Journal of Functional Foods 27 (2016) 131139

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Effect of fermentation on the content of bioactive

compounds in tofu-type products

Ylenia Riciputi a, Diana Isabella Serrazanetti b, Vito Verardo c,*,

Lucia Vannini a,b, Maria Fiorenza Caboni a,b, Rosalba Lanciotti a,b
a
Department of Agri-Food Science and Technology, Alma Mater Studiorum, University of Bologna, Piazza
Goidanich 60, 47521 Cesena, FC, Italy
b
Interdepartmental Centre for Agri-Food Industrial Research (CIRI Agroalimentare), Alma Mater Studiorum,
University of Bologna, Piazza Goidanich 60, 47521 Cesena, FC, Italy
c
Department of Chemistry and Physics (Analytical Chemistry Area) and Research Centre for Agricultural and
Food Biotechnology (BITAL), Agrifood Campus of International Excellence, ceiA3, University of Almera,
Carretera de Sacramento s/n, E-04120 Almera, Spain

A R T I C L E I N F O A B S T R A C T

Article history: Tofu is part of Asian cuisine and may be the most popular food made from soy. Due to the
Received 11 May 2016 influence of processing on some compounds (fatty acids, tocopherols, phospholipids, sterols
Received in revised form 18 August and phenolic compounds) content of soy food products, soybean flour, traditional tofu and
2016 tofu obtained by soymilk fermentation were studied. Fermentation was performed using
Accepted 27 August 2016 lactic acid bacteria, such as Lactobacillus casei and Lactobacillus acidophilus.
Available online Fermentation by lactic acid bacteria plays an essential role in the reduction of isoflavones
glycosides/aglycone ratio (from 0.14 to 0.89 in traditional and fermented tofu, respectively)
Keywords: and in the higher bioavailability of isoflavones, however it does not significantly affect soybean
Soybean fatty acids and tocopherols. This behaviour suggests the use of these microorganisms to
Fermented tofu enhance the added value of soybean products, and promote the selection of tailor made
Isoflavones starter culture able to growing well in soy milk and contemporary to produce bioactive com-
Tocopherols pounds for specific consumers category.
Phytosterols 2016 Elsevier Ltd. All rights reserved.
Phospholipids

increase in annual production. The increase in soybean pro-

1. Introduction duction can be attributed to increased crop yield and demand
as food and fuel needs (Orf, 2010).
Since the past centuries, soybean has been one of the most One of the major reasons is its unique chemical composi-
important agricultural commodities in the world, with a gradual tion; thus, for many years soybean has been recognised as a

* Corresponding author. Department of Chemistry and Physics (Analytical Chemistry Area) and Research Centre for Agricultural and Food
Biotechnology (BITAL), Agrifood Campus of International Excellence, ceiA3, University of Almera, Carretera de Sacramento s/n, E-04120
Almera, Spain. Fax: +39-(0)547-382348.
E-mail address: vito.verardo@unibo.it (V. Verardo).
Chemical compounds: -Tocopherol (PubChem CID: 14985); Sitosterol (PubChem CID: 222284); Campesterol (PubChem CID: 173183); Stig-
masterol (PubChem CID: 5280794); Phosphatidylethanolamine (PubChem CID: 6437392); Phosphatidylinositol (PubChem CID: 86289142);
Phosphatidylcholine (PubChem CID: 24778933); Daidzin (PubChem CID: 107971); Genistin (PubChem CID: 5281377); Glycitin (PubChem
CID: 187808).
http://dx.doi.org/10.1016/j.jff.2016.08.041
1756-4646/ 2016 Elsevier Ltd. All rights reserved.
132 Journal of Functional Foods 27 (2016) 131139
great source of nutrients. On average, dry soybean contains
about 4041% protein, 824% oil, 35% carbohydrate, and 5% ash
(Medic, Atkinson, & Hurburgh, 2014). Protein and fatty com-
ponents in soybeans are high in quality as well as in quantity.
In particular, soy protein contains all the essential amino acids,
most of which are present in amounts that closely match those
required by humans or animals. Moreover, soybean lipids
contain a high proportion of unsaturated fatty acids, includ-
ing oleic, linoleic, and linolenic acids and minor lipid
components as phospholipids, phytosterols and tocopherols
with a significant impact on human health (Medic et al., 2014).
The tradition of preparing soy foods, typical of Asian culture,
has been spread to the world in the last years, thanks to ag-
ricultural and processing innovation, cultural exchanges and
the influence of Chinese and other Asian immigrants. Thou-
sands of studies (in vivo and in vitro, with animals and human
subjects) have shown that soybeans and soy components have
many health-promoting effects, including hypocholesterolemic,
anticancer and antioxidant properties (Sugano, 2006). In par-
ticular, since FDAs approval of soy health claim, many studies
have further demonstrated the link between soy product con-
sumption and lowering the risk of coronary heart disease (CHD)
(He & Chen, 2013). Moreover, regular consumption of soy can
help prevent breast and prostate cancers, improve bone health
and memory and alleviate menopausal symptoms in some
women.
Many types of biologically active components, including
isoflavones, lecithin, saponins, lectins, oligosaccharides, and
trypsin inhibitors, have shown to be partially responsible for
these effects (Anderson, Smith, & Washnock, 1999; Barnes, 1998;
Liu, 2004; Messina, 1999; Messina, Gardber, & Barnes, 2002; Shu
et al., 2001).
Soymilk is the liquid extract of soybeans and it is used to
produce tofu (Liu & Lin, 2000). Tofu, a fundamental compo-
nent of Asian food culture, is a traditional oriental food,
composed principally by proteins and lipids and produced di-
rectly by coagulation of soymilk. Historically, tofu is a major
source of proteins in the Chinese diet and calcium-coagulated
tofu also serves as an important source of calcium, an essen-
tial macro-mineral. Unfortunately, tofu products vary greatly
in their composition even within the same variety because there
is no established standard of identity (He & Chen, 2013).
Lactic acid bacteria (LAB) have been applied as starter cul-
tures in fermented foods and beverages for a long time, because
they can improve their nutritional, organoleptic, technologi-
cal and shelf-life characteristics (Leroy & De Vuyst, 2004). Despite
recent advances, the study of LAB and their functional activi-
ties in soy based products is still an emerging field of research
that has to realise its full potential yet (Florou-Paneri, Christaki,
& Bonos, 2013).
The ability of LAB to rapidly grow in soymilk showed that
this is a suitable habitat for these microorganisms, as already
demonstrated by several authors (Serrazanetti, Ndagijimana,
Miserocchi, Perillo, & Guerzoni, 2013; Wang, Yu, Yang, & Chou,
2003). In fact, many probiotic strains possess -galactosidase
activity, which allows their growth in soymilk (Fung & Liong,
2010). Moreover, some Lactobacilli are known to hydrolyse
-glucosides, increasing the bio-availability of isoflavones. In
fact, it is known that LAB fermentation can increase the nu-
tritional values of soy-derived products; in fact it causes
isoflavone conversion from glycosylated and/or acylated to agly-
cone, consequently, promoting their absorption (Hendrich, 2002;
Pyo, Lee, & Lee, 2005). It is reported by Chien, Huang, and Chou
(2006) that fermentation of soymilk with LAB and Bifidobacterium
transforms acetyl (A) and malonyl (M)-glucoside (G) isoflavones
to aglycones, the biologically active oestrogen-like isoflavones
(Setchell & Cassidy, 1999) which are absorbed faster and in
higher amounts than their glucosides in humans (Izumi et al.,
2000). In fact, it has been showed that fermentation of soy-
beans enhanced the bioavailability of isoflavones due to the
faster absorption of soy isoflavones aglycones (Izumi et al., 2000).
Genistein-rich products such as fermented whole-soy foods and
their extracts may be useful in preventing osteoporosis, meno-
pausal symptoms, coronary heart disease and cancers.
Moreover, Lactobacillus acidophilus was reported as present-
ing the ability to metabolise oligosaccharides during the
fermentation of soymilk (Donkor, Henriksson, Vasiljevic, & Shah,
2005).
In this context, the overall aim of this research was to evalu-
ate the potential of selected strains of LAB to produce functional
tofu-type product having an increased concentration of bioactive
compounds including isoflavone aglycones, phenolic com-
pounds and unsaturated long chain fatty acids. In particular,
traditional tofu obtained through coagulating soy milk with
chemical agents and tofu obtained through the fermentation
with selected lactic acid bacteria strains (L. casei and L. aci-
dophilus) before the addition of coagulating agents were
compared and characterised in terms of lipidic and phenolic
profiles, focusing mainly on isoflavone content.
2. Materials and methods
2.1. Solvents and reagents
All solvents were purchased from Merck KGaA (Darmstadt,
Germany). Reagents were provided by Sigma Aldrich (St. Louis,
MO, USA).
2.2. Samples and samples preparation
The samples studied were represented by soybean flour, tra-
ditional tofu and fermented tofu. Soybean flour was provided
by Company ConBio (Santarcangelo, Italy) and it was ob-
tained from soybeans (Glycine max (L.) Merrill) belonging to the
PR91M10 variety. Also the two different kinds of tofu were pre-
pared by Company ConBio (Santarcangelo, Italy). Traditional tofu
was prepared according to the company flow chart (Fig. 1),
instead, fermented tofu was obtained by fermentation of
soymilk with L. casei and L. acidophilus according to Serrazanetti
et al. (2013).
The strains were co-inoculated in soymilk (6 log CFU mL1)
and then incubated at 32 C for 15 hours. Coagulation agent
Nigari (Nigari composition: MgCl2 + 6H2O: more than 95%; Zn:
less than 70 ppm; As2O3: less than 4 ppm; provided by Soluzione
Naturale, Brescia, Italy) was added in order to standardise the
curd formation. For Nigari preparation, 243 g of Nigari salt was
solved in 1 L of distilled water; 24 mL of that solution was added
for every litre of soymilk after the heating step (80 C). The curd
 Journal of Functional Foods 27 (2016) 131139 133

2.5. Phytosterol analysis of soybean flour, tofu and

Traditional tofu Fermented tofu fermented tofu

Hot soymilk (80 C) Soymilk + LAB The lipidic fraction was saponified at room temperature using
(106 CFU/ml) 10 mL of methanolic 0.5 M KOH for 18 h in the dark under con-
stant stirring. After saponification the organic fraction was
Addition of coagulant
(Nigari) Incubation (32 C, 15 washed with deionised water and the unsaponifiable matter
hrs) till pH 5.5 was extracted three times with diethyl ether. The organic frac-
Curd formation tions were pooled together and solvent was removed under
Curd formation vacuum. The unsaponifiable matter was stored in n-hexane/
Filtration and 2-propanol (4/1 v/v) at 18 C until GC analysis.
centrifugation Heating (80 C) The previous extract was analysed by GC after silylation.
The trimethylsilyl derivatives (TMS) of sterols were analysed
Vacuum packaging Addition of coagulant as reported by Iafelice, Verardo, Marconi, & Caboni (2009) by
(Nigari) GC-FID. The analysis was carried out in triplicate for each
Pasteurization sample.
(75 C, 10 min) Vacuum packaging

Pasteurization 2.6. Determination of tocopherols in soybean flour, tofu

Storage
(4-10 C) (75 C, 10 min) and fermented tofu

Storage One gram of fat sample was dissolved in 10 mL of n-hexane

(4-10 C)
Fig. 1 Process flow diagram of traditional tofu and tofu
produced after soymilk fermentation (L. casei and
L. acidophilus).
was pressed mechanically with a filter press in order to obtain
firm and regular tofu, which has been cut in small portions
(125 g). The tofus produced were vacuum-packaged and
pasteurised. The samples were freeze dried and the analysis
was performed on dry matter.
2.3. Lipid extraction from soybean flour, tofu and
fermented tofu
Lipid fraction was extracted according to a modified version
of the method described by Folch, Lees, & Sloane-Stanley (1957),
as reported previously by Boselli, Velazco, Caboni, & Lercker
(2001).
The milled samples (20 g) were homogenised with 200 mL
of chloroform/methanol solution (1/1, v/v) in a glass bottle with
screw cap. The bottle was kept in an oven thermostated at 60 C
for 20 min before adding 100 mL chloroform. After 3 min of
homogenisation, the content of the bottle was filtered. The fil-
trate was mixed thoroughly with a 1 M KCl solution and left
overnight at 4 C in order to allow phase separation. The lower
phase was collected, dried with a rotary evaporator, dis-
solved in 5 mL hexane/isopropanol 4/1 (v/v) and stored at 18 C
until analysis.
and then, it was filtered through a 0.45 m nylon filter. The to-
copherols were determined by HPLC equipped with a
fluorimeter detector. The excitation wavelength was 290 nm
and the emission one was 325 nm. The used column was a Luna
Hilic (250 mm 4.6 mm i.d., 5 m particle size) Phenomenex
column in isocratic conditions as reported by Gomez-Caravaca,
Verardo, & Caboni (2010). The calibration curves were con-
structed with standard solutions of each compound and they
were used for quantification.
2.7. Phospholipids determination of soybean flour, tofu
and fermented tofu
Phospholipids analysis in tofu samples was carried out ac-
cording to the method of Verardo et al. (2013). Briefly, the
extracted lipids were weighed (100 mg), dissolved in 1.5 mL of
chloroform/methanol (88/12 v/v) and transferred into capped
test tubes for HPLC analysis.
Phospholipids separation was performed on an Agilent
HP 1200 Series liquid chromatograph (Agilent Technologies)
with a PL-ELS1000 evaporative light-scattering detector
(Polymer Laboratories, Church Stretton, UK). A silica column
(150 mm 3 mm, 3 m particle diameter; Phenomenex) main-
tained at room temperature (25 C) was used. The HPLC system
was controlled by Agilent ChemStation software (Agilent Tech-
nologies, Santa Clara, CA, USA), while chromatogram registration
and data processing were assessed by ClarityLite Version
2.4.0.190 (DataApex, Prague, Czech Republic). Each extract was
injected three times.
2.8. Extraction and analysis of isoflavones and bound
phenolic compounds

2.4. Total fatty acid composition analysis
The FA composition of soybean and tofu samples was
determined from the lipid fraction as FAMEs by capillary
gas chromatography analysis as reported by Verardo,
Gmez-Caravaca, Gori, Losi, & Caboni (2013).
Briefly, free phenolic extractions were performed following a
protocol of Verardo, Riciputi, Garrido-Frenich, & Caboni (2015).
4 g of soybean flour or dried tofu was extracted by a Starsonic
90 Liarre ultrasonic bath (Bologna, Italy), at 40 C for 10 minutes,
with 40 mL of methanol/water mixture 4/1 (v/v). After cen-
trifugation at 1000 g for 5 min, the surnatant was removed and
134 Journal of Functional Foods 27 (2016) 131139

the extraction was repeated twice more. The surnatants were Similar results were showed by Caprioli et al. (2016); accord-
collected in a flask, evaporated at 50 C under vacuum, dried ing to their data we can affirm that soybean is, with lentil,
under nitrogen stream and reconstituted with 5 mL of water/ chickpea and fava bean, a good legume source of linolenic acid.
methanol 1/1 (v/v). In fact, the -3 fatty acids content accounted for 4.59 mg/
The residues from free phenolic extraction were treated by 100 mg of fat. The presence both of linolenic acid and -3 fatty
alkaline hydrolysis. acids confirms the health promoting effects of soy based prod-
The extracts were then stored at 18 C until analysis. Each ucts. In fact, the role of these substances as important
extraction was replicated three times. component of all cell membranes is recognised and more-
Phenolic compounds analysis was performed, in tripli- over they are essential in growth and development throughout
cate, by HPLC-DAD-ESI-MS according to a previous work (Verardo the life cycle (Omoni & Aluko, 2005; Simopoulos, 1991).
et al., 2015). The technology used to transform flour to traditional tofu
did not significantly affect fatty acids composition. In fact, the
2.9. Statistical analysis main fatty acids were those described previously in soybean
flour, and also in traditional tofu, and the ratio saturated/
The results reported in this study are the averages of three rep- unsaturated fatty acids was 0.20. Comparing LAB-fermented
etitions (n = 3). LSD Fisher significant difference multiple tofu with traditional tofu and with soybean flour, it can be
comparison (one-way ANOVA) was evaluated using Statistica noticed that fermentation and other technological steps do not
6.0 software (2001, StatSoft, Tulsa, OK, USA). influence the fatty acid composition of the raw material. In par-
ticular, the samples studied were characterised by the same
fatty acid composition where linoleic and oleic acid repre-
sented the first and second fatty acid (34.18 and 22.2 mg/
3. Results and discussion
100 mg fat for fermented tofu, respectively) and their
contribution to the total amount was higher than 75%, in agree-
3.1. Fatty acid composition
ment with literature (Prstamo & Fontecha, 2007). However,
production process significantly affected the content of C18:3
As reported in Table 1 soybean flour fatty acid composition was
3 (4.24 and 4.25 mg/100 mg in traditional and fermented tofu,
characterised by C16:0, C18:1, C18:2, C16:0 and C18:3 3 as the
respectively) compared to soybean flour (4.59 mg/100 mg); these
major compounds (7.78, 22.49, 34.52, 7.78, and 4.59 mg/100 mg,
results showed that the C18:3 3 content was not affected by
respectively) with the predominance of unsaturated fatty acids,
the LAB fermentation.
as confirmed by the saturated to unsaturated fatty ratio (0.19).
In all the products analysed, unsaturated fatty acids (UFA)
(sum of mono and polyunsaturated fatty acids) accounted for
more than 80% of total fatty acid and the UFA/SFA (saturated
Table 1 Fatty acids composition (mg/100 mg fat) of fatty acids) ratio in all samples showed values much higher
soybean flour and tofu samples (traditional and (UFA/SFA~5) than 2, that is the right ratio for a correct diet, as
fermented by L. casei and L. acidophilus). Different letter
suggested by the Italian Society of Human Nutrition.
in a row means significant differences (p < 0.05).
In this way, tofu should be considered a good source of UFA
Fatty acid Soybean Traditional Fermented
fatty acids that can report several beneficial effects on cardio-
flour tofu tofu
vascular disease including improved blood lipid profile,
C8:0 nd 0.08 0.00 a 0.09 0.00 b improved insulin sensitivity, lower incidence of type 2 diabe-
C10:0 0.06 0.00 a 0.06 0.03 a 0.12 0.02 a
tes and anti-arrhythmic effects (Caprioli et al., 2016).
C11:0 nd 0.03 0.01 a 0.03 0.01 a
C11:1 0.03 0.01 a 0.07 0.03 a 0.05 0.01 a
C14:0 0.06 0.01 a 0.06 0.01 a 0.06 0.00 a 3.2. Sterols analysis
C16:0 7.78 0.28 a 7.48 0.19 a 8.06 0.41 a
C16:1 0.06 0.00 a 0.06 0.01 a 0.11 0.05 a Sterol composition of soybean flour showed -sitosterol as the
C17:0 0.07 0.01 a 0.06 0.00 a 0.07 0.00 a main compound (172.23 mg/100 g fat), accounting for 57.7% of
C17:1 0.04 0.01 a 0.04 0.00 a 0.05 0.01 a the total content. In soybean flour campesterol (60.12 mg/
C18:0 3.29 0.28 a 3.19 0.01 a 3.41 0.04 a
100 g fat), stigmasterol (50.82 mg/100 g) and 7-avenasterol
C18:1t 0.13 0.03 a 0.11 0.03 a 0.20 0.03 a
C18:1c11 22.49 1.02 a 19.89 0.48 a 22.20 2.03 a
(15.11 mg/100 g) were also identified and quantified. As
C18:1c12 0.96 0.16 a 0.98 0.01 a 1.06 0.01 a reported in Table 2, -sitosterol was also the most abundant
C18:2 34.52 0.97 a 33.70 0.78 a 34.18 0.34 a sterol in tofu, ranging from 55.2 to 57.1% of total sterols
C18:3 3 (ALA) 4.59 0.13 a 4.24 0.06 b 4.25 0.07 b (156.2168.2 mg/100 g of fat), followed by campesterol (20.2
C20:0 0.30 0.05 a 0.30 0.00 a 0.33 0.02 a 21.3%, 59.560.4 mg/100 g of fat), stigmasterol (16.818%, 49.5
C20:1 0.15 0.05 a 0.15 0.02 a 0.14 0.00 a
51.1 mg/100 g of fat) and 7-avenasterol (5.55.8%, 15.517.1 mg/
C22:0 0.35 0.03 a 0.35 0.01 a 0.37 0.00 a
100 g of fat). The concentration of 7-avenasterol was higher
C24:0 nd 0.04 0.01 b 0.04 0.01 b
C24:1 nd 0.04 0.00 b 0.04 0.00 c than the results reported by Ozawa et al. (2001) indepen-
C22:5 nd 0.06 0.00 b 0.05 0.00 c dently on the presence of fermentation process probably due
Total fatty acids 74.88 70.99 74.89 to the use of whole soybean flour.
UFA/SFA 5.29 5.09 4.96 The sterol composition of fermented tofu was not so dif-
Trans-fatty acids 0.13 0.03 a 0.11 0.03 a 0.20 0.03 a ferent compared to soybean flour and traditional tofu except
-3 Fatty acids 4.59 0.13 a 4.24 0.06 a 4.25 0.07 a
for -sitosterol (its content was significantly lower compared
 Journal of Functional Foods 27 (2016) 131139
Table 2 Sterols composition (mg/100 g fat) of soybean
flour and tofu samples (traditional and fermented by
L. casei and L. acidophilus). Different letter in the same
column means significant differences (p < 0.05).
Soybean Traditional Fermented
flour tofu tofu
Campesterol 60.12 0.25 a 60.42 0.17 a 59.53 0.45
Stigmasterol 50.82 0.85 a 51.11 0.06 a 49.55 0.53
-Sitosterol 172.23 0.35 a 156.22 0.45 c 168.23 0.19
7-Avenasterol 15.11 0.14 a 15.46 0.19 a 17.08 0.06
Total 298.27 1.58 a 283.21 0.87 b 294.39 1.12
to soybean flour but higher than traditional tofu) and
7-avenasterol (17.08 mg/100 mg of fat with respect to
15.11 mg/100 mg of fat in soybean flour and 15.46 mg/100 mg
of fat in traditional tofu) (Table 2). The higher concentration
of -sitosterol and 7-avenasterol in fermented tofu com-
pared to traditional tofu suggested that the fermentation by
LAB could increase the bioavailability of this compound in the
final product. In general sterols are considered prebiotic com-
pounds which can improve the colonisation of probiotic bacteria
in the gut (Tsai et al., 2014). Moreover, since phytosterols and
phytostanols are structurally similar to cholesterol (except that
they possess different side chains and the latter is devoid of
a double bond) they affect the absorption of cholesterol in the
body and hence may reduce the total LDL cholesterol levels
in the blood (Borrelli & Ernst, 2010). Thus, these substances are
proposed as complementary and alternative medicine (CAM)
for menopausal women (Borrelli & Ernst, 2010).
Looking at the content of total free sterols, they ranged
from 283.2 to 298.3 mg per 100 g of fat with no significant
differences between soybean flour and fermented tofu (298.3
and 294.4 mg/100 g fat, respectively), while those samples were
significantly different if compared with traditional tofu
(283.2 mg/100 g fat).
3.3. Tocopherol evaluation
As reported in Table 3, no significant differences were ob-
served in the content of total tocopherols among the three
samples, which ranged from 463.4 to 509.9 g/g of fat. These
concentrations were in agreement with those reported previ-
ously by Guzman and Murphy (1986). Alpha-tocopherol,
-tocopherol and -tocopherol were the tocopherols detected
in the samples analysed independently on the production
process adopted. They represent, according to literature data,
the main tocopherols of soybean flour and products (Tavva et al.,
2007). In particular, 67.9% of total tocopherols in soybean flour
corresponded to -tocopherol and the other 32.1% was the sum
of -tocopherol (9.8%) and -tocopherol (22.3%). The tradi-
Table 3 Tocopherols composition (g/g fat) of soybean flour and tofu samples (traditional and fermented by L. casei and
L. acidophilus). Different letter in the same column means significant differences (p < 0.05).
-Tocopherol
Soybean flour 50.06 5.85 a
Traditional tofu 32.91 0.11 b
Fermented tofu 48.88 4.82 a
135
tional production process decreased the content of -tocopherol
compared to soybean flour, while the fermentation process
counteracted this reduction. In fact, -tocopherol was the only
tocopherol that significantly decreased in traditional tofu.
Soybean -tocopherol, the biosynthetic precursor to
-tocopherol, is the predominant form found in the bean,
whereas -tocopherol is the most bioactive component (Tavva
a
a
et al., 2007). In fact, among the tocopherols, -tocopherol has
b a well-recognised key physiological role and exerts the most
b potent biological activity compared to the others (Jiang, 2014;
a Yoshida, Saito, Sargent Jones, & Shigeri, 2007). Consequently,
the results obtained showed the positive role of lactic acid bac-
teria in maintaining the natural occurring level of vitamin E
of soybean flour in traditional and fermented tofu.
3.4. Phospholipids determination
The phospholipids in soybean flour were characterised by three
main compounds (phosphatidylinositol (PI), phosphatidyle-
thanolamine (PE) and phosphatidylcholine (PC), 12.5, 32.9 and
54.6% of the total content, respectively) in agreement with data
previously reported by Lee et al. (2010). Phospholipids are one
of the important constituents of the cellular membrane bilayer;
both the polar head groups and fatty chains in phospholip-
ids not only affect the physicochemical properties of the
membrane, but also play distinctive roles in biological events
(Stubbs & Smith, 1984). In detail, phospholipids and particu-
larly PC play a positive role in cholesterol homeostasis,
stabilisation of the membrane functions and supporting the
hepatic functions (Ipatova, Prozorovskaia, Torkhovskaia,
Baranova, & Guseva, 2004; Lee et al., 2010).
The total content and the contribution of single phospho-
lipids varied significantly in relation to the fermentation process
(Table 4). Soybean flour total phospholipid content (87.05 g/g
of fat) was 12% higher than in traditional tofu (76.60 g/g of
fat) and 52.4% than in fermented tofu (41.45 g/g of fat). In tofu
samples the distribution was: 10.9/38.7/50.4% (traditional tofu)
and 14.2/44.1/41.7% (fermented tofu) for PI, PE and PC, respec-
tively. No lyso-forms were found in the samples analysed.
However, according to Serrazanetti et al. (2013), the fermen-
tation process resulted in tofu having different textural
properties, lower pH values and higher syneresis compared to
traditional one. These features can contribute to the loss of
phospholipids in fermented tofu, compared to the traditional
one and to soybean flour, during whey release. In fact, a less
solid structure seemed to enhance syneresis of whey and, thus,
the loss of molecules such as phospholipids that constitute the
membrane fat globules (Burgain et al., 2014).
To our knowledge, no previous studies have been done about
the effect of LAB fermentation on phospholipids in soybean
and soybean products.
-Tocopherol -Tocopherol Total
346.40 12.90 a 113.40 4.19 a 509.85 22.93 a
313.21 0.13 a 112.60 9.61 a 458.72 9.60 a
313.83 30.08 a 100.70 12.52 a 463.42 47.43 a
136 Journal of Functional Foods 27 (2016) 131139

Table 4 Phospholipids composition (g/g of fat) of soybean flour and tofu samples (traditional and fermented by L. casei
and L. acidophilus). Different letter in the same column means significant differences (p < 0.05).
PI PE PC Total
Soybean 10.85 0.35 a 28.65 0.35 a 47.55 0.49 a 87.05 0.49 a
Traditional tofu 8.35 0.64 b 29.65 0.07 b 38.60 0.28 b 76.60 0.99 b
Fermented tofu 5.90 0.14 c 18.30 0.28 c 17.25 0.64 c 41.45 1.06 c
PI, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine.

3.5. Determination of free and bound phenolic
compounds
The regular consumption of isoflavones may help reduce the
risk of certain diseases. In fact, recent studies on the effects of
isoflavones on heart health, bone health and post-menopausal
symptoms indicate positive effects that are highly attractive
to the functional foods market (Brouns, 2002). In this study the
analysis of isoflavones allowed the identification of fifteen com-
pounds, eight aglycones and seven glycosylated derivatives, with
an increased aglycones/glycosylated ratio in fermented tofu com-
pared to soybean flour and traditional tofu (0.53 in fermented
tofu vs. 0.03 and 0.12 in soybean and traditional tofu, respec-
tively) (Table 5). In detail, genistein-G-M and genistin were the
most abundant isoflavones in soybean, contributing for 28.2
and 27.4% of the total isoflavone content, followed by daidzin
(12.73 mg/g dry matter, 15.7% of the total content) and an isomer
of genistein-G (9.35 mg/g dry matter, 11.5% of the total content).
Moreover, different genistein-G-A isomers were identified and
quantified in soybean, contributing for 6% of the total content
(4.88 mg/g dry matter). These results are in part in agreement
with other studies about isoflavone composition of soybean
and soybean products (Lee, Chung, Kim, & Jung, 2015; Shao et al.,
2011). Some differences in the results could be due to envi-
ronmental and botanic characteristics and process technologies
of analysed samples (Zhang, Chang, & Liu, 2015).
Soybean flour and tofu samples showed significant differ-
ences in terms of total isoflavones content. Moreover, glucoside
concentration was lower in fermented tofu if compared with
soybeans flour and traditional tofu.
Comparing the two kinds of processed product with soybean
flour used to produce them, traditional tofu showed a strong
increment of daidzin (20.68 mg/g dry matter), genistein-G
(17.38 mg/g dry matter) and daidzein (7.38 mg/g dry matter),
but the content of genistein-G-M and genistin decreased sig-
nificantly to 11.7 and 12% of total isoflavone content. The same
trend can be seen in fermented tofu isoflavone profile, com-
pared to soybean flour. It has been reported that the not
conjugated isoflavone aglycones were the predominant chemi-
cal forms in fermented soy products (Oh, Hwang, & Lim, 2012).
However, among tofu, the fermented one showed a hard dec-
rement of daidzin and genistein-G but fermentation increased
the content of genistein-G-M (10.26 mg/g dry matter) and in
particular daidzein (21.17 mg/g dry matter), the major isoflavone
in fermented tofu (34.5% of total isoflavones content).
As concerning total isoflavones content, traditional and fer-
mented tofu showed a reduction of isoflavones of 13.4% and
24.2% (70.08 and 61.36 mg/g dry matter respectively) com-
pared to soybean flour, confirming the study of Lee et al. (2015).
The present data confirmed that some isoflavones were lost
in the processing steps (Jackson et al., 2002) and they were re-
covered in the wastes of production process (i.e. whey).
The difference between the two types of tofu could be due
to production technology; fermented tofu had a less compact
structure and, therefore, some compounds may have been lost
during curd breaking or purge step.

Table 5 Isoflavones composition (mg/g dry matter) of soybean flour and tofu samples (traditional and fermented by
L. casei and L. acidophilus). Different letter in a row means significant differences (p < 0.05).
Compound Soybean Traditional tofu Fermented tofu
Genistein-G <LOQ <LOQ <LOQ
Daidzin 12.73 0.46 b 20.68 0.20 a 2.86 0.07
Glycitin 3.20 0.17 b 3.65 0.04 a 0.25 0.03 c
Genistein-G-A 1.44 0.01 a 0.43 0.01 b 0.49 0.01 b,c
Genistein-G-A 0.92 0.01 b 1.01 0.02 a 0.84 0.02 c
Glycitein-G 0.47 0.04 a 0.02 0.00 b
Genistein-G 9.35 0.42 b 17.38 0.06 a 2.12 0.02 c
Genistin 22.22 0.24 a 8.17 0.16 c 9.88 0.05 b
Dihydroxy-methoxyisoflavone 2.87 0.47 a 0.88 0.07 c 1.223 0.002 b
Genistein-G-A 0.20 0.01 <LOQ <LOQ
Genistein-G-A 2.32 0.03 a 0.772 0.001 b 0.85 0.02 b
Genistein-G-M 22.84 0.19 a 8.432 0.003 c 10.26 0.03 b
Daidzein 2.09 0.02 c 7.26 0.41 b 21.17 0.42 a
Glycitein <LOQ <LOQ 1.92 0.16
Genistein 0.31 0.01 c 1.39 0.14 b 9.50 0.03 a
Total (mg/g dry matter) 80.96 0.26 a 70.08 0.06 b 61.36 0.62 c
Aglycones/glycosylated isoflavones ratio 0.05 0.14 0.89
A = acetyl; G = glycosyl; M = malonyl.
 Journal of Functional Foods 27 (2016) 131139 137

Table 6 Bound phenolic compounds (mg/g dry matter) in soybean and tofu samples. Different letter in a row means
significant differences (p < 0.05).
Compound Soybean Traditional tofu Fermented tofu
p-Hydroxybenzoic acid 0.19 0.01 c 0.26 0.02 b 0.41 0.04 a
Vanillic acid 1.84 0.08 a 1.29 0.05 b 1.87 0.07 a
Syringic acid 9.32 0.16 c 13.68 0.21 b 20.95 0.19 a
p-Hydroxybenzaldehyde 0.69 0.06 c 0.93 0.05 b 1.39 0.07 a
Ferulic acid derivative 3.04 0.13 b 3.71 0.08 a 3.92 0.12 a
p-Coumaric acid 5.17 0.05 b 5.70 0.10 b 8.40 0.14 a
Ferulic acid 3.37 0.12 c 4.49 0.10 b 7.69 0.22 a
Synapic acid 1.35 0.05 b 1.12 0.02 c 3.17 0.09 a
Phloridzin 2.12 0.12 b 1.92 0.10 c 3.29 0.09 a
p-Coumaric acid hexose derivative 2.94 0.17 c 4.06 0.21 b 6.33 0.29 a
Total (mg/g dry matter) 30.04 1.10 c 37.16 1.56 b 57.42 1.47 a

In terms of aglycones and glycosides, isoflavones aglycones
were higher in fermented tofu compared to the traditional
one. Glycosides decreased from 74.4% to 28.2% and aglycones
increased from 25.6% to 71.8% in fermented tofu compared to
traditional tofu, confirming the role of fermentation on agly-
cone increasing in soy products (Oh et al., 2012). Daidzein and
genistein protect the cells from the damaging effects of free radi-
cals that are known to promote ageing and to be involved in a
number of disease states like atherosclerosis, joint inflamma-
tion and diabetic complication. Moreover, the consumption of
aglycones has been recommended to women in order to relieve
the main symptoms of post-menopausal life phase (Brouns, 2002).
Table 6 reports the content of bound phenolic compounds
identified in soybean and tofu samples.
Total bound phenolic content was 30.0, 37.2 and 57.4 mg/g
dry matter for soybean flour, traditional tofu and fermented
tofu respectively. As reported for total bound phenolics, except
for vanillic and ferulic acid derivatives, all the bound pheno-
lics reported higher amounts in fermented tofu compared with
traditional tofu and soybean. The principal bound phenolic com-
pound was the syringic acid and it represented the 3136.8%
of total bound phenolic compounds. p-coumaric, ferulic and
p-cumaroyl-hexose derivatives represented the other princi-
pal bound phenolic compounds.
Dietary polyphenols can be metabolised by probiotic or-
ganisms to release aromatics and small phenolic acids, which
alter the bioactivity and bioavailability of flavonoids (Cardona,
Andrs-Lacueva, Tulipani, Tinahones, & Queipo-Ortuo, 2013).
The formation of bioactive polyphenol-derived metabolites
and the modulation of colonic microbiota may both contrib-
ute to host health benefits (protection against gastrointestinal
disorders and pathogens, nutrient processing, reduction of serum
cholesterol, reinforcement of intestinal epithelial cell-tight junc-
tions and increased mucus secretion and modulation of the
intestinal immune response through cytokine stimulus) (Gianotti
et al., 2011; Gotteland et al., 2008; Vitali et al., 2010).
(sterols and phospholipids), isoflavones and other phenolic com-
pounds compared to soybean or traditional tofu.
The reduction of glycosides/aglycone ratio and conse-
quently the higher bioavailability of isoflavones, the increase
of antioxidant polyphenols, sterols and bioactive phospholip-
ids in fermented tofu suggest the use of these microorganisms
to enhance the added value, in terms of bioactive com-
pounds of soybean products. Sterols, phospholipids, aglycones
and polyphenols are key substances that can positively improve
and help womens metabolism specifically in the post-
menopausal period of life. In particular, aglycone-rich products
such as tofu obtained by fermented soy milk may be useful
in preventing menopausal symptoms and other diseases.
Lactic acid bacteria are very promising sources for novel
products and applications, especially those that can satisfy the
increasing consumers demands for natural products and func-
tional foods. They can be used in the diet of humans and
animals, with particular role in their health status.
In this context, the specific selection of tailor made starter
culture of lactic acid bacteria strains able to grow well in soy
milk and contemporary to produce bioactive compounds for
a specific consumers categorycould be suggested.
Acknowledgements
The author Vito Verardo thanks the Spanish Ministry of
Economy and Competitiveness (MINECO) for Juan de la Cierva
post-doctoral contracts.
This work was performed in the framework of the Italian
project MIAOVER50 focused on healthy diets for over-50-year-
old consumers.
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