic content by Fast Blue BB.
b
FRAP e Ferric ion reducing power.
c
ABTS e ABTS radical scavenging capacity (%).

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