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Received: 8 March 2018    Revised: 12 November 2018    Accepted: 15 November 2018

DOI: 10.1111/jfpp.13856

ORIGINAL ARTICLE

Effect of blueberry flower pulp on sensory, physicochemical

properties, lactic acid bacteria, and antioxidant activity of
set-type yogurt during refrigeration

Dong Liu  | Xin Xia Lv

Technology, Anqing Vocational & Technical

Department of Food Processing
College, Economic and Technological
Development Zone, Anqing, P.R. China
Correspondence
Xin Xia Lv, Department of
Food Processing Technology, Anqing
Vocational & Technical College, Economic
and Technological Development Zone,
Tianzhushan Road 99, Anqing 246003,
P.R. China.
Email: lvxinxia2015@aqvtc.cn
Funding information
Anhui Provincial Quality Project,
Grant/Award Number: 2016jyxm0639;
2015zdjy181; Excellent Young Talents Fund
Program of Higher Education Institutions
of Anhui Province, Grant/Award Number:
gxyqZD2018120
Abstract
Blueberry flowers were rich in polyphenols and eight essential amino acids. They were
used to prepare blueberry flowers yogurt (BFY) with milk and probiotics. The effects
of blueberry flower pulp on sensory and physicochemical properties, lactic acid bacte‐
ria, and antioxidant of set‐type yogurt were evaluated. Results showed that blueberry
flower pulp compatible with yogurt well. Syneresis rate of BFY ranged from 20.7% to
23.9%, and viscosity ranged from 2.5 to 3.8 pa.s. Blueberry flower pulp reduced the
rate of syneresis at different degrees and significantly improved the viscosity of yo‐
gurt. It could also promote the growth of Lactobacillus plantarum and Streptococcus ther‐
mophilus, and the production of amino acids. During the 29 days’ refrigerated storage,
the total number of probiotics in BFY was more than 1 × 107 cfu/mL. Compared to the
control yogurt, IC50 values of BFY scavenging DPPH (21.06–34.05 mg/ml), ABTS
(20.12–34.57 mg/ml), and OH radical (19.87–33.99) reduced at varying degrees. The
antioxidant activities of BFY were meaningfully enhanced by blueberry flower pulp
and relatively stable.
Practical applications
In recent years, blueberry flower has been used as a scented tea in China, and it is popu‐
lar among consumers. In this study, we added 1%–5% of blueberry flower pulp to de‐
velop BFY, and its effects on physicochemical properties, probiotics, and antioxidant
activity of yogurt were evaluated. Blueberry flower pulp could remarkably improve the
quality and antioxidant activities of yogurt.

1 |  I NTRO D U C TI O N shelf‐life is focused on by the manufacturers and researchers, especially

Yogurt is a fermented dairy product, which is rich in nutrition and ac‐
tivity probiotics. It is usually produced with Streptococcus thermophilus
and Lactobacillus delbrueckii subsp. bulgaricus, as well as other probi‐
otics, such as Lactobacillus acidophilus, and Bifidobacterium (Ye, Liu,
Zhang, Yang, & Wang, 2012). To keep the qualities of yogurts relatively
stable, the yogurt was usually stored at refrigerated about 1 month
(Tseng & Zhao, 2013). If the refrigeration temperature is higher, the lac‐
tic acid bacteria will continue to metabolize and apply nutrients lead‐
ing to post‐acidification so that the sensory quality of yogurts declines
(Aryana & Olson, 2017). How to keep the qualities stable during the
in the development of yogurts with new functional addition. Recently,
polyphenol from fruits and fruit seed, dietary fiber from fruits and fruit
processing wastes, and botanical extracts have been added to yogurt
to improve the quality and bioactive of yogurt (Aryana & Olson, 2017;
Pereira, Cavalcanti, et al., 2016; Pereira, Faria, et al., 2016). Polyphenol
from apple, berry, and grape seed enhanced the antioxidant activity of
yogurt, while the color differences between fortified yogurts and the
yogurt without addition were detected (Chouchouli et al., 2013; Sun‐
Waterhouse, Zhou, & Wadhwa, 2012). What’s more, supply of pine‐
apple peel powder had significantly reduced fermentation time, and
firmness decreased of yogurt (Sah, Vasiljevic, McKechnie, & Donkor,

J Food Process Preserv. 2018;e13856. wileyonlinelibrary.com/journal/jfpp © 2018 Wiley Periodicals, Inc.  |  1 of 10
https://doi.org/10.1111/jfpp.13856
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2016). Wine grape pomace had improved viscosity, kept syneresis,
and lactic acid percentage stable during refrigeration, and it was a
good functional antioxidant dietary fiber to improve physicochemical
quality and antioxidant activity (Tseng & Zhao, 2013). Plant extracts
were also added to yogurt, and their effects on yogurt were evaluated.
Multiple research results have indicated that the use of herbal prepa‐
rations (basically aqueous extracts) not only increases the bioactive
compounds and antioxidant activity of yogurts and fermented milks,
but also increases considerably the sensory acceptance of these new
fermented dairy products (Granato, Santos et al., 2018; Ramos et al.,
2017). Yogurt that enhanced with peppermint, dill, basil, green mate,
clove, and lemongrass extracts had faster rates of pH reduction than
plain yogurt (Amirdivani & Baba, 2011; Ramos et al., 2017). However,
extracts from green, black, and Pu`er tea did not remarkably influence
lactic acid levels during refrigeration (Jazir, Slama, Mhadhbi, Urdaci, &
Hamdi, 2009; Najgebauer‐Lejko, Sady, Grega, & Walczycka, 2011). In
addition, some plant extracts, such as extracts from apple and grape
seed could enhance the viability of probiotic bacteria for storage peri‐
ods (Chouchouli et al., 2013; Sun‐Waterhouse et al., 2012). Blueberry
yogurts were also developed, and blueberry could improve the physi‐
cochemical and sensory properties of yogurts (Cinbas & Yazici, 2008;
Ścibisz, Ziarno, Mitek, & Zaręba, 2012). Blueberry (Vaccinium spp.) was
one of the most popular berries and was well‐liked by consumers for
its nutrients. Blueberry leaves and flowers were rich in phenolic com‐
pounds, and showed good antioxidant activity (Routray & Orsat, 2014;
Wan, Yuan, Cirello, & Seeram, 2012). In recent years, blueberry flowers
had been used as a scented tea in China, and it was popular among
consumers. The purpose of this study was to analyze the nutrients of
blueberry flowers. We used it as functional raw materials to develop
blueberry flower yogurts (BFY), and the effect of blueberry flower pulp
on physicochemical properties, probiotics, and antioxidant activity of
set‐type yogurt during refrigeration.
2 |  M ATE R I A L S A N D M E TH O DS
2.1 | Materials
Yogurt starter, which composed of L. plantarum, S. thermophilus, and B.
bifidum, was purchased from Harbin Meihua Biotechnology Co., Ltd.
(Harbin, China). UHT milk (fat 4.4 g/l00 ml, protein 3.6 g/l00 ml, car‐
bohydrates 5.0  g/l00  ml, Hefei Yili dairy Co., Ltd., Hefei, China) and su‐
crose was purchased from the supermarket. Blueberry flowers (Misty)
were collected from Anqing Guiwen Agricultural Technology Co.,
Ltd. ABTS (2,2‐azino‐bis‐3‐ethylbenzothiazoline‐6‐sulfonic acid),
DPPH (1,1‐Diphenyl‐2‐picrylhydrazyl radical), Folin–Ciocalteu’s
phenol reagent, and epigallocatechin gallate (EGCG) standards were
purchased from Sigma‐Aldrich (Shanghai, China). All the other used
chemicals and solvents were up to analytical grade.
2.2 | The determination of nutrients and amino
acid of blueberry flowers and yogurt
Crude protein, crude fat, crude fiber, ash, total solids, and nonfat solids
were tested according to standard methods of Association of Official
Analytical Chemists (2003) procedures. The content of polyphenols
were determined by Folin–Ciocalteu method at 765 nm, which de‐
scribed by Liu and Tan (2016), and polyphenols were presented as
EGCG. The standard curve was: A765 nm = 0.0122 CEGCG + 0.0072
(R2 = 0.9989), when the concentration of EGCG ranged from 10 to
50 (μg/mL). Determination of amino acid was tested according to the
method described by Liu and Tan (2016). Briefly, one gram of fresh
flowers was acidolysis with 10 ml HCL (6 mol/L) at 140°C for 8 hr,
and acidolysis mixtures were diluted to 10 ml with 0.02 mol/L HCL.
Samples (20 μl) were injected into the automatic amino acid analyzer
L‐8900 (Hitichi, Japan) and test conditions were setup according to
the method reported by Ye, Ren, Wu, Wang, and Liu (2013). The ex‐
periments were repeated 3 times, and results expressed as mean  ±  SD.
2.3 | Preparation of BFY
BFY was prepared according to Jaziri, Slama, Mhadhbi, Urdaci, and
Hamdi (2009) with minor modification. Based on preliminary experi‐
ments, 600 ml of UHT milk and 1%–5% (w/v) of fresh blueberry flowers
were put into a Philips (HR2084) food processor, and the mixtures were
mashed intermittently for 2–5 min. The mixtures were filtered with six
layers of clean gauze to remove flowers residue, and 30 g sucrose was
added. The mixtures were heated to 94°C for 10 min. The mixtures were
cooled to 42°C, and 2.0 g/L of yogurt starters were inoculated for cultur‐
ing at 42°C for 8 hr. Then, BFYs (BFY1%, BFY2%, BFY3%, BFY4%,and BFY5%)
were got, and yogurts without blueberry flowers pulp were the control.
All the yogurts were cooled and stored at 6°C for 29d. The experiments
were repeated ten times with each test carried out in five copies.
2.4 | Consumer test and sensory analysis
Consumer tests were carried out according to the method of Dantas
et al. (2016). The degree of liking of yogurt samples were evalu‐
ated in terms of their appearance, aroma, taste, texture, and over‐
all acceptance by 112 participants (50 men and 62 women, aged
18–52 yr), who randomly worked at Anqing Vocational &Technical
College. Fifty milliliter yogurt was presented to recruited panelists,
and a 9‐point scale was used to assess the yogurts (1 = disliked im‐
mensely, 9 = liked immensely).
The sensory properties of the control yogurt and BFY were
explored with quantitative descriptive analysis (Janiaski, Pimentel,
Cruz, & Prudencio, 2016). Thirty volunteers who consumed yogurt
over 4 times a week, about 2 L, were recruited, and they were the
students and teachers from department of food processing tech‐
nology of the Anqing Vocational &Technical College. Volunteers’s
sensory acuity was tests through the recognition of basic tastes
and odors (Janiaski et al., 2016). The descriptive sensory vocab‐
ulary, definitions, and references of the control yogurt and BFY
(Table 1) were generated by the volunteers according to Janiaski et
al. (2016) and Pimentel, Cruz, and Prudencio (2013). The evaluation
sheet was prepared in consensus and consisted of 9 cm nonstruc‐
tured scales, anchored at the two terminals by “absent” (anchored
at 0) or “weak” (anchored at 0.5 cm) to the left and “intense,” or
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TA B L E 1   Attributes, definitions, and reference samples developed by descriptive sensory panel

Descriptor Definition Reference sample

Appearance
Color Intensity of color ranging from white color Weak: Yili natural nonfat yogurt; intense:
to yellow 100 ml of Yili fermented milk + 150 ml of
pasteurization reconstituted milk (Wangzai)
Brightness Reflection of light on the surface of A little: Vigor curd milk; a lot: Yili full‐fat plain
fermented dairy products yogurt
Syneresis Presence of superficial liquid Absent: Vigor curd milk without liquid surface;
a lot: Vigor curd milk
Particles Presence of milk white petals residue in the Absent: 100 ml of Yili full‐fat plain yogurt; a lot:
yogurts 100 ml of fermented natural whey beverage
(Yili) + 2.5 g of gelatin (Cargill)
Aroma
Acid Acid aroma profiles of fermented milk Weak: 400 ml of Yili plain full‐fat yogurt 
products + 600 g of Yili UHT milk (final pH =5);
intense: 1,000 ml of Yili plain full‐fat yogurt
kept at 42°C for 3–5 hr (final pH = 4)
Artificial blueberry Artificial blueberry aroma of dairy Weak: 400 ml of UHT milk (Yili) + 10 drops of
products blueberry essence (Zhejiang jubang high‐tech
co. LTD); intense: 400 ml of UHT milk (Yili)
with 60 drops of blueberry essence (Zhejiang
jubang high‐tech co. LTD)
Flavor
Acid taste Acid taste of fermented dairy products Weak: 400 ml of Yili plain full‐fat yogurt 
+ 600 g of Yili UHT milk (final pH = 5);
intense: 1,000 ml of Yili plain full‐fat yogurt
kept at 42°C for 3–5 hr (final pH = 4)
Sweet taste Sweet taste associated with sugar Weak: 400 ml of plain yogurt (Yili) with 10 g of
presence refined sugar (Taikoo); intense: 400 ml of plain
yogurt (Yili) with 60 g of refined sugar (Taikoo)
Artificial blueberry taste Artificial blueberry taste of dairy products Weak: 400 ml of UHT milk (Yili) + 10 drops of
blueberry
essence (Zhejiang jubang high‐tech co. LTD);
intense: 400 ml of UHT milk (Yili) with 60
drops of blueberry essence (Zhejiang jubang
high‐tech co. LTD)
Texture
Firmness Ease of cutting the yogurt when manipu‐ Weak: Yili plain full‐fat yogurt; Strong:
lated with a spoon Danoninho (Danone) petit suisse with
banana pulp
Viscosity Fluid resistance to falling off when Weak: 50 ml of Yili full‐fat plain yogurt + 50 ml
transferred Yili UHT milk;
Strong: 90 g of Yili full‐fat plain yogurt + 10 ml
of dissolved pectin (10%, Cargill)

“a lot” or “strong” to the right (anchored at 8.5 cm) described by
Janiaski et al. (2016). The panelists, who were trained for 1 hr,
participated in sensory properties evaluation of three yogurts
(control yogurt, BFY2% , and BFY4% ,) with the evaluation sheet.
The experiments were implemented in three replicates. A 2‐factor
analysis of variance (ANOVA) and an F test were conducted based
on data from each panelist to screen out more qualified panelists
who could discriminate among yogurts (P value of Fsamples < 0.5),
exhibited repeatability (P value of Frepetitions ≥ 0.05), and reached
agreements with the other members (Janiaski et al., 2016). Finally,
20 panelists (10 men and 10 women, aged 18–46 yr), 10 students
and 10 teachers were considered trained, and 6 samples was sup‐
plied to them. Fifty milliliter yogurt of each sample were served in
containers coded with random 3‐digit numbers, at a temperature
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12 ± 1°C, and presented in a random order. The evaluation sheet The smaller IC50 value was, the stronger antioxidant activity gained
developed during the training sessions was used, and the data (Chaikham, 2015). All of the measurements were obtained by con‐
were collected. ducting seven replications.

2.5 | Measurement of titratable acidity, pH, 2.8 | Statistical analysis

syneresis, and viscosity
Control and HTY were taken samples on 1, 8, 15, 22, and 29 days.
Titratable acidity, as percent lactic acid, was measured according to
Tseng and Zhao (2013). PHS‐3C pH meter (Shanghai Rex Instruments
Factory, shanghai, China) was used to determine the pH of yogurts.
Syneresis and viscosity were measured according to the method re‐
ported by Yangilar and Yildiz (2018). All the experiment repeated 7
times, and results expressed as mean ± SD.
2.6 | Probiotics counts
S. thermophilus and B. bifidum were measured according to Ye et al.
(2012). S. thermophilus was enumerated onto M17‐lactose agar plates,
and cultured under aerobic conditions at 37°C for 72 hr. Enumeration
of B. bifidum was performed with MRS‐raffinose agar plate containing
0.05% LiCl, and L. plantarum was enumerated onto MRS agar plates.
Plates were cultured in a carbon dioxide incubator MCO‐18AIC
(SANYO, Japan) at 37°C for 72 hr. Milky spot colonies of 0.3–0.7 mm
diameter were B. Bifidum, and milky or light yellow spot colonies of
2.1–3 mm diameter were L. plantarum (Brinques & Ayub, 2011). And
Gram’s staining methods were used for further confirmation. The
colony count was expressed by log cfu/ml and results were the means
of seven replications.
SPSS16 was used for ANOVA for data and significant differences
(p < 0.05) among means were determined by the least significant
difference test. To perform principal component analysis (PCA), the
average data of each attribute was used and an original data ma‐
trix, X = (Xij) n × p, had 6 rows (the 6 yogurts) and 12 columns (the
12 sensory attributes). And the Kaiser–Meyer–Olkin and Bartlett’s
test of sphericity were used to test whether data are suited for PCA
with SPSS16 (Granato, Putnik, et al., 2018). The original data were
̄
(Xij −X)
standardized according to a formula, Zij = SDj
(1). Zij represents the
standardized value of j index of the i yogurt. X̄ j and SDj represents
n
the mean and standard deviation of j index, respectively. X̄ j = 1n Xij,
∑
n
i=1
and SDj = n−1
2 1 ∑
(Xij − Xj ) . Then, the standardized data matrix,
̄ 2
i=1
Z = (Zij)n × p, was got, and the covariance matrix C is obtained with a
formula, C = 1
n−1
Z� Z (Granato, Putnik, et al., 2018). Eigenvalue and
eigenvector of matrix C, and principal component scores were com‐
puted by XLSTAT 2018. According to Janiaski, Pimentel, Cruz, and
Prudencio (2016) and Granato, Putnik et al. (2018), the number of
principal components were determined according to the accumula‐
tive contribution rate of variance of principal components (over 75%)
and the eigenvalue (greater than 1).
3 | R E S U LT S A N D D I S CU S S I O N

3.1 | Main nutritional ingredient of blueberry

2.7 | Antioxidant activity
To investigate the antioxidant activities of BFYs, ABTS, DPPH, and
OH radical scavenging capacity was assayed. The ABTS radical scav‐
enging activity was carried out according to the method described
by Dudonné, Vitrac, Coutiere, Woillez, and Mérillon (2009). The
DPPH and OH radical scavenging activity assay described by Ye et
al. (2012) was employed. All activities were expressed as IC50, which
indicated the ability to remove 50% oxidants or oxidizing substances.
flowers and yogurt
As shown in Table 2, the main components of fresh blueberry
flowers include crude protein, crude fat, crude fiber, phenolic sub‐
stances, and minerals. The contents of them in fresh blueberry flow‐
ers were 7.68 g/100 g, 0.97 g/100 g, 6.33 g/100 g, 280 mg/100 g,
and 0.76 g/100 g, respectively. Protein, crude fiber, and minerals
might enhance the nutrition of yogurt. Meanwhile, polyphenols
could improve the antioxidant activity of yogurt (Routray & Orsat,

TA B L E 2   Main nutrients of blueberry flowers and yogurts

Main components BF Control BFY1% BFY2% BFY3% BFY4% BFY5%

Crude protein (g/100 g) 7.68 ± 1.36 3.14 ± 0.07 3.18 ± 0.10 3.21 ± 0.14 3.25 ± 0.06 3.28 ± 0.09 3.32 ± 0.21
Crude fat (g/100 g) 0.97 ± 0.06 4.06 ± 0.13 4.06 ± 0.22 4.05 ± 0.16 4.01 ± 0.09 3.98 ± 0.03 3.96 ± 0.11
Crude fiber (g/100 g) 6.33 ± 0.07 ND ND ND ND ND ND
Polyphenol(mg/100 g) 280 ± 1.20 ND 2.67 ± 0.25 5.26 ± 0.12 7.85 ± 0.19 10.36 ± 0.10 12.82 ± 0.22
Ash (g/100 g) 0.76 ± 0.08 0.73 ± 0.05 0.74 ± 0.09 0.74 ± 0.04 0.73 ± 0.06 0.76 ± 0.05 0.75 ± 0.02
Total solids (g/100 g) ND 13.73 ± 0.08 14.07 ± 0.14 14.55 ± 0.21 15.03 ± 0.26* 15.40 ± 0.11* 15.85 ± 0.05**
Not‐fat solids (g/100 g) ND 9.67 ± 0.14 10.01 ± 0.10 10.50 ± 0.13 11.02 ± 0.19* 11.42 ± 0.21** 11.89 ± 0.09**

Note. ND, not detected; BF, blueberry flowers.

*Significant differences were detected at p < 0.05 compared to the control. **Significant differences were detected at p < 0.01 compared to the
control.
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2014; Wan et al., 2012). In BFY, the content of polyphenols ranged
from 2.67 to 12.82 mg/100 g, and they increased as the amount of
blueberry flower pulp increased. The contents of crude protein, fat,
and ash were not markedly changed between BFY and the control.
The contents of total solids and nonfat solids in BFY3% , BFY4% , and
BFY5% were over 15.03 and 11.02 g/100 g, respectively, which were
notably (p < 0.05, p < 0.01) higher than those of the control, and
they were positively correlated to the concentration of blueberry
flower pulp. The main components of BFY were consistent with the
requirements of the codex standard for fermented milks, CODEX
STAN 243.
3.2 | Amino acid composition and content of
blueberry flowers and yogurt
Blueberry flowers contain 18 kinds of amino acids, and their total con‐
tent was 7,422.1 μg/g (Table 3). Eight essential amino acids were de‐
tected in blueberry flowers, and the content of them was 3,200.2  μg/g,
accounting for 43.12% of the total amino acid content. The contents
of essential amino acids, threonine (Thr), valine (Val), methionine
(Met), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), lysine (Lys),
tryptophan (Trp) was 325.4, 428.8, 86.2, 348.5, 607.9, 389.7, 450.8,
and 562.9 μg/g, respectively (Table 2). In BFY, 18 kinds of amino acids
were detected including 8 essential amino acids. The total content of
18 kinds of amino acids was in the range of 14,641.4–15,017.5 μg/g,
and eight essential amino acids were over 7,670.8 μg/g. The contents
of 18 kinds of amino acids were improved to different degrees, which
were positively correlated to the addition of blueberry flower pulp.
The content of glutamate, glycine, cystein, phenylalanine, arginine,
and tryptophan in BFY4% and BFY5% were significantly (p < 0.05,
p < 0.01) higher than those of the control, whereas there was no
significant difference among the other amino acids. Some of amino
acids from blueberry flowers stimulated the lactic acid bacteria’s
growth, which causes proteolysis and produces amino acids. This
might account for the increasing of amino acids (Tamime & Robinson,
2007).
3.3 | Consumer test and sensory properties of BFY
In Table 4, the acceptance score of the color between control yogurt
and BFY was not significant difference. Aroma, taste, and texture ac‐
ceptance scores of BFY2%, BFY3%, BFY4%, and BFY5% were obviously
(p < 0.05, p < 0.01) higher than that of the control, and the aromas
were positively correlated with the concentration of blueberry flower
pulp. The texture of BFY was fine and uniform, and the texture score
was over 7.38. These indicated that the added 2%–5% blueberry
flowers pulp improved the texture quality of yogurt. Like pineapple
peel powder and wine grape pomace could improve the texture qual‐
ity of yogurt (Sah et al., 2016; Tseng & Zhao, 2013), dietary fiber from
blueberry flower pulp might play an important role on these positive
effects. BFY had the typical flavor of yogurt, and had the unique fla‐
vor of the blueberry flowers. BFY was delicious, and it was well‐liked
by the panelists. The overall evaluation scores were larger than 7.5,
which was higher than that of the control in varying degrees.

TA B L E 3   Amino acid composition of blueberry flowers and yogurt

Amino acid (μg/g) BF Control BFY1% BFY2% BFY3% BFY4% BFY5%

Aspartate (Asp) 730.5 ± 1.6 1,420.0 ± 7.7 1,426.8 ± 9.1 1,432.9 ± 8.5 1,441.0 ± 8.8 1,446.2 ± 6.8 1,455.1 ± 6.2

●
Threonine (Thr) 325.4 ± 8.6 560.5 ± 6.2 563.6 ± 11.3 566.4 ± 7.5 569.2 ± 10.2 572.0 ± 12.3 574.3 ± 9.9
Serine (Ser) 352.2 ± 10.8 825.4 ± 10.4 828.2 ± 11.6 830.2 ± 7.8 835.6 ± 13.1 837.9 ± 8.7 842.8 ± 17.2
Glutamate (Glu) 1,101.7 ± 10.6 512.3 ± 8.5 522.6 ± 12.8 534.0 ± 10.2 542.9 ± 8.9 554.2 ± 10.2* 566.5 ± 11.3*
Glycine (Gly) 372.5 ± 1.5 270.6 ± 9.6 284.2 ± 10.5 297.2 ± 6.6* 310.5 ± 7.8* 313.7 ± 7.2** 317.9 ± 5.6**
Alanine (Ala) 463.9 ± 3.3 721.0 ± 10.4 725.5 ± 9.3 730.0 ± 13.3 734.6 ± 10.5 738.5 ± 15.2 744.0 ± 9.7
Cystein (Cys) 33.2 ± 1.1 141.2 ± 6.6 154.4 ± 18.1 167.5 ± 8.2* 170.8 ± 6.1* 174.0 ± 6.5* 177.3 ± 5.7*
●
Valine (Val) 428.8 ± 4.3 1,076.2 ± 9.1 1,080.3 ± 8.5 1,084.1 ± 6.1 1,008.0 ± 7.7 1,092.9 ± 8.9 1,098.1 ± 5.8
Methionine (Met)● 86.2 ± 2.9 452.3 ± 15.8 453.2 ± 10.6 454.0 ± 11.2 455.1 ± 10.2 455.8 ± 7.6 456.7 ± 6.6
●
Isoleucine (Ile) 348.5 ± 2.1 931.6 ± 12.2 934.5 ± 10.1 938.7 ± 9.8 941.7 ± 14.5 945.2 ± 8.4 949.5 ± 9.0
Leucine (Leu)● 607.9 ± 1.4 1802.7 ± 3.7 1806.5 ± 9.5 1812.8 ± 8.8 1820.2 ± 7.7 1825.9 ± 9.0 1830.6 ± 5.8
Tyrosine (Tyr) 244.6 ± 6.0 826.1 ± 5.9 828.5 ± 5.8 830.7 ± 15.2 833.2 ± 11.3 836.1 ± 12.2 838.9 ± 8.5
Phenylalanine(Phe)● 389.7 ± 10.2 922.7 ± 10.1 936.5 ± 11.2 950.3 ± 8.1 954.2 ± 12.5 968.0 ± 21.5* 971.6 ± 8.9*
●
Lysine (Lys) 450.8 ± 11.0 1,130.2 ± 8.3 1,134.7 ± 8.9 1,139.5 ± 9.9 1,143.6 ± 7.6 1,148.5 ± 6.8 1,152.2 ± 6.5
Histidine (His) 57.2 ± 3.2 526.4 ± 8.5 526.9 ± 12.3 527.5 ± 10.5 528.0 ± 8.7 528.3 ± 18.4 529.1 ± 10.2
●
Arginine (Arg) 181.6 ± 2.2 622.6 ± 10.5 644.5 ± 14.2 656.1 ± 10.7 658.0 ± 5.5* 659.7 ± 7.9* 662.3 ± 11.3*
Proline (Pro) 684.5 ± 1.3 1,052.3 ± 8.3 1,059.0 ± 9.3 1,065.1 ± 8.8 1,070.4 ± 7.0 1,079.2 ± 4.9 1,086.5 ± 8.6
Tryptophan(Trp)● 562.9 ± 1.1 716.0 ± 8.7 731.5 ± 15.2 737.6 ± 10.4 752.2 ± 20.2 758.4 ± 8.8* 764.1 ± 11.3*
●
Essential amino acid; BF, blueberry flowers. *Significant differences were detected at p < 0.05 between BFY and control yogurt. **Significant differ‐
ences were detected at p < 0.01 compared to the control.
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TA B L E 4   Sensory acceptance of yogurts

Sensory properties Control BFY1% BFY2% BFY3% BFY4% BFY5%

Appearance 7.56 ± 0.06 7.62 ± 0.12 7.65 ± 0.13 7.68 ± 0.19 7.59 ± 0.21 7.62 ± 0.11

Aroma 7.20 ± 0.22 7.67 ± 0.16 7.94 ± 0.28* 8.01 ± 0.21* 8.08 ± 0.31* 8.21 ± 0.48**
Taste 7.37 ± 0.46 7.56 ± 0.06 8.18 ± 0.29** 8.11 ± 0.54* 7.93 ± 0.25* 7.89 ± 0.36*
Texture 7.15 ± 0.22 7.38 ± 0.32 7.85 ± 0.16* 8.06 ± 0.23* 8.02 ± 0.12* 7.99 ± 0.11*
Overall acceptance 7.21 ± 0.28 7.54 ± 0.16 7.96 ± 0.07* 7.98 ± 0.12* 7.85 ± 0.23* 7.72 ± 0.08
*Significant differences were detected at p < 0.05 compared to the control. **Significant differences were detected at p < 0.01 compared to the
control.

The intensity of descriptive attributes data was analyzed with
XLSTAT 2018, and the first two principal components (PC1 and
PC2) were gotten when eigenvalue was over one. In Figure 1, the
PC1explained 73.18% of the variability contained in the original vari‐
ables, and the PC2 explained 17.91%, respectively, totaling 91.08%.
Therefore, the attributes were considered important in a principal
component because its correlation with the axis was over than 0.7
(Janiaski et al., 2016). PC1 separated the yogurts in relation to the
content of blueberry flower pulp (the left side presents control yo‐
gurt, BFY1% andBFY2% and the right side contains BFY3% , BFY4%, and
BFY5%). PC1 was represented by particles, color, firmness, viscosity,
artificial blueberry taste, and aroma, in the positive part of the axis,
and syneresis, and sweet taste in the negative part of the axis. PC2
was mainly represented by acid aroma and acid taste. These results
suggest that the addition of blueberry flower pulp to the yogurt
led to different sensory properties. In Table 5, the mean intensity
values for the sensory attributes are given. The color, brightness,
and particles of BFY were stronger than the control, while syneresis
was less than the control. In BFY1% , BFY2%, and BFY3% , acid aromas
were stronger compared to the control; however, acid tastes were
no significant change, except BFY2% , BFY3% and BFY5% . BFY had a
more intense artificial strawberry aroma and taste, but sweet taste
was not significantly changed. In addition, blueberry flower pulp had
1.2
BFY2%
1
0.8 Acid aroma
Acid taste
0.6
0.4
PC2 (17.91 %)
positively effects on the texture of yogurt, and it could increase the
intensity of firmness and viscosity of yogurt. The phenomenon could
be related to crude fiber from blueberry flower pulp (Table 2).
3.4 | Titratable acidity and pH of BFY
Titratable acidity and pH were detected weekly. As shown in Figure 2,
in the 29 days refrigeration, Lactic acid bacteria kept metabolizing
slowly to produced lactic acid and other organic acids (Tamime &
Robinson, 2007), which led to titratable acidity increasing (Figure 2a).
The pH kept on decreasing (Figure 2b) during refrigerated storage,
and similar phenomena were observed in previous studies (Chaikham
2015; Tamime & Robinson, 2007). pH and titratable acidity of BFY1%,
BFY2%, BFY3%, and BFY4% were in the range of 4.1–4.8 and 0.76%–
1.37%, respectively, which were not significant differences from the
control. It indicated that 1%–4% of blueberry flowers pulp had a little
effect on pH and titratable acidity during 29 days of cold storage. In
addition, the similar results were also found in other yogurts, which
enhanced with peppermint, dill, basil, green tea, black tea, Hawk tea,
Fuzhuan brick tea, and Plantago psyllium extracts (Amirdivani & Baba,
2011; Granato, Santos et al., 2018; Jaziri et al., 2009).
3.5 | Syneresis and viscosity of BFY
BFY3%
Syneresis and viscosity were an important property of set‐type yo‐
gurts (Najgebauer‐Lejko et al., 2011). Syneresis rate of BFY ranged
from 20.7% to 23.9%. It decreased at different degrees during the
29 days of cold storage (Figure 3a). At the same storage period, the
syneresis of BFY was lower than those of the control, and it sug‐

0.2 Particles gested that the water‐holding capacity increased compared to the

Firmness
0
Syneresis
Brightness
Viscosity
control. The main reason might be that some active ingredients of
Artificial blueberry aroma
-0.2 BFY1%
Artificial blueberry taste
Color blueberry flower pulp, such as crude fiber interacted with protein
-0.4 Sweet taste from the yogurt, and they established network structure resulted in

-0.6 C BFY5% the declining of syneresis (Sahan, Yasar, & Hayaloglu, 2008). Viscosity
of BFY ranged from 2.5 to 3.8 pa.s, and it increased as the amount of
-0.8 BFY4%
blueberry flower pulp raising (Figure 3b). Viscosity of BFY3% , BFY4%,
-1
-1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 and BFY5% were remarkably (p < 0.05) higher than those of the con‐
PC1 (73.18 %) trol, and nonfat milk solids and dietary fiber from blueberry flower
pulp were possible the reasons to account for the phenomenon (Sah
F I G U R E 1   Principal component analysis plots. Principal
component 1 = PC1, principal component 2 = PC2; attributes et al., 2016). In addition, the viscosity of the control and BFY was
(red vectors) and yogurts (blue circles) relatively stable during 29 days of cold storage (Figure 3b).
LIU and LV |
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TA B L E 5   The mean intensity of sensory attributes for yogurts

Attribute Control BFY1% BFY2% BFY3% BFY4% BFY5%

Appearance
Color 3.90 ± 0.21 3.92 ± 0.11 4.10 ± 0.07 4.50 ± 0.12* 4.71 ± 0.20** 4.84 ± 0.23**
Brightness 3.30 ± 0.11 3.41 ± 0.21 4.20 ± 0.25* 4.62 ± 0.22** 5.03 ± 0.15** 5.06 ± 0.58**
Syneresis 5.01 ± 0.14 4.73 ± 0.37 4.52 ± 0.56* 4.16 ± 0.12** 4.10 ± 0.11** 3.92 ± 0.17**
Particles 0.21 ± 0.07 0.60 ± 0.05** 1.09 ± 0.22** 1.22 ± 0.06** 1.50 ± 0.21** 1.33 ± 0.10**
Aroma
Acid aroma 3.50 ± 0.23 3.80 ± 0.14 4.31 ± 0.29** 4.22 ± 0.76** 3.52 ± 0.15 3.39 ± 0.34
Artificial blueberry 0.09 ± 0.06 1.10 ± 0.03** 2.02 ± 0.05** 3.10 ± 0.29** 3.91 ± 0.68** 4.49 ± 0.35**
aroma
Flavor
Acid taste 5.30 ± 0.41 5.41 ± 0.23 5.65 ± 0.49* 5.68 ± 0.52* 4.92 ± 0.06 4.85 ± 0.45*
Sweet taste 4.61 ± 0.12 4.72 ± 0.32 4.55 ± 0.45 4.56 ± 0.98 4.75 ± 0.66 4.49 ± 0.33
Artificial blueberry 0.08 ± 0.02 1.10 ± 0.10** 2.02 ± 0.06** 2.91 ± 0.07** 3.70 ± 0.22** 4.51 ± 0.06**
taste
Texture
Firmness 3.93 ± 0.26 4.40 ± 0.19* 4.91 ± 0.37** 5.32 ± 0.54** 5.70 ± 0.26** 5.72 ± 0.78**
Viscosity 3.19 ± 0.12 3.52 ± 0.45* 4.30 ± 0.06** 4.57 ± 0.38** 5.09 ± 0.25** 5.20 ± 0.32**
*Significant differences were detected at p < 0.05 compared to the control. **Significant differences were detected at p < 0.01 compared to the
control.

F I G U R E 2   pH and titratable acidity of

BFY during refrigerated storage

3.6 | Numbers of probiotics in BFY
The number of lactic acid bacteria in yogurt during cold storage was
shown in Table 6. During the same cold storage period, the number of
L. plantarum in BFY was significantly (p < 0.05) higher than the con‐
trol, except to BFY1%. The number of S. thermophilus in BFY increased
to a different level compared to the control, and the number of S.
thermophilus in BFY2%, BFY3%, BFY4%, and BFY5% was significantly
(p < 0.05) higher than those of the control during 29 days of cold stor‐
age. This indicates that blueberry flower pulp, like green tea, Litsea
coreana L. and fermented tea extracts, could promote the growth
of L. plantarum and S. thermophilus to varying degrees (Amirdivani &
Baba, 2011; Granato, Santos et al.,2018; Jaziri et al., 2009). And the
maximum values of L. plantarum and S. thermophilus were 7.88 and
7.86log cfu/mL, respectively. However, blueberry flower pulp had
a little effect on B. bifidum. During the storage period, the number
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F I G U R E 3   Syneresis and viscosity

of BFY during refrigerated storage.
*
Significant differences were detected
at p < 0.05 compared to the control;
**
Significant differences were detected at
p < 0.01 compared to the control

TA B L E 6   Numbers of probiotics in BFY during refrigerated storage

Probiotics (log
cfu/mL) Day Control BFY1% BFY2% BFY3% BFY4% BFY5%

1 7.23 ± 0.02 7.59 ± 0.18 7.82 ± 0.05* 7.83 ± 0.07* 7.86 ± 0.11* 7.85 ± 0.04*

8 7.20 ± 0.06 7.57 ± 0.09 7.84 ± 0.11* 7.86 ± 0.13* 7.85 ± 0.08* 7.88 ± 0.17*
L. plantarum 15 7.15 ± 0.07 7.57 ± 0.12 7.79 ± 0.06* 7.78 ± 0.03* 7.77 ± 0.03* 7.73 ± 0.10*
22 7.10 ± 0.14 7.45 ± 0.21 7.70 ± 0.08* 7.76 ± 0.04* 7.70 ± 0.15* 7.65 ± 0.18*
29 7.02 ± 0.03 7.39 ± 0.11 7.72 ± 0.09* 7.75 ± 0.08* 7.68 ± 0.06* 7.60 ± 0.14*
1 7.20 ± 0.21 7.55 ± 0.23 7.76 ± 0.16* 7.82 ± 0.09* 7.78 ± 0.05* 7.79 ± 0.06*
8 7.19 ± 0.07 7.56 ± 0.15 7.76 ± 0.21* 7.86 ± 0.28* 7.80 ± 0.09* 7.78 ± 0.04*
S. thermophilus 15 7.15 ± 0.15 7.46 ± 0.10 7.74 ± 0.17* 7.72 ± 0.09* 7.75 ± 0.03* 7.70 ± 0.02*
22 7.04 ± 0.13 7.40 ± 0.11 7.68 ± 0.20* 7.61 ± 0.22* 7.71 ± 0.08* 7.62 ± 0.19*
29 7.06 ± 0.09 7.37 ± 0.11 7.62 ± 0.04* 7.56 ± 0.13* 7.66 ± 0.12* 7.58 ± 0.20*
1 7.03 ± 0.17 7.05 ± 0.14 7.07 ± 0.21 7.06 ± 0.22 7.07 ± 0.16 7.08 ± 0.09
8 7.02 ± 0.06 7.06 ± 0.25 7.04 ± 0.14 7.03 ± 0.08 7.02 ± 0.17 7.04 ± 0.10
B. bifidum 15 6.91 ± 0.08 6.96 ± 0.06 7.03 ± 0.10 7.00 ± 0.11 7.01 ± 0.05 6.99 ± 0.13
22 6.77 ± 0.12 6.94 ± 0.18 6.98 ± 0.14 7.01 ± 0.32 7.00 ± 0.07 6.96 ± 0.07
29 6.62 ± 0.22† 6.70 ± 0.05† 6.77 ± 0.04* 6.95 ± 0.18* 6.93 ± 0.07* 6.90 ± 0.31
*Significant differences were detected at p < 0.05 compared to the control. †Indicate the mean was significantly different at p < 0.05 compared to the
sample shown in the same column at the beginning of refrigeration.

of L. plantarum and S. thermophilus decreased, but not significantly. 3.7 | Antioxidant activity of BFY
The number of B. bifidum did not obviously change in BFY3%, BFY4%,
and BFY5%, while it decreased significantly (p < 0.05) in the control, DPPH, ABTS, and OH radical scavenging capacities were usually

BFY1%, and BFY2%. This suggests that oxygen toxicity on probiotic applied to determination of the antioxidant activities of blueberry

strains might be limited and their growth environment improved leaves, flowers, fermented dairy (Routray & Orsat, 2014; Wan et al.,

(Gaudreau, Champagne, Remondetto, Bazinet, & Subirade, 2013; 2012). The antioxidant activity of BFY was evaluated and showed

Molan, Flanagan, Wei, & Moughan, 2009), when the concentration of in Table 7. During the same refrigeration period, the IC50 values of

blueberry flower pulp more than 3%. In the whole refrigerated time, BFY scavenging DPPH, ABTS, and OH radical were meaningfully

the total number of probiotics in BFY was more than 1 × 107 cfu/mL, (p < 0.05; p < 0.01) lower than those of the control, that meant, the

which was consistent with the requirements of the codex standard antioxidant activity was significantly higher than that of the control.

for fermented milks, CODEX STAN 243 (Tamime & Robinson, 2007). Therefore, like other antioxidant plant extracts, blueberry flower
LIU and LV |
      9 of 10

TA B L E 7   Antioxidant activity of BFY during refrigerated storage

IC50 value of yogurt (mg/mL)

Antioxidant Indicator Day Control BFY1% BFY2% BFY3% BFY4% BFY5%

1 37.12 ± 0.22 32.69 ± 0.32* 31.05 ± 1.30* 27.51 ± 0.92** 23.38 ± 0.30** 21.06 ± 1.03**

8 37.51 ± 0.35 32.71 ± 0.32* 31.08 ± 0.97* 27.50 ± 0.88** 23.50 ± 0.26** 21.62 ± 0.32**
DPPH 15 38.17 ± 1.21 33.33 ± 1.12* 32.19 ± 1.65** 28.12 ± 2.11** 24.13 ± 1.55** 23.56 ± 2.11**
22 38.86 ± 1.21 33.45 ± 1.21* 32.20 ± 0.82** 28.16 ± 0.59** 24.54 ± 0.77** 23.53 ± 1.03**
29 39.23 ± 1.76 34.05 ± 2.30* 32.80 ± 0.93* 30.30 ± 1.41** 26.08 ± 0.83** 25.19 ± 0.51**
1 41.61 ± 2.03 33. 50 ± 0.62* 30.21 ± 1.05* 23.04 ± 1.21** 22.21 ± 0.67** 20.12 ± 1.33**
8 41.66 ± 1.12 33. 52 ± 0.13* 30.31 ± 0.65* 23.71 ± 0.36** 22.65 ± 0.61** 20.89 ± 1.52**
ABTS 15 42.53 ± 1.29 33.95 ± 0.51* 31.26 ± 2.51* 25.67 ± 2.02** 23.96 ± 1.06** 22.27 ± 0.37**
22 43.50 ± 2.95 34.06 ± 0.67* 31.98 ± 1.87** 26.57 ± 3.36** 24.23 ± 1.45** 22.77 ± 0.19**
29 43.72 ± 0.98 34.57 ± 1.72* 32.78 ± 1.97* 26.52 ± 1.80** 25.65 ± 2.03** 23.56 ± 1.14**
1 38.46 ± 2.12 31.11 ± 1.63* 28.25 ± 1.65* 22.65 ± 1.49** 20.85 ± 1.22** 19.87 ± 0.33**
8 39.05 ± 3.30 31.79 ± 2.12* 28.18 ± 0.53* 22.86 ± 2.11** 21.08 ± 2.29** 19.96 ± 0.18**
OH 15 39.68 ± 1.25 32.76 ± 0.50 29.66 ± 0.91** 23.89 ± 1.02** 22.66 ± 0.93** 20.90 ± 1.20**
22 39.85 ± 2.02 32.77 ± 2.69 29.67 ± 2.05** 24.26 ± 2.21** 22.97 ± 1.16** 21.88 ± 1.85**
29 40.91 ± 0.28 33.99 ± 2.02* 33.18 ± 2.89* 25.69 ± 2.71** 23.28 ± 2.07** 22.11 ± 1.02**
*Significant differences were detected at p < 0.05 compared to the control. **Significant differences were detected at p < 0.01 compared to the
control.

pulp could enhance the antioxidant activity of yogurt (Granato, AC K N OW L E D G M E N T S

Santos et al., 2018; Najgebauer‐Lejko et al., 2011; Ramos et al.,
The present work has been supported by Excellent Young Talents
2017). The antioxidant activity of BFY was higher than hickory‐
Fund Program of Higher Education Institutions of Anhui Province
black soybean yogurt (Ye et al., 2013), and it mainly attributed to
(Grant No. gxyqZD2018120), and Anhui Provincial Quality Project
the phenolic compounds which came from blueberry flower pulp.
(Grant No. 2016jyxm0639; 2015zdjy181).
What’s more, yogurt contains inherent antioxidants, including cer‐
tain peptides and free amino acids, as well as the exopolysaccharides
of lactobacillus (Sah, Vasiljevic, McKechnie, & Donkor, 2014). During
C O N FL I C T O F I N T E R E S T
the 29 days refrigerated storage, the IC50 values of BFY scaveng‐
ing DPPH, ABTS, and OH radical was not significantly (p > 0.05) in‐ The authors have declared no conflicts of interest for this article.
creased, and the antioxidant activities of BFY were relatively stable.
Similar phenomena had been observed in previous studies, they
had found that the antioxidant activities slightly dropped in grape ORCID
seed, peppermint, dill, and basil extracts enhanced yogurt (Granato,
Dong Liu  https://orcid.org/0000-0001-5846-3196
Santos et al., 2018; Najgebauer‐Lejko et al., 2011; Ramos et al.,
2017). Polyphenols declined slowly due to the bacterial metabolic
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How to cite this article: Liu D, Lv XX. Effect of blueberry
flower pulp on sensory, physicochemical properties, lactic
acid bacteria, and antioxidant activity of set‐type yogurt
during refrigeration. J Food Process Preserv. 2018;e13856.
https://doi.org/10.1111/jfpp.13856