Waste Biomass Valor (2017) 8:1343–1350
DOI 10.1007/s12649-016-9654-6
ORIGINAL PAPER
Evaluation of Waste of the Cheese Industry for the Production
of Aroma of Roses (Phenylethyl Alcohol)
L. Conde-Báez1 • J. Castro-Rosas1 • J. R. Villagómez-Ibarra1 • J. B. Páez-Lerma2
C. Gómez-Aldapa1
Received: 17 October 2015 / Accepted: 16 August 2016 / Published online: 30 August 2016
Ó Springer Science+Business Media Dordrecht 2016
ABSTRACT The whey is a by-product obtained during
the manufacture of cheese. It includes three types of whey;
sweet (SW), acid (AW) and curd (CW). These residues are
rich in lactose and proteins. Practically, there are no studies
on the use of the three types of whey, as a substrate of
micro-organisms for the production of phenylethyl alcohol
(PEA). We assessed the production of PEA by Kluyver-
omyces marxianus using SW, AW and CW as substrate.
Whey pH was adjusted to 4.8, pasteurized at 63 °C/30 min
and inoculated with K. marxianus (1 9 106 CFU mL-1).
The inoculated whey was embedded in agitation (180 rpm)
at 30 °C for 96 h. The identification and quantification of
PEA was performed by gas chromatography. In addition, it
was determined the percentage of (a–b) lactose by
molecular structure with nuclear magnetic resonance. K.
marxianus was capable of producing PEA in the three
types of whey (SW, AW and CV) in maximum concen-
trations of 0.96, 0.70, and 0.47 g L-1, respectively. For SW
(maximum concentration of PEA), it was found a con-
centration of b-lactose 82.35 %. Produced by the cheese
industry, whey could be used as an alternative for the
production of PEA by K.marxianus.
& C. Gómez-Aldapa
cgomeza@uaeh.edu.mx
L. Conde-Báez
lau_iam@hotmail.com
1
Área Académica de Quı́mica (AAQ), Instituto de Ciencias
Básicas e Ingenierı́a (ICBI), Ciudad del Conocimiento,
Universidad Autónoma del Estado de Hidalgo (UAEH),
Carretera Pachuca-Tulancingo Km 4.5,
42186 Mineral de la Reforma, Hidalgo, Mexico
2
Departamento de Ingenierı́a Quı́mica-Bioquı́mica, Instituto
Tecnológico de Durango, 34080 Durango, Mexico
•
Keywords Phenylethyl alcohol  Dairy waste  NMR 
Lactose  Kluyveromyces marxianus
Introduction
Whey is the remaining liquid produced after the curd
separation during the cheese manufacturing process and it
contains about 50 % of the nutrients in the milk [1, 2].It is
estimated that producing 1 kg of cheese generates around
9 L of whey [3]. The worldwide whey production is around
160 million tons each year, being Europe (86 million tons)
and United States (48 million tons) the main producers [4].
The dairy industry produces two types of whey, com-
monly known as sweet and acid whey. The first one is
obtained from cheese making where the enzymatic coag-
ulation is used to a pH of about 5.6. Another is the acid
whey, which is produced when coagulation is by acidifi-
cation to pH 5.1 or less [5, 6]. However, a third type of
whey, resulting from the production of curd with a pH of
about 6.5 occurs. Regardless of the type of whey,
approximately 47 % of it is discarded in bodies of water, or
it is discharged into soils worldwide [7]. The high volumes
generated and its content of organic matter, mainly lactose
(40–60 g L-1), proteins (1.4–33.5 g L-1) and fat
(0.08–10.6 g L-1), makes it a highly contaminating resid-
ual by itself when dumped untreated into the environment
[8]. The values of biological oxygen demand (BOD)
(30–50 g L-1) and chemical oxygen demand (COD) (60-
80 g L-1) present in whey are above standards [9]. Its
continuous discharge causes a rapid consumption of oxy-
gen, eutrophication phenomena, and fats of flotation, the
formation of foam, salinization, acidification, generation of
unpleasant odors, presence of biogenic, among other ele-
ments [10]. However, the bioconversion of whey,
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important for its lactose content, is considered an economic
substrate, and it is of great interest to produce value added
compounds such as bioethanol, biohydrogen, biogas,
enzymes, biopolymers, organic acids, etc. [11–13]. PEA
(C8H10O), which is a volatile alcohol with aroma of roses,
may occur among these compounds.
PEA is widely used in the food industry, for modification of
the organoleptic compositions of the taste of: fruit formulas,
ice cream, candy, soft drinks, gelatins, puddings, rubber gum,
among others [14]. In the pharmaceutical industry it has
applications as antiseptic and local anesthetic [15]. PEA is also
used in the manufacture of perfumes and cosmetics [16]. It
should be noticed that currently, a greater production of PEA
is carried out by chemical methods, which involve the use of
toxic reactives, like benzene and styrene; it also needs a high
temperature process ([300 °C) [17]. Fortunately, the demand
for PEA obtained by biochemical processes is increasing
[18, 19]. Through these biochemical processes, environmental
pollution is decreased significantly. PEA can be synthesized
by certain yeasts through two separate routes as precursor of L-
phenylalanine [20, 21]. It has been reported that K. marxianus
has the ability to produce high concentrations of PEA and uses
the lactose as carbon source [22, 23].
The production of PEA using K. marxianus, has been
carried out using various substrates, mostly synthetic
(molasses, malt extract, yeast extract nitrogenous base,
base dextrose peptone—yeast extract) [14, 16, 18, 24, 25].
However, the use of crude whey obtained directly from
the production of cheese as a substrate for PEA produc-
tion has not been reported. On the other hand, using the
lactose and proteins in the whey as a substrate for the
production of this metabolite, would allow the reduction
of high a contaminant load in this residual [26]. The
objective was to assess the production of PEA and the
reduction of lactose by K. marxianus from whey from the
cheese industry.
Materials and Methods
Cheese Whey (SW, AW and CW) Characterization
and Preparation
Three types of whey preparation were used; sweet whey (SW),
acid (AW) and curd cheese (CW), provided by the cheese
industry PROUNILAC in the Hidalgo State, Mexico. Whey
was taken directly from the cheese production line; placed in 4
L polyethylene containers, transported in containers with ice
to the laboratory and stored at 4 °C until use. The pH was
determined in accordance to the norm NMX-F-317-S-1978.
For these analyses, fermented whey samples were centrifuged
at 6000 rpm/10 min. The ability of K. marxianus to assimilate
lactose depends on the presence of two genes; LAC12P and
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Waste Biomass Valor (2017) 8:1343–1350
LAC4. LAC12P has an optimal pH for lactose uptake of *4.7
[27]. In order to use the three types of whey (SW, AW and
CW) in the study, their pH was adjusted to 4.8 using 1 N
H2SO4, and then pasteurized at 63 °C for 30 min. Several
flasks were prepared with 100 mL of each type of whey. Also,
COD was determined according to the APHA standard
methods [28]. For these analyses, fermented whey samples
were centrifuged at 6000 rpm/10 min.
The protein content was determined according to the
Kjeldahl method, of the AOAC 930.52. 25 mL of whey
was settled in digestion (250 mL) tubes, with 15 mL of
H2SO4 (98 %) this took place in a block of digestion
(420 °C/90 min). Subsequently, 70 mL of water and
10 mL of NaOH (40 % w/w) were added. Released NH3
was distilled into a solution of boric acid (4 % w/v), and
titrated with HCl (0.1 M), to a colorimetric endpoint, with
methylene 1:1 red indicator solution.
Biological Material
The microorganism utilized was K. marxianus ITD00262
[29]. It was preserved in agar plates with standard methods
(SMA) [standard methods (Bioxon, Mexico)-Agar] at 4 °C
until use.
Phenyl Ethyl Alcohol (PEA)
The strain of K. marxianus was grown in a Petri dish with half
LPY and incubated at 28 °C/24 h (Arsa, Mexico). A sample
of the crop was transferred to an Erlenmeyer flask containing
100 mL of broth lactose-peptone and yeast extract (LPY) at
pH 4.8. The flask was incubated under agitation (120 rpm,
Shaker Thermo Scientific) for 12 h at 28 °C. Subsequently, it
was diluted in peptone (Bioxon, Mexico) in decimal dilutions
to have an initial inoculum of 1 9 106 cells mL-1. This
inoculum was added to flasks containing whey at pH 4.8; the
flasks were incubated in agitation (180 rpm) at 30 °C for
72 h. Every 24 h, aliquots of 1 mL were taken from each flask
and placed in sterile plastic (Eppendorf, Mexico) tubes and
stored at -20 °C until being analyzed by gas chromatography
to detect the production of PEA. All fermentations were
carried out in duplicate. The results presented in this study
were the average of a minimum of two independent
experiments.
Count of Yeasts
Periodically, a yeasts count from each flask was done using
the technique of direct counting to the microscope (Motic,
Canada) using a Neubauer (Marienfeld) Chamber, by the
count in each sample volume of 20 lL. Blue stained cells
were considered to be viable.
Waste Biomass Valor (2017) 8:1343–1350 1345

Quantification of PEA, in Fermented SW, AW
and CW
For detection and quantification of PEA in samples of the
three types of fermented whey, the sterile tubes of plastic
(Eppendorf, Mexico) containing 1 mL of whey samples
were centrifuged at 6000 rpm/3 min. The floating cover was
removed and leaked with membranes of cellulose with a
diameter of pore of 0.5 lm; once filtered, it was directly
analyzed with gas chromatography. We used a Perkin Elmer
gas chromatograph N931-6403 (Norwalk, USA), equipped
with a detector FID, and a DB WAX J&W Scientifics column
(60 m 9 0.25 mm 9 0.25 lm). Injector and detector tem-
peratures were 250 and 300 °C, respectively. The oven
temperature was increased from 35 to 210 °C with a heating
rate of 20 °C/min for 4 min. Nitrogen was used for all
samples as the carrier gas, at a flow rate of 1 mL min-1. The
injected summary was 2 lL; the compounds were identified
and quantified by comparison with patterns. The injection of
the samples was performed in duplicate.

Table 1 Comparative table of

Parameters SW AW CW
results of the kinetics of SW,
AW and CW at pH 4.8, 30 °C/ PEA (g L-1) 0.96 ± 0.008 0.70 ± 0.026 0.47 ± 0.010
180 rpm -1
Ethanol (g L ) 15.59 ± 1.92 45.90 ± 5.73 25.33 ± 2.740
rp (g L-1 h-1) 0.009 ± 0.02 0.007 ± 0.12 0.006 ± 0.07
-rs (g L-1 h-1) 0.52 ± 0.05 0.55 ± 0.21 0.32 ± 0.09
lm (h-1) 0.18 ± 0.03 0.22 ± 0.15 0.15 ± 0.22
tg (h) 1.67 ± 0.25 1.20 ± 0.13 2.02 ± 0.33
Yx/s (g g-1) 0.19 ± 0.09 0.21 ± 0.05 0.17 ± 0.11
yeast (CFU mL-1) 1.13 x109 ± 8.6x107 1.48x109 ± 3.1 x108 1.02x109 ± 9.0 x108
Lactose initial (g L-1) 55.10 ± 1.48 46.36 ± 0.803 50.13 ± 3.40
Lactose residual (g L-1) 1.50 ± 2.38 2.54 ± 0.131 3.94 ± 2.38
CODa initial (mgO2 L-1) 51,833 ± 3790.96 53,500 ± 3663.57 53,444 ± 3663.57
COD final (mgO2 L-1) 16,472 ± 277.31 25,666 ± 759.03 25,666 ± 759.03
Protein final (%) 0.71 ± 0.01 0.57 ± 0.12 0.83 ± 0.08
tg time of generation, Yx/s biomass, rp rate of formation of product performance, -rs substrate consumption
speed
a
Chemical oxygen demand

Fig. 1 Setting the Gompertz

model to the fermentation of
acid whey at pH 4.8, 30 °C/
180 rpm, where a, xc and k are R^2 = 0.98386
μmax=0.22h-1
mathematical parameters that λ = 0.12 h-1
describe the sigmoidal curve tg=1.33 h
Gompertz
Log10 (CFU/mL)

R^2 = 0.99881
μmax=0.25h-1
λ = 1.15 h-1
tg=1.20 h

Richards

R^2 = 0.98575
μmax=0.22h-1
λ = 0.12 h-1
tg=1.33 h
Logistic

Time (h)

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1346 Waste Biomass Valor (2017) 8:1343–1350

Nuclear Magnetic Resonance (NMR) of Lactose (Yx (biomass)/s (lactose)) and the rate of consumption of substrate
for SW, AW and CW (-rs). The rate of formation of product was determined to:
rp ¼ rgYps ð1Þ
The three types of whey samples contained in plastic (Ep-
pendorf, Mexico) sterile tubes were centrifuged at 6000 rpm/ where rg is cell growth rate (g L-1 h-1), Yp/s is product
6 min. Then, they were heated to 60 °C/30 min, to evaporate yield (Yp/s, lactose base) Cell growth rate (rg) was calcu-
excess moisture. The samples were dissolved in 1 mL of heavy lated as:
water (D2O). The NMR spectra were obtained using a nuclear
rg ¼ lCC ð2Þ
magnetic resonance (NMR Varian, 400 MHz) spectrometer.
Being l the specific growth rate (h-1) and Cc the cell
Mathematical Models concentration (g L-1 h-1) substrate consumption rate
(-rs) was calculated as:
The Monod model equations were used to calculate the rate of rs ¼ Yxs rg þ mCc ð3Þ
formation of product (rp), the performance of biomass

doble signal doble signal

(J=3.5 Hz) (J=7.9 Hz)

5.08 5.04 4.5 4.4 4.3

ppm ppm

OH OH
OH H
6 6
OH OH 4 H 4 H
OH H O O
6' 6 5
4' H
O
4 H
O
H O HO
5 OH
HO
2
H 5 O HO
5 H 3
2
OH 1' 3 H OH
1
HO H H H
2 2 H
3 OH
1 3 H OH
1
H H H
OH

α-lactose
β-lactose

5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9
ppm

Fig. 2 Spectrum of NMR 1H at 400 MHz in D2O of the lactose of SW

123
Waste Biomass Valor (2017) 8:1343–1350 1347

doble signal
(J=8.1 Hz)

OH OH 4.40 4.35 4.30 4.25 4.20 4.15 4.10 4.05

OH H (ppm)
6' H 6 H
4' O 4 O
H 5' 5 OH
O HO
HO
3' 2'
OH
1' 3 H2 OH 1
H H H H

β-lactose

4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7
(ppm)

Fig. 3 Spectrum of NMR 1H at 400 MHz in D2O of the lactose of AW

Statistical Analysis
Yx/s is biomass yield (Yx/s, lactose base), rg is cell growth
rate (g L-1 h-1), m is cell maintenance (h-1) and Cc is
The data were analyzed with an analysis of variance of a
cell concentration (g L-1 h-1).Yeast generation time (tg)
single via ANOVA, by comparison of averages by LSD
was calculated following [30] Gompertz (a, xc, k),
(Least Squares Deviation) with a confidence level of 95 %,
Richards (a, xc, d and k) and Logistic (a, xc, k) model
using STATISTICA software version 8.0.
parameters were also calculated to measure maximum
specific growth rate (lm). Experimental data were adjus-
ted independently in Gompertz, Richards and Logistic
mathematical equations by non-linear regression accord- Results and Discussion
ing to [31] Marquardt algorithm. All calculations were
done with a 95 % confidence interval and using the It was observed that K. marxianus was capable of pro-
OriginÒ 8.0 software package. Once values of a, k, and ducing PEA in the three types of whey (SW, AW and CW)
xc, were calculated and applied as solution model, values (Table 1). The maximum concentrations of PEA obtained
A, lm and k were obtained using the corresponding were 0.96, 0.70, and 0.47 g L-1 at 24 h for each type of
equation solution [31]. whey. The rate of formation of product (rp) was slightly

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482
doble signal doble signal
(J=3.5 Hz) (J=7.9 Hz)483

5.09 5.07 5.05 5.03 5.01 4.99

(pmm)
4.50 4.45 4.40 4.35 4.30 4.25
(pmm)

OH OH
OH H
6' 6
OH OH 4' H 4 H
OH H O O
6 6 5'
4' H 4 H H 5
' O O O HO OH
HO
2' 1' 2 1
H 5' O HO 5 H 3' OH 3 H OH
HO H H H H
2' 2 1
3' OH 1' 3 H OH
H H H
OH

β-lactose
α-lactose

5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0
(pmm)

Fig. 4 Spectrum of NMR 1H at 400 MHz in D2O of the lactose of CW

higher for SW (0.009 g L-1 h-1) compared to AW and who reported a maximum reached 5.10 9 108 CFU mL-1
CW fermented (Table 1). These results adjusted with the in diluted whey; which reflects the affinity of the native
model of Monod (R2 0.98) indicate that the formation of strain of K. marxianus of the substrate under study.
the product is associated with exponential growth accom- The specific maximum speed of growth of K.marxianus
panied by the formation of PEA throughout fermentation. (lm) using the adjustment of Richards, Gompertz and
This behaviour has already been described by some authors Logistic models was similar to values ranging from 0.22 to
[14, 32, 33]. However, the concentration consisting of 0.25 h-1 with a correlation coefficient (R2) of 0.98 (Fig. 1),
ethanol in SW (15.59 g L-1), AW (45.90 g L-1) and CW in the fermented where the maximum biomass concentra-
(25.33 g L-1), indicates that this was not used as a source tion was obtained. These values are slightly below those
of carbon for the path that surrounds the glyoxylate, reported by Banat et al. [35], who obtained a 0.29 h-1.
favouring the production of PEA as it has been previously These differences could be due to the complexity of the
reported [18]. This explains in some way the concentra- substrate used.
tions obtained from PEA. In addition, in the present study, On the other hand, for this type of whey (AW), the time
substrates were worked in crude. of generation (tg) was 1.20 h, this result is consistent with
Regarding the growth, it is observed a maximum what was reported for K. marxianus in nutrient media
increase in the concentration of K. marxianus of [36–39]. However, the retrieved growth, determined by the
1.13 9 109, 1.48 9 109 and 1.02 9 109 CFU mL-1 for generation time (tg 1.20 h) is faster than that reported by
SW, AW and CW, respectively. These cell count results, Eun and Chang [39], who obtained a tg of 1.65 h in
are higher than what it was reported by Dragone et al. [34] deproteinized whey.

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The content of protein in each type of whey (SW, AW
and CW) is similar to that reported by Araujo et al. [40]
(Table 1). Regarding the efficiency of chemical oxygen
demand removal, it was 68, 52 and 50 % ([50 g L-1
lactose initial) for SW, AW and CW, respectively. These
results are superior to those reported by Aktas et al. [1],
who achieved a 70 % removal in a medium with only
10 g L-1of lactose.
Regarding the rate of consumption of substrate (-rs) for
SW and the fermented concentrations were similar with
0.52–0.55 g L-1 h-1, respectively. These results are
reflected in the production of PEA, not in the CW
(0.47 g L-1). This indicates the formation of PEA through
the catabolic pathway involving sugars. However, the top
count of yeast concentration for AW could have influenced
the production of the metabolite.
In addition, the formation of lactose present in each
whey observed in the NMR spectra of 1H for the SW and
AW (Figs. 2, 3) presented among its most significant sig-
nals, two double signals (J = 7.9 Hz) in d 4.49 and
4.28 ppm, properties of anomeric protons, assigned to H-1
and H-10 , corresponding to b-lactose. Among d 3.8 and
3.0 ppm, there was the rest of the signals present in the
structure. Additionally, in d 5.06 ppm, there was a double
signal (J = 3.5 Hz), which could correspond to a-lactose
anomeric proton. However for the spectrum of NMR 1H
(Fig. 4) presented among its most significant signals, two
double signals (J = 8.1 Hz) in d 4.36 and 4.15 ppm,
properties of anomeric protons, assigned to H-1 and H-10 ,
corresponding to b-lactose. Among d 3. 7 and 2.9 ppm
were the rest of the signals present in the structure.
These signals were able to determine that the SW,
showed a higher content of b-lactose (82.35 %) versus AW
(72.51 %) and CW (65.25 %). These results could explain
the consumption of b-lactose by K. marxianus that triggers
in the increase of the production of PEA in this type of
whey. In addition, other nutrients such as easily assimilable
nitrogen (EAN) could have influenced the formation of
PEA. It has been reported that a source of nitrogen for the
production of PEA is necessary [25, 41, 42].
Conclusions
K. marxianus was capable of producing PEA from the
waste of the cheese industry, presented as SW, AW and
CW. However, SW was the one that presented a potential
for the production of PEA. SW, showed a higher content of
b-lactose (82.35 %) versus AW (72.51 %) and CW
(65.25 %). This study could represent an alternative use to
this type of disposal and thereby reduce their environ-
mental impact.
1349
Acknowledgments Thanks to the National Council for Science and
Technology (CONACYT) for the scholarship given (263643) and the
producer of dairy PROUNILAC for the support access to biological
samples.
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