CyTA - Journal of Food

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Profiles of free fatty acids, free amino acids, and

volatile compounds of milk bases fermented by
Lactobacillus casei GBHM-21 with different fat
levels

Zhining Bao, Jian Xiong, Weifeng Lin & Jun Ye

To cite this article: Zhining Bao, Jian Xiong, Weifeng Lin & Jun Ye (2016) Profiles of
free fatty acids, free amino acids, and volatile compounds of milk bases fermented by
Lactobacillus�casei GBHM-21 with different fat levels, CyTA - Journal of Food, 14:1, 10-17, DOI:
10.1080/19476337.2015.1035673

To link to this article: https://doi.org/10.1080/19476337.2015.1035673

© 2015 The Author(s). Published by Taylor & Published online: 18 Jun 2015.
Francis.

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CyTA – Journal of Food, 2016
Vol. 14, No. 1, 10–17, http://dx.doi.org/10.1080/19476337.2015.1035673

Profiles of free fatty acids, free amino acids, and volatile compounds of milk bases fermented by
Lactobacillus casei GBHM-21 with different fat levels
Perfiles de tres ácidos no grasos, aminoácidos no grasos y compuestos volátiles de bases de leche
fermentadas con Lactobacillus casei GBHM-21 con diferentes niveles de grasa
a
Zhining Bao , Jian Xionga,b, Weifeng Lina* and Jun Yea
a
College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China; bState Key Laboratory
of Polymer Materials Engineering, Sichuan University, Chengdu 610065, China
(Received 1 December 2014; final version received 25 March 2015)

Milk fat content is an important factor affecting the flavor of fermented dairy products. The effects of milk fat concentration (0–90) g·L−1 on
the changes of flavor-related compounds in the milk base fermented by Lactobacillus casei GBHM-21 were investigated. The amount of
some of the free fatty acids (FFAs) and free amino acids (FFAs) increased as the fat concentration increased, accompanied by a concomitant
increase of the quantities and intensities of volatile compounds (VCs). A substantial increase in long-chain fatty acids was observed,
whereas no significant effect on short-chain fatty acids was observed. The amount of the total FFA was linearly and positively correlated
with the total FAA. The most abundant group of VCs was ketones for low-fat samples and carbonyl acids for high-fat samples. The level of
FFA-derived ketones increased dramatically first and then reached a plateau, while FFA-derived carbonyl acids continuously increased.
These results provided valuable informative utilization of milk fat in the fermented industry.
Keywords: fermented milk base; free fatty acid; free amino acid; volatile compound; Lactobacillus casei

El nivel de grasa en la leche es un factor importante en el sabor de los productos lácteos fermentados. Se investigaron los efectos del nivel
de grasa en la leche (0–90 g·L−1) en los cambios en los compuestos relacionados con el sabor en bases de leche fermentadas con
Lactobacillus casei GBHM-21 durante 72 h. Los ácidos no grasos (FFA) y los aminoácidos no grasos (FAA) de forma selectiva, además de
la calidad e intensidad de los compuestos volátiles (VC) aumentaron significativamente con el aumento de la concentración de grasa. No se
observó ningún efecto significativo en los ácidos grasos de cadena corta, mientras que sí se observó un aumento sustancial en los ácidos
grasos de cadena larga. Se encontró que la relación positiva entre el total de ácidos no grasos y aminoácidos no grasos era lineal. El grupo
más abundante de compuestos volátiles resultó ser las cetonas para las muestras bajas en grasa, mientras que los ácidos carbonilos para las
muestras altas en grasa. El aumento en las cetonas derivadas de ácidos no grasos primero fue dramático y después cónico, mientras que el
aumento en los ácidos carbonilos derivados de ácidos no grasos fue lineal. Estos resultados resultarían útiles para una mayor expansión de
las aplicaciones de la grasa en la industria de la fermentación.
Palabras clave: Base de leche fermentada; ácido no graso; aminoácido no graso; compuesto volátil; Lactobacillus casei

1. Introduction proteolysis. Additionally, lipolysis leads to the formation of free

Fermented milk bases are widely used as ingredients in a variety
of foods, such as fermented dairy beverages (Gomes et al., 2013)
and bakery products(Hassan, El-Shazly, Sakr, & Ragab, 2013), for
flavor enhancement. Milk fat content is an important factor influ-
encing the flavor of fermented dairy products. Increasing fat
content for better taste and flavor has been demonstrated in cul-
tured buttermilk, which was usually made from skim milk (White,
2013). The flavor-related compounds in fermented dairy products,
such as those associated with the taste and aroma, are primarily
derived from microorganism-mediated glycolysis, proteolysis,
and lipolysis (Collins, McSweeney, & Wilkinson, 2003; Hassan,
Mona, El-Gawad, & Enab, 2013; Smit, Smit, & Engel, 2005). The
addition of milk fat could enhance the flavor of fermented milk by
affecting the metabolism of the microorganisms.
However, there is only limited report concerning the effect of
milk fat concentration on fermented dairy products. Researchers
(Shakerian et al., 2014, 2013) investigated the effects of different
levels of fat on characteristic indices in yogurt and found that
different fat levels significantly affected the production of organic
acids but not the cell counts of probiotic bacteria or the degree of
fatty acids (FFAs) and their oxidation products such as ketones, and
therefore contributes to the development of a favored fermented
aroma (Hassan, Mona, et al., 2013). Meanwhile, proteolysis leads
to the production of free amino acids (FAAs), which are crucial for
the characteristic taste of fermented milk and also are important
precursors in the synthesis of essential volatile compounds (Bassoli,
Borgonovo, Caremoli, & Mancuso, 2014; Mikulec, Habuš,
Antunac, Vitale, & Havranek, 2010). However, the interrelation
among the concentrations of these flavor-related compounds is
relatively less studied.
The LAB L. casei has a multi-enzyme system including
esterases, cell-envelope proteinases, peptidases, and amino
acid-converting enzymes (Choi & Lee, 2001; Martínez-Cuesta,
Fernández de Palencia, Requena, & Peláez, 2001). All those
enzymes could contribute to the generation of flavor-related
compounds during fermentation. Therefore, the strain plays a
prominent role in the flavor of fermented dairy products and
exhibits great potential for the fermentation of milk bases.
In this study, the changes in microbial growth and flavor-
related compounds in milk bases fermented with L. casei

*Corresponding author. Email: linwf@scut.edu.cn

© 2015 The Author(s). Published by Taylor & Francis.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

GBHM-21 following the addition of anhydrous butterfat
(ABF) were studied. The aim of this work was to determine
whether the fat level significantly affects microbial metabo-
lism and flavor profile and to better expand the application of
fat. Changes in levels of FFAs, FAAs, and volatile compounds
during fermentation were studied to evaluate the effects of the
fat level on flavor-related compounds and on the relationships
among these compounds.
2. Materials and methods
2.1 Strain and reagents
The GBHM-21 bacterial strain has been widely identified as L.
casei by the China General Microbiological Culture Collection
Center (CGMCC). Bacteria were freeze-dried at a concentration
of 1.66 ± 0.24 × 1010 cfu·g−1. The skim milk powder (SMP),
containing 342.5 ± 5.2 g·kg−1 protein and 32.7 ± 0.3 g·kg−1
water, was purchased from Fonterra (Auckland, New Zealand).
ABF, containing 998.0 g·kg−1 fat, less than 3.0 g·kg−1 FFAs, and
less than 1.0 g·kg−1 water, was purchased from Nestle
(Shuangcheng, China).
2.2 Preparation of the fermented milk base
Five standardized milk bases (1–5) were prepared. Milk base 1–
milk base 5 (CK1–CK5) were standardized according to SMP
and ABF levels to contain 29 g·L−1 protein and 0, 15, 30, 60,
and 90 g·L−1 fat, respectively. The standardized milk bases were
hydrated by stirring at 60 C for 20 min, homogenized at 20–25
MPa, heated at 95 C for 5 min, and finally cooled to 37 C in a
water bath before inoculation. The inoculation rate was 0.04%
(w/w). The milk bases were incubated separately at 37 C for
72 h. The resulting samples were termed sample 1–sample 5
(S1–S5). Fermentation was stopped by rapidly cooling the milk
bases to 4 C in an ice-water bath. All samples were prepared in
triplicate.
2.3 Acidity
The pH of the milk bases was monitored before (CK) and after
(S) fermentation using a digital pH meter (pHS-25; INESA.CC,
Shanghai, China). The titration acidity was determined according
to the Association of Official Analytical Chemists (AOAC)
920.124. Neutralization titration was performed using standard
0.1 M NaOH with phenolphthalein as an indicator. The result
was expressed as lactic acid (g·L−1).
2.4 Viable cell counts
The number of viable probiotic cells was counted before and
after fermentation as previously described (Fonteles, Costa, de
Jesus, & Rodrigues, 2012). Serial decimal dilutions of each
sample were plated in triplicate onto MRS (de Man, Rogosa
and Sharp) Agar and incubated at 37 C for 72 hours. The results
were expressed as cfu·g−1.
2.5 FFAs
Milk base lipid extraction and gas chromatographic analysis
were performed as described by Güler and Gürsoy-Balci
(2011), with minor modifications. Briefly, 5 g milk base was
mixed with 1 mL of 2.5 M H2SO4 and 10 mL internal standard
CyTA – Journal of Food 11
solution, which contained 400.0 mg·L−1 pentanoic (5:0) and
heptadecanoic (17:0) acids (Sigma, USA) in diethyl ether. The
mixtures were shaken and mixed for 1 min and then centrifuged
(Eppendorf, DE, USA) at 1776 × g for 10 min at 0 C, then the
samples were allowed to stand for 1 h. The upper layer was
transferred to a centrifuge tube containing 3.0 g anhydrous
Na2SO4, which was then added to 10 mL hexane.
After centrifugation at 1776 × g for 10 min, the lipid extracts
were applied to an aminopropyl-bonded phase column (Strata
NH2, Phenomenex, USA) that had been conditioned with 10 mL
hexane/diethyl ether (1:1, v/v). One milliliter of extract was then
passed through the column (twice). Next, 5 mL hexane/diethyl
ether (1:1, v/v) was passed through the column (twice) to remove
all triacylglycerols. The FFAs were then eluted using 2 mL
diethyl ether containing 2% formic acid and purged with nitro-
gen to concentrate the samples; 1 μL of the concentrated solution
was used for gas chromatography–mass spectrometry (GC–MS;
Agilent 7890A, USA) analysis. All extractions and GC analyses
of FFAs were performed in duplicate.
FFAs were analyzed using a GC–MS column
(30 m × 0.25 mm inner diameter × 0.25 μm film thickness).
Under the GC operating conditions, helium was used as the carrier
gas at a constant flow rate of 1 mL·min−1. The GC oven tempera-
ture was set to 50 C for 5 min, raised to 230 C at a rate of
5 C·min−1, and then held at 230 C for 20 min. The injector
temperature was 250 C, and the run time was 58 min. An
Agilent 5975 model quadrupole mass selective detector was oper-
ated in the scan mode with the mass range of 33–330 m·z−1 and a
scan range of 1 scan·s−1. The interface line to the MS was set at
280 C. The MS was operated in the electron impact (EI) mode
with an electron energy of 70 eV and was calibrated by auto-
tuning. Peak identification was performed by comparing the reten-
tion times and ion spectra with those of authentic standards
(Aldrich Chemical Co., Steinheim, Germany) and spectra from
the mass spectral database (Nist, 2005, Agilent, Wiley Number,
USA). The final FFA concentrations were expressed in mg·L−1
milk base.
2.6 FAAs
FAA extraction was performed as described by Bütikofer, Fuchs,
Bosset, and Gmür (1991), with some modifications. All samples
were frozen before grinding. About 15 g of grated milk base was
weighed, 50 mL n-hexane was added, and the mixture was
homogenized for 30 s using a mixer (TM-767II, Haipan
Instrument Co. Ltd, Zhongshan, China). The milk base was left
to precipitate for 1 min, and the hexane was carefully aspirated
off. The degreasing procedure was repeated twice under the
same conditions. The degreased residue was transferred to a
250 mL pear-shaped flask in a fume hood, and the residual
hexane was removed using a vacuum with an attached water
pump for 1 h. FAAs were extracted from the sample by the
addition of 1 mL 5% w/v sulfosalicylic acid solution to 1.0 g
of each sample; the resulting mixture was centrifuged at
20,000 × g for 10 min at 4 C.
FAAs in the supernatants were quantified by high perfor-
mance liquid chromatography (HPLC) analysis, as described by
Cui, Zhao, Li, Zhao, and Sun (2014). Supernatants were
vacuum-dried, derivatized using phenyl isothiocyanate, and
then dried again. The derivatized dried samples were dissolved
in diluent (phosphoric acid–acetonitrile water solution, pH 7.4)
and then centrifuged at 3552 × g. The supernatants were then
analyzed using an HPLC system (Waters, Boston, MA, USA)
12 Z. Bao et al.

equipped with a Waters Pico-Tag amino acid column (150 m)
and a Waters 486UV detector. The parameters used were as
follows: column temperature, 38 C; measurement wavelength,
254 nm; mobile phase elutes (binary gradient elutions) of solvent
A (sodium acetate–acetic acid buffer solution, pH 6.4,
1.0 mL·min−1) and solvent B (60% acetonitrile solution in pure
water, 1.5 mL·min−1). The injection volume was 10 μL. The
concentrations of FAAs were calculated using a standard con-
taining 18 different amino acids (Waters).
2.7 Solid-phase microextraction (SPME)-GC–MS
Volatile compounds were extracted and analyzed as described by
Feng et al. (2014) with some modifications. An SPME TriPlus
automated sampler equipped with a 75 μm carboxen/polydi-
methylsiloxane fibre (CAR/PDMS, Supelco, Inc., Bellefonte,
PA, USA) was employed to extract volatile compounds from
the fermented milk bases. Two-gram samples were placed in 20
mL gas-tight vials (Supelco). After the samples were equilibrated
at 60 C for 20 min, they were subjected to extraction using CAR/
PDMS fibres for 40 min with continuous heating and agitation.
After extraction, the fibres were inserted into the GC injector for
3 min to desorb the analytes. Each fermented milk base sample
was extracted in duplicate. The fibre was conditioned before use
by insertion into the GC injector port for 1 h at 230 C. It was
then desorbed for 3 min at 230 C between injections to prevent
contamination.
Volatile compounds were analyzed using a Trace GC–MS
system equipped with an Ultra GC, a TriPlus automated sampler,
and a quadrupole DSQ II MS (Thermo Finnigan, San Jose, CA,
USA). Separation was performed using a TR-Wax column
(30 m × 0.32 mm × 0.25 μm; J&W Scientific, Folsom, CA,
USA). The GC–MS conditions used were consistent with those
described by Feng et al. (2013). Briefly, helium was used as the
carrier gas with a flow rate of 1.0 mL·min−1 and a split ratio of
10:1. The temperature of the column was maintained at 40 C for
3 min, increased to 120 C at 5 C·min−1, and held for 2 min. It
was then raised to 250 C at a rate of 7 C·min−1 and held at 250 C
for 5 min. The injection temperature was 230 C, and the ion
source temperature was set at 250 C. The mass spectrometer was
operated in the EI mode. The ionization energy, detector voltage,
scan range, and scan rate applied for analyses were as follows:
70 eV; 350 V; 35–350 m·z−1; and 3.00 scans·s−1, respectively.
Chromatograms and mass spectra were evaluated using Xcalibur
software version 2.0 (Thermo Finnigan). Volatile compounds
were identified by comparing the retention indices (RIs) obtained
with those described previously under the same conditions and
by comparing the mass spectra with those included in the
Nist2005 and Varian libraries. The volatile compounds identified
were also compared to previously reported aroma compounds
produced during milk fermentation. The volatile compounds
were compared as area units (AU × 106) and relative
percentages.
2.8 Statistical analysis
The results are shown as means, standard deviations, and relative
standard deviations. One-way analysis of variance was used to
assess the homogeneity of variances. Statistical analyses of
recorded results were performed using the Statistical Program
for Social Science for Windows 16.0 (StatSoft. Inc., USA), and p
values of less than 0.05 were considered to be statistically
significant.
3. Results and discussion
3.1 Fermentation characteristics
The effect of different fat levels on viable cell counts, pH, and
titrimetric acidity during fermentation was analyzed (Table 1).
The viable cell counts were increased consistently for S1–S5
after fermentation, reaching 1–2 × 109 cfu·g−1 (up from
1–2 × 107 cfu·g−1 after inoculation). These increases in viable
cell counts suggested that increasing the fat concentration to
90 g·L−1 did not inhibit the proliferation of L. casei
GBHM-21. Shakerian et al. (2014) also found no significant
effects of fat levels (0.5–5.0%) on cell counts (higher than × 106
cfu·mL−1) of probiotics in yogurts. This was probably due to the
concentration and constitution of FFAs, especially short-chain
fatty acids (SCFAs) shown in 3.2. The number of viable cells
within the five bases remained high, despite the high acidity

Table 1. Effects of different fat concentrations on the viable cell count and acidity of fermented milk bases before (CK) and after (S) 72 h.
Tabla 1. Efectos de las diferentes concentraciones de grasa en el recuento viable de células y acidez de las bases de leche fermentadas antes (CK) y después
(S) de 72 h.

Viable cell count (cfu·g−1) pH Titrimetric acidity (g·L−1)

Sample* CK S CK S CK S

1 (1.12 ± 0.05) × 107 (1.10 ± 0.15) × 109a 6.40 ± 0.01a 3.75 ± 0.01a 0.01 ± 0.00a 12.61 ± 0.15a
2 (1.12 ± 0.05) × 107 (1.20 ± 0.13) × 109a 6.40 ± 0.01a 3.65 ± 0.01a,b 0.20 ± 0.02b 12.70 ± 0.11a,b
3 (1.12 ± 0.05) × 107 (1.17 ± 0.18) × 109a 6.41 ± 0.01a 3.61 ± 0.01a,b 0.36 ± 0.04c 12.82 ± 0.13a,b
4 (1.12 ± 0.05) × 107 (1.62 ± 0.20) × 109b 6.43 ± 0.01b 3.55 ± 0.01a,b 0.60 ± 0.03d 13.08 ± 0.12b
5 (1.72 ± 0.35) × 107 (1.79 ± 0.24) × 109b 6.46 ± 0.01c 3.54 ± 0.01b 0.79 ± 0.01e 13.13 ± 0.11b

Note: *Samples 1–5 are defined for subsets before (CK) and after (S) fermentation as below.
1: milk base containing 29 g·L−1 protein and 0 g·L−1fat; 2: milk base containing 29 g·L−1 protein and 15 g·L−1 fat; 3: milk base containing 29 g·L−1 protein and 30 g·L−1 fat; 4:
milk base containing 29 g·L−1 protein and 60 g·L−1 fat; 5: milk base containing 29 g·L−1 protein and 90 g·L−1 fat.
a–e
Mean values ± standard deviation, with different letters in the same row, are significantly different (p ≤ 0.05).
Nota: *Las muestras 1 hasta 5 son definidas por el subgrupo antes (CK) y después (S) de la fermentación como a continuación.
1: base de leche de 29 g·L−1 de proteína y 0 g·L−1fat; 2: base de leche de 29 g·L−1 de proteína y 15 g·L−1 de grasa; 3: base de leche de 29 g·L−1 de proteína y 30 g·L−1 de grasa;
4: base de leche de 29 g·L−1 de proteína y 60 g·L−1 de grasa; 5: base de leche de 29 g·L−1 de proteína y 90 g·L−1 de grasa.
a–e
Los valores promedio ± desviación estándar, con diferentes letras en la misma fila son significativamente distintos (p ≤ 0,05).
 CyTA – Journal of Food 13

values, implying that the viability and metabolic activity of L. C6:0 (Choi & Lee, 2001). And the observation that SCFAs and
casei GBHM-21 during the fermentation were considerable. methyl mercaptan can react to form methyl thioester, which also
Following the increase in fat content, the titrimetric acidity results in fermented aromas (Collins et al., 2003).
of CK2–CK5 increased slightly compared with that of CK1. We The quantity of unsaturated fatty acids (USFAs)
expected that is because a small amount of SCFAs in the ABF increased, particularly C18:1 (3.70 mg·L−1 in S2 and
(Xu & Qiu, 2001) dissolved in the aqueous phase during titri- 12.22 mg·L−1 in S5). The changes in FFA profiles suggested
metric acidity measurements. There were no significant differ- that the increased fat content enhanced the synthesis of
ences (p > 0.05) in pH and titrimetric acidity among S1–S5, lipases, esterases, and other relevant enzymes in the lag
which was consistent with the changes in viable cell counts. phase in L. casei GBHM-21. Therefore, the aroma-related
compounds formed from FFA catabolism are important for
evaluating the effects of FFAs on the flavor of fermented
3.2 FFAs milk bases.
Figure 1 shows the effects of different fat concentrations on FFAs in
the milk bases before and after L. casei GBHM-21 fermentation. As
expected, the total FFA concentrations of CK1–CK5 and S1–S5 all 3.3 FAAs
increased along with the increase in fat concentration (the inset Figure 2 shows the effects of increasing fat concentrations on
figure in Figure 1). Obviously, the increase of the fat concentration FAAs in milk bases fermented with L. casei GBHM-21. The
in the milk base brought more FFAs after fermentation. The highest total FAA contents of CK1–CK5 increased from
concentration of an individual FFA in S4 and S5 was C8:0, while 7.55 ± 0.09 mg·100 g−1 to 10.62 ± 0.02 mg·100 g−1 along
that in S2 and S3 was C10:0. Notably, there was no significant with the increase in the fat level.
difference in SCFAs (C4:0–C6:0), while middle-chain fatty acids As shown in the inset figure in Figure 2, there were clearly
(C8:0–C10:0) and long-chain fatty acids (LCFAs, C12:0–C18:0) positive relationships (R2 = 0.964) between the amount of fat in the
exhibited the greatest increase along with the fat concentration milk base and the total FAA contents after fermentation. However,
increase. No significant difference in SCFAs also could be related there were obviously different trends in the concentrations of indi-
to no significant effects on the viable cell count because FFAs, vidual FAAs (Figure 2). With the increase in fat concentration, the
particularly SCFAs, inhibited bacterial growth (Nair et al., 2005). concentration of some FAAs, such as Glu, Leu, Trp, Phe, and Lys,
Furthermore, the composition ratio of LCFAs was higher among were significantly increased. In contrast, the levels of Ser, Gly, His,
samples with high fat concentrations (S4 and S5), while the percen- Thr, and Pro were the highest in S3. Additionally, some FAAs, such
tage of SCFAs was higher in samples with low fat concentration. as Ala and Cys, were not significantly affected by the fat concen-
This may be due to the strong esterolytic activity of L. casei for tration. The different changes in the levels of various FAAs could be
Total free fatty acid concentration (mg · L–1)

120 S5

100
S4
80

60 S3
24 S2 CK5
40
CK4
22 20 CK3
CK2
Free fatty acid concentration (mg · L–1)

S1
20 0
CK1

18 c 0 20 40 60 80 100
c
Fat concentration (g · L–1)
16 c
d

d
d
14 b
b
12 c
b c b
d c
10
b
8 a,b c
a,ba,b b a,b a b

6 a a a
b b b S2
4 a
a c
d S3
a

a b
a a a
S4
2 a
S5
0
C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C16:1 C16:0 C18:1 C18:0

Figure 1. Effects of different fat concentrations on FFA concentrations in milk bases fermented with L. casei GBHM-21. C4:0, butanoic acid; C6:0,
hexanoic acid; C8:0, octanoic acid; C10:0, decanoic acid; C12:0, dodecanoic acid; C14:0, tetradecanoic acid; C16:1, palmitoleic acid; C16:0, hexadecenoic
acid; C18:1, oleic acid; C18:0, octadecanoic acid. S2–S5: milk bases containing 29 g·L−1 protein and 15, 30, 60, or 90 g·L−1 fat, respectively, fermented
for 72 h.
Figura 1. Efectos de diferentes concentraciones de grasa en concentraciones de ácidos no grasos en bases de leche fermentadas con L. casei GBHM-21.
C4:0, ácido butírico; C6:0, ácido hexanoico; C8:0, ácido octanoico; C10:0, ácido decanoico; C12:0, ácido dodecanoico; C14:0, ácido tetradecanoico;
C16:1, ácido palmitoleico; C16:0, ácido hexadecenoico; C18:1, ácido oleico; C18:0, ácido octadecanoico. S2–S5: bases de leche de 29 g·L−1 de proteína y
15, 30, 60, o 90 g·L−1 de grasa, respectivamente, fermentadas durante 72 h.
14 Z. Bao et al.

20 S1 S2 S3 S4 S5 100

95

90

FAA (mg · 100g–1)

85
c
e b 80
a
b 75
b FAA
a 70
Linear fit for FAA
d y = 0.232x + 71.781
Free amino acids (mg · 100g–1)

65 2
R = 0.964
60
d c 0 20 40 60 80 100
b e Fat concentration (g · L–1)
a
10
d

d c

e
e c
c
cc d d
c c bc aaaaa d
b c a ab b
bc c c
b aba a a a ab aaa c
c ab b
b d a d aaaaa
b c
aaa c a
a b bb
b bb a
a a a
aaa
bc
aaa
0
Asp Glu Ser Gly His Arg Thr Ala Pro Tyr Val Met Cys Ile Leu Trp Phe Lys

Figure 2. Effects of different fat concentrations (0, 15, 30, 60, and 90 g·L−1 fat for S1–S5) on FAA concentrations in milk bases fermented with L. casei
GBHM-21.
Note: a–eMean values ± standard deviation with different letters in the same row that are significantly different (Duncan’s test, p ≤ 0.05). Asp: Asparagine; Glu:
glutamic acid; Ser: serine; Gly: glycine; His: histidine; Arg: Arginine; Thr: threonine; Ala: Alanine; Pro: proline; Tyr: tyrosine; Val: valine; Met: methionine; Cys:
cystine; Ile: isoleucine; Leu: leucine; Trp: tryptophan; Phe: phenylalanine; Lys: lysine.
Figura 2. Efectos de diferentes concentraciones de grasa (0, 15, 30, 60 y 90 g·L−1 de grasa para S1–S5) en concentraciones de ácidos no grasos en bases
de leche fermentadas con L. casei GBHM-21.
Nota: a–eLos valores promedio ± desviación estándar con letras distintas en la misma fila son significativamente diferentes (Test Duncan, p ≤ 0,05).Asp: Asparagina;
Glu: ácido glutámico; Ser: serina; Gly: glicina; His: histidina; Arg: Arginina; Thr: treonina; Ala: Alanina; Pro: prolina; Tyr: tirosina; Val: valina; Met: metionina; Cys:
cistina; Ile: isoleucina; Leu: leucina; Trp: triptófano; Phe: fenilalanina; Lys: lisina.

attributed to the improved proteolysis and FAA catabolism for L. 95

casei GBHM-21 influenced by the increase of fat.
In S1, S2, and S3, the major FAAs were Pro (sweet), Thr, 90
and His (bitter), while Glu (fresh, sour), Pro, and Trp (bitter)
FAA (mg · 100g–1)

were predominant in S4 and S5. Because FAAs have been 85

shown to be associated with different tastes (Bassoli et al.,
2014), these results suggested that the taste of the milk base 80
fermented by L. casei GBHM-21 obviously affected when the
fat content was increased to a certain extent, i.e., up to 75
FAA
60 g·L−1 in our study. FAAs can also be converted via a Linear fit for FAA
variety of pathways due to the catalytic activity of enzymes, 70
y = 0.195x + 70.067
contributing to malty, fruity, and sweet flavors (Ardö, 2006; R2 = 0.991
Fox, Singh, & McSweeney, 1995). Since FAAs significantly 65
0 20 40 60 80 100 120
changed, the aroma formation was expected to be quite FFA (mg · L–1)
different.
If the total FFA and FAA contents of S1–S5 were studied Figure 3. Linear relationship between the total FFA concentration and
jointly, a linear, positive relationship (R2 = 0.991) was estab- the total FAA concentration in milk bases fermented with L. casei
GBHM-21.
lished (Figure 3). In contrast, Hernández et al. (2009) found
a linear, inverse relationship between the total FFA and FAA Figura 3. Relación lineal entre la concentración total de ácidos no
contents because the FFAs, particularly SCFAs, hydrolyzed grasos y la concentración total de aminoácidos no grasos en bases de
leche fermentadas con L. casei GBHM-21.
by lipase could inhibit bacterial growth (Nair et al., 2005).
These data showed that there was no inhibition of bacterial
growth (Table 1) and that both total FAA and total FFA
contents increased. This was because the percentage of 3.4 Volatile compounds
SCFAs decreased with the increase in fat concentration in Both the relative amounts and types of volatile compounds were
our study. Therefore, the content and composition of FFAs significantly affected (p < 0.05) by the increase in fat (Table 2).
should be considered when assessing the effects of FFAs on Fat addition brought markedly larger and considerably more
bacterial growth and FAAs. complex volatile compounds in S2–S5 than those in S1. The
Table 2. Effects of different fat concentrations (0, 15, 30, 60 and 90 g·L−1 fat for S1–S5) on volatile compounds in milk bases fermented with L. casei GBHM-21 for 72 h.
Tabla 2. Efectos de diferentes concentraciones de grasa (0, 15, 30, 60 y 90 g·L−1 de grasa para S1–S5) en los compuestos volátiles en bases de leche fermentadas con L. casei GBHM-21 durante 72 h.

S1 S2 S3 S4 S5
Classification Compound CAS Area (AU × 106) Area (AU × 106) Area (AU × 106) Area (AU × 106) Area (AU × 106) Metabolism origin **
a b b c d
Ketones 91.7% 50.9% 50.3% 42.4% 31.6%
2,3-Butanedione 431-03-8 77.4a 194.5b 235.6b 309.7c 317.5c Lactate
2-Pentanone 107-87-9 – 6.4a 27.8b 31.0b 35.4b FFA
3-Hydroxy-2-butanone 513-86-0 111.0a 349.1b 386.5c 385.6c 412.4c Lactate
2-Hentanone 110-43-0 – 22.4a 394.0b 441.7c 465.5c FFA
2-Nonanone 821-55-6 – 12.6a 48.2b 53.7b 59.6b FFA
2-Undecanone 112-12-9 – 4.0a 6.8b 8.1b,c 9.3c FFA
Carbonyl acids 6.3%a 43.1%b 43.2%b 53.1%c 62.4%d
Acetic acid 64-19-7 12.8a 249.9b 495.6c 736.4d 911.1e Lactate, FFA, FAA
Butyric acid 107-92-6 – 43.1a 44.5a 63.0b 69.9b FFA
Isobutyric acid 79-31-2 – – – 53.7a 63.2b FFA
Isovaleric acid 503-74-2 – – – 122.4a 988.2b FFA
Hexanoic acid 142-62-1 – 101.2a 273.5b 339.2c 346.3c FFA
Octanoic acid 124-07-2 – 76.2a 101.2b 190.7c 159.3d FFA
Phthalic acid 85-44-9 – 27.7a 30.2a 34.2a 24.6a FAA
Aldehydes 2.1%a 5.7%b 3.1%c 2.0%a 1.5%d
Acetaldehyde 75-07-0 4.2a 63.6b 47.9c 58.6b,c 55.0b,c Lactate, FAA
Trans-2-decenal 3913-81-3 – – – – 6.1 FFA
33-Methylbutyraldehyde 590-86-3 – 2.8a 19.7b – – FAA
Others 0%a 0.2%b 2.5%c 2.5%c 4.5%d
Ester Ethyl caproate 123-66-0 – 1.8a 6.1b 7.0b 9.1c FFA
Sulphide Methyl disulphide 624-92-0 – – – – 2.5 FAA
Furan 2-Ethylfuran 3208-16-0 – – – 3.90a 38.6b Combined pathways
Alkenes 1,4-Pentadiene 591-93-5 – – 32.6a 44.2a 79.6b Combined pathways
D-Limonene 5989-27-5 – – 9.0a 8.7a 13.9b Combined pathways
Aromatic hydrocarbons Toluene 108-88-3 – – – – 3.6 Combined pathways
Naphthalene 91-20-3 – – 7.3a,b 12.2b 40.3c Combined pathways
Total 205.4a 1155.3b 2159.2c 2904.0d 4121.0e

Note: *A/A, the ratio of the area of each compound to the total area of each sample.
**Details about the origin of metabolism were taken mainly from Hassan, Mona, et al. (2013) and Ziadi et al. (2010).
a–e
Different letters in the same row indicate significant statistical differences (Duncan’s test, p < 0.05).
Nota: *A/A, proporción del área de cada compuesto sobre el área total de cada muestra.
**Los detalles sobre el origen del metabolismo fueron obtenidos principalmente de Hassan, Mona, et al. (2013) y Ziadi et al. (2010).
a–e
Las distintas letras en la misma fila indican diferencias estadísticamente significativas (Test de Duncan, p < 0.05).
CyTA – Journal of Food
15
16 Z. Bao et al.
total area and the types of volatile compounds detected increased
along with the fat concentration increase; for example, the total
area of S5 was 3.6-fold of that of S2, and eight more volatile
compounds detected in S5 were found than those in S3. Zaręba,
Ziarno, Ścibisz, and Gawron (2014) identified 14 types of vola-
tile compounds in L. casei DN-114 001 fermented milk. Of these
14 compounds, seven were consistent with compounds identified
in our study. Changes in the amounts and types of compounds
suggested that the concentration of milk fat had a crucial influ-
ence on the formation of fragrant aromas in fermented dairy
products.
In this study, the identified volatile compounds were divided
into ketones, carboxylic acids, aldehydes, and others. The pro-
portion of these different types of compounds was different
among samples; the dominant volatile compounds in S1, S2,
and S3 were ketones (91.7%, 50.9%, and 50.3%, respectively),
while those in S4 and S5 were carbonyl acids (53.1% and 62.4%,
respectively). The most common compounds in S2 and S3 were
acetic acid, 2-heptanone, and 3-hydroxy-2-butanone, whereas the
major compounds in S4 and S5 were isovaleric acid, acetic acid,
and 2-heptanone, predominantly metabolites from FFAs.
Acetic acid has contributed a sharp vinegar-like flavor to
milk, and butyric acid has an essential role in the formation of
basic fermented dairy aromas (Frank, Owen, & Patterson, 2004).
As the fat content increased, enzymes related to fat metabolism
increased, resulting in significant changes in the types and
amounts of carboxylic acids, particularly branched-chain (BC)
isovaleric acid and isobutyric acid. A number of ketones are
important in determining the aroma of fermented dairy products:
2,3-butanedione (sweet buttery), 2-heptanone (musty, sweet,
moldy, varnish), and 2-nonanone (floral fruity, peach) (Frank
et al., 2004; García-Quintáns, Blancato, Repizo, Magni, &
López, 2008). Methyl ketones are produced by the oxidation of
polyunsaturated fatty acids (Hassan, Mona, et al., 2013), and
methyl ketones generated through this process are generally odd-
chain ketones (Collins et al., 2003). Consistent with these path-
ways, the ketone present at the highest concentration in both S2
and S3 was 2-heptanone.
As presented in Figure 4, the simultaneous increase in rela-
tive total amounts of ketones and carbonyl acids was associated
with the increases in FFAs and fat. For ketones derived from
FFAs, the relative amount increased sharply when the fat con-
centration increased from 0 to 30 g·L−1 and gently when the fat
concentration increased from 30 to 90 g·L−1. However, for
carbonyl acids metabolized from FFAs, the relative amount
increased linearly (R2 = 0.946). Therefore, increasing the fat
content affected the production of volatile ketones and carbonyl
acids. From these data, we concluded that the metabolism of
FFAs by L. casei GBHM-21 was limited, particularly the pro-
duction of ketones.
Besides FFA metabolism, various volatile compounds are
also produced from the biochemical pathways leading to FAA
metabolism. Ziadi et al. (2010) concluded that aminotransferase
could catalyze the conversion from α-ketoglutarate and BC
amino acids into BC keto acids. We observed difference in
these metabolites concurrent with the elevated FAA levels in
the fermented milk base (Figure 2). Methional, broken down
from Met (Fox et al., 1995), was present only in S2 and S3.
Dimethyl disulfide, the decomposition product of methional (Fox
et al., 1995), was only found in S5. Although additional volatile
compounds were present at relatively low levels, these types of
compounds have previously been shown to play important roles
in the flavor of the fermented milk base by providing a rich and
1800
600 1600
1400
Carbonyl acids (AU × 106)
500
Ketones (AU × 106)
1200
400
1000
300 800
600
200
400
100 Ketones
200
Carbonyl acids
---- Line fitting carbonyl acids 0
0 2
y = 17.25x – 65.72 R = 0.946
–200
0 20 40 60 80 100
Fat concentration (g · L–1)
Figure 4. Effects of fat concentrations on the relative total amount of
ketones and carbonyl acids derived from FFAs in milk bases fermented
with L. casei GBHM-21.
Note: Ketones derived from FFAs, including 2-pentanone, 2-hentanone,
2-nonanone, and 2-undecanone; carbonyl acids derived from FFAs,
including butyric acid, isobutyric acid, isovaleric acid, hexanoic acid,
and octanoic acid.
Figura 4. Efectos de la concentración de grasa en la cantidad total
relativa de cetonas y ácidos carbonilos derivados de ácidos no grasos
en bases de leche fermentadas con L. casei GBHM-21.
Nota: Cetonas derivadas de ácidos no grasos, incluyendo 2-pentanona, 2-
hentanona, 2-nonanona y 2-undecanona; ácidos carbonilos derivados de
ácidos no grasos, incluyendo ácido butírico, ácido isobutírico, ácido
isovalérico, ácido hexanoico y ácido octanoico.
soft casein aroma (Frank et al., 2004). Furthermore, 2-ethylfuran
is considered to represent a lipid oxidation product and could
evoke a powerful burnt, sweet, and coffee-like flavor. This
compound can influence the course of the Maillard reaction
with amino acids (Adams, Bouckaert, Lancker, Meulenaer, &
Kimpe, 2011), which were also enhanced in S4 and S5, as
discussed earlier. Therefore, fermented milk bases prepared
with increased fat content may have a more fragrant aroma
characteristic of fermentation and may be more useful for food
applications, such as baking.
4. Conclusions
In conclusion, increasing the fat concentration in L. casei
GBHM-21 fermented milk base to 90 g·L−1 had a significant
impact on both GBHM-21 fermentation characteristics and
the levels of FFAs, FAAs, and volatile compounds. The
increase in fat not only enhanced bacterial lipolysis but
also modulated proteolysis and the synthesis of volatile
compounds by exogenously stimulating the L. casei
GBHM-21 enzyme system. Additionally, the relationships
among fat concentration, total FFAs, total FAAs, and the
volatile compounds derived from FFAs were established.
The results of ketones and carbonyl acids are probably two
important parameters to explain the difference in volatile
compounds. The study could contribute to a better under-
standing of fat application in the fermented dairy industry.
Future studies are required to establish a flavor model, eval-
uate the flavor of the fermented milk base, and identify the
active enzymes in L. casei GBHM-21.
Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the Opening Project of State Key
Laboratory of Polymer Materials Engineering of Sichuan
University [grant number KF201301]; the National Natural
Foundation of China [grant number 31270617]; and the
National 973 project of China [grant number 2010CB732201].
ORCID
Zhining Bao http://orcid.org/0000-0002-6120-1119
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