Animal Feed Science and Technology 225 (2017) 173181

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Animal Feed Science and Technology

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

Optimization of key aroma compounds for dog food

attractant
Maoshen Chen, Xuemei Chen, John Nsor-Atindana, Kingsley George Masamba,
Jianguo Ma, Fang Zhong
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue,
214122, Wuxi, Jiangsu Province, China

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

Article history: Although several investigations devoted to dog food attractants (DFAs) have been con-
Received 9 May 2016 ducted, these studies fell short in identifying the key aroma compounds that could improve
Received in revised form 5 December 2016 the palatability performance of pet foods. The aim of this study was to identify the key aroma
Accepted 11 December 2016
compounds in the DFA using headspace-solid phase micro-extraction, gas chromatography-
mass spectrometry and Partial Least Squares Regression (PLSR) analysis. Seven DFAs with
Keywords: different aroma compounds obtained by Maillard reaction were employed in the study. A
Dog food attractant
total of 53 aromatic compounds were identied categorized as follows: 7 alcohols, 11 alde-
Volatile aroma compounds
hydes, 4 ketones, 4 organic acids, 7 esters, 2 ethers, 10 heterocycle compounds, 4 phenols,
Palatability
Plsr 2 terpenes and 2 polycyclic aromatic hydrocarbons. Eight adult beagle dogs were used for
the preference test (two-pan test) and acceptance test (one-pan test) to rank these seven
DFAs. PLSR was performed to correlate the instrumental data with the preference results.
Twenty-three aroma compounds of hexanoic acid, acetaldehyde, heptanone, butyl hex-
anoate, heptyl formate, methyl pyrazine, 2,5-dimethyl pyrazine, 2-heptanone, pentanal,
ethyl decanoate, heptanal, octanal, pentanol, acetone, ethyl caprylate, 3-methyl butanal,
anisole, 2-ethyl hexanol, 2-pentyl furan, 2,3-butanediol, benzaldehyde, ethyl vanillin and
vanillin correlated with the preference data. Benzaldehyde, vanillin and 2, 5-dimethyl
pyrazine were added to dry dog foods in order to validate the PLSR results. The presence
of three aroma compounds signicantly increased the preferences of the samples (dry dog
foods). The validation study involving the three compounds suggests that the 23 compounds
selected by the PLSR model are aroma compounds which inuence palatability. Therefore
the manipulation of these avor compounds could improve the palatability of dog foods.
2016 Elsevier B.V. All rights reserved.

1. Introduction

Typically, dog foods are categorized based on their moisture: wet, semi-dry and dry. Apart from its highest nutritional
content on a weight basis, dry dog foods not only cost the least to package, but also are the most convenient as far as
handling and storage are concerned (Gierhart and Hogan, 1993). These associated advantages make dry dog food attractive
and the most preferred among most pet owners (Li et al., 2012). However, dry dog foods are least palatable among the three
categories (Gierhart and Hogan, 1993). When an unpalatable food is offered, a dog may pass it up and thereby not take

Corresponding author.
E-mail address: fzhong@jiangnan.edu.cn (F. Zhong).

http://dx.doi.org/10.1016/j.anifeedsci.2016.12.005
0377-8401/ 2016 Elsevier B.V. All rights reserved.
174 M. Chen et al. / Animal Feed Science and Technology 225 (2017) 173181

advantage of its nutritional value (Boudreau and White, 1981). In order to improve the palatability of dry dog food, dog food
attractants (DFAs) have been developed and incorporated into a number of dry dog foods to enhance the avor and other
organoleptic properties, and consequently improve acceptability (Gierhart and Hogan, 1993; Chen and Trivedi, 2005; Shi
et al., 2007).
The importance of the Maillard reaction in avor chemistry has long been recognized as evidenced by a number of studies
for reaction product avorants of DFA. (Lee and Tandy, 1991). For example, Lugay et al. (1978) found that the palatability of
the animal foods could be increased by incorporating an effective palatability enhancer formulated by reacting an aqueous
mixture of a reducing sugar, animal blood, yeast and fat with lipase and protease at moderate temperature and pH, and
subsequently heating the resulting mixture to an elevated temperature to fully develop the avor. Similarly, palatability
enhancer produced by heating reducing sugar and sulfur-containing compound at temperature about 90130 C and pH
between 6 and 9 could signicantly improve the palatable performance of pet foods (Shi et al., 2007). Nevertheless, the
literature survey so far revealed that no publications are available committed to the analysis and identication of the key
aroma compounds in the attractants responsible the palatability performance. However, the identication of the key aroma
compounds is a prerequisite for understanding the palatability and preference of DFA. Moreover, such information could be
relevant for manufacturers of DFA during formulations.
The aim of this study was to identify the key avor compounds of the DFA that play major role in avor enhancement
of dry dog foods. By employing headspace-solid phase micro-extraction and GCMS techniques, the volatile compounds
of seven DFAs were successfully determined. Additionally, the preference ranking for seven DFAs was obtained through
preference tests. Finally, the preference data was correlated to the volatile compounds using partial least square regression
(PLSR). In order to validate the PLSR results, the preferences of dog foods with the addition of several key avor compounds
were tested.

2. Materials and methods

2.1. Materials

Seven DFAs (DFA1, DFA2, DFA3, DFA4, DFA5, DFA6 and DFA7) with different aroma compounds were obtained by Maillard
reaction. Brewers yeast, chicken meal and soybean meal were used as protein source and xylose was used as reducing sugar.
The pH of Maillard reaction was from 6 to 9 and the temperatures were in the range of 100140 C. The ingredients of
the basal dog food contained rice, maize, sh meal, soybean meal, wheat, brewers yeast, soybean oil, dicalcium phosphate,
sodium chloride, vitamins, minerals, potassium sorbate, etc (Chen et al., 2016). The basal dog food contained all nutrients
in amounts sufcient to meet the requirement of an adult dog as described in the Association of American Feed Control
Ofcials (AAFCO, 1997). The basal dry dog food was produced by extrusion using a twin-screw food extruder (Jinan Saixin
Machinery Co., Ltd). Seven dry dog foods (DF1, DF2, DF3, DF4, DF5, DF6 and DF7) were nally obtained by uniformly spraying
seven DFAs onto the basal dog food.

2.2. Extraction of volatile aroma compounds

Headspace-solid phase microextraction (HS-SPME) was used to determine the volatile components in the seven DFAs.
Each sample was put in a 15 mL screw-cap vial equipped with a polytetrauoroethylene/silicone septum. The internal
standard was 0.01 mL cyclohexanone dissolved in distilled water, with nal concentration in the sample to be 1.3 g g1 .
A 75 m carboxen/polydimethyl-siloxane ber (ANPEL Laboratory Technologies Inc., Shanghai, China) was exposed to the
sample headspace for 30 min at 50 C. The ber was chosen for its high capacity of trapping compounds in food products.
After sampling, the volatiles were desorbed from the SPME ber coating to the injection port of the gas chromatograph (GC)
at 230 C for 7 min in splitless mode.

2.3. Chromatographic analyses

The isolation, tentative identication, and semi-quantication of the volatile compounds were performed on a gas
chromatograph (Trace GC Ultra, Thermo Fisher Scientic, USA), coupled with a Varian mass spectrometer (MS) detec-
tor (TSQ Quantum Ultra EMR, Thermo Fisher Scientic, USA). The GCMS system was equipped with a TG-WAXMS
(30 m 0.53 mm 0.25 m) column (Thermo Fisher Scientic, USA). The initial temperature of the column was 40 C and
was held at that temperature for 2 min. The temperature was then increased by 4 C per min to 230 C, and held at that
temperature for 5 min. All samples were analyzed in triplicate. Tentative identication of compounds was conducted by
matching the mass spectra of unknowns with those in the mass library. The retention times for C6 -C30 n-alkanes standards
(Supelco Analytical, Bellefonte, PA, USA) were determined. Experimental retention indices (RIexp. ) were calculated accord-
ing to Ven Den Dool and Kratz (Van den Dool and Kratz, 1963) and compared to literature values (RIlit. ) for identication
purposes.
 M. Chen et al. / Animal Feed Science and Technology 225 (2017) 173181 175

2.4. Determination of palatability

Eight adult beagle dogs (four males and four females) with weight ranging from 7.5 to 15.7 kg were used. Dogs were indi-
vidually housed in pens measuring approximately 1.2 2.5 m indoors at Laboratory Animal Center in Nantong University,
Nantong, Jiangsu, China. The procedures of this study were carried out in accordance with the European Community guide-
lines (Directive 2010/63/EU) for the care and use of experimental animals. An acceptance test (one-pan test) and preference
tests (two-pan test and free-choice test) were performed. For the acceptance test, only one diet was placed in front of each
dog. The dog was offered 500 g each samples for 24 h daily for 5 days. The intake ratio (IR) of each food was calculated by
dividing the grams consumed of the diet by the total grams provided (500 g). For the preference tests, two different diets,
sample A and sample B, were placed in front of each dog. All dogs were allowed free access to water. Dogs were offered
500 g each of sample A and sample B in separate bowls for 24 h daily for 5 days. The placement of the bowls was alternated
each day to eliminate any bowl placement bias by the dogs. First-choice and rst-approach data were collected. At the end
of the hour, any refused food was weighed to determine food intake of each diet. The consume ratio (CR) was calculated
by dividing the weight consumed one of each diet by the total weight consumed of both diets. Data were analyzed using
IBM SPSS Statistics Version 19.0 software using t-Tukey test. A probability of p < 0.05 was considered statistically signicant
(Dust et al., 2005).

2.5. Data analysis

Partial least square regression (PLSR) was used to create external preference maps for determining the relationships
between instrumental volatile data (X-matrix) and acceptability data (Y-matrix). This procedure was carried out using
SIMCA-P 11.5 Demo (Umetrics AB; USA). This analysis has been described in detail by Song et al. (Song et al., 2010).

2.6. Validation experiment

Several avor compounds identied by the PLSR model were chosen and added to dog food for palatability tests. The
amount of avor compounds added was based on the difference between the maximum and minimum values among the 7
DFAs.

3. Results

3.1. The volatile aromatic composition of the seven dog food attractants

The aroma compounds of the 7 DFAs are presented in Table 1. A total of 53 aromatic compounds were tentatively identied
in the 7 DFAs. These aromatic compounds were grouped as follows: alcohols (seven compounds), aldehydes (eleven com-
pounds), ketones (four compounds), organic acids (four compounds), esters (seven compounds), ethers (two compounds),
heterocycle compounds (ten compounds), phenols (four compounds), terpenes (two compounds) and polycyclic aromatic
hydrocarbons (two compounds).
Alcohols were derived mainly from the oxidative decomposition of fat (Wettasinghe et al., 2001). Quantitatively, 1,2-
propanediol was the most abundant alcohol in both sample DFA1 and sample DFA2 followed by 4-butylbenzyl alcohol.
Total alcohols of sample DFA1 and sample DFA2 reached 23.29 g g1 and 16.77 g g1 , respectively. For sample DFA5, only
4.43 g g1 was recorded as total alcohols. In the case of sample DFA3, alcohols were conspicuously undetected. Although
the amounts of alcohols detected in DFA4 and DFA6 were relatively small, the two samples had higher preference values.
In this study, the abundant aldehydes were hexanal, nanonal and benzaldehyde, and their contents were more than
1 g g1 in some samples. Heptanal and octanal, which are common lipid oxidation products, were also found in most
samples. However, ethyl vanillin and vanillin were only found in sample DFA4. These two volatile compounds are common
avoring substances. Vanillin is the major component of natural vanilla and is one of the most widely used and important
avoring compounds worldwide (Walton et al., 2003).
Four organic acids at high concentrations in most samples were detected in this study. DFA2 had the highest organic
acid content (19.73 g g1 ), while DFA4 had the lowest amount (4.27 g g1 ) Acetic acid has a strong irritating smell and is
known for its important avoring role on DFAs.
Ten heterocycle compounds were detected in this study, including 3 furans, 2 pyrans, 3 pyrazines, 1 pyrimidine and 1
pyrrole. Furfural, which is an oxygenated furan (Koppel et al., 2013) was detected in four samples. Although 3 pyrazine
compounds were detected in the study, the highest amount was only 0.44 g g1 . Ethyl maltol was a common avoring
substance and was detected in six samples. Its content in sample DFA4 reached 15.51 g g1 , however, it was not detected
in sample DFA5.

3.2. Palatability tests

The measure of palatability is most important in the pet food industry since palatability largely determines the price of
pet food and is a critical element in the manufacture of pet food. Because animals are unable to declare preferences directly,
176 M. Chen et al. / Animal Feed Science and Technology 225 (2017) 173181

Table 1
The aroma compounds in 7 dog food attractants (g g1 ).

Compound RI(Exp. ) RI(Lit. ) DFA1 DFA2 DFA3 DFA4 DFA5 DFA6 DFA7
Alcohols

A1 ethanol 928 934 0.18 n.d. n.d. 0.24 0.19 0.17 0.52
A2 pentanol 1239 0.14 0.23 n.d. 0.32 1.01 0.35 n.d.
A3 2-ethyl hexanol 1486 1488 0.15 0.53 n.d. 0.76 n.d. 0.28 0.14
A4 1-octen-3-ol 1455 1460 n.d. 0.52 n.d. 0.41 1.15 n.d. 0.23
A5 (S)-1,2-propanediol 1590 13.03 8.95 n.d. n.d. n.d. n.d. n.d.
A6 2,3-butanediol 1532 1542 n.d. n.d. n.d. 0.11 0.23 n.d. n.d.
A7 4-butylbenzyl alcohol 1915 1920 9.79 6.54 n.d. n.d. 1.85 n.d. n.d.

Aldehydes
B1 acetaldehyde 714 n.d. n.d. n.d. 0.14 n.d. 0.14 n.d.
B2 pentanal 967 968 0.16 0.18 0.21 0.38 0.62 0.37 0.25
B3 3-methyl butanal 913 911 n.d. 0.15 0.14 0.17 0.65 0.27 n.d.
B4 hexanal 1080 1077 1.03 1.78 1.26 1.83 0.29 1.55 1.45
B5 heptanal 1176 1174 0.18 0.34 n.d. 0.40 0.56 0.41 0.38
B6 octanal 1279 1,282 0.25 0.52 n.d. 0.44 0.37 0.45 0.35
B7 nonanal 1388 1390 2.52 2.96 n.d. 1.21 1.42 1.01 3.20
B8 trans-2,4-decadienal 1819 1820 0.12 0.13 0.16 n.d. n.d. 0.08 n.d.
B9 benzaldehyde 1525 1530 0.89 1.05 0.46 0.89 2.29 1.27 1.01
B10 ethyl vanillin 2517 2514 n.d. n.d. n.d. 3.77 n.d. n.d. n.d.
B11 vanillin 2548 2560 n.d. n.d. n.d. 8.90 n.d. n.d. n.d.

Ketones
C1 acetone 809 n.d. n.d. n.d. 0.12 0.39 0.25 0.11
C2 heptanone 1176 n.d. n.d. n.d. 0.24 0.31 0.24 n.d.
C3 2-heptanone 1172 1172 n.d. 0.13 n.d. n.d. 0.31 0.24 n.d.
C4 2-piperidone 2054 2060 0.21 n.d. 0.10 n.d. 0.36 0.17 0.14

Organic acids
D1 acetic acid 1474 1488 3.25 10.31 3.94 1.15 5.88 3.85 2.72
D2 propanoic acid 1560 1574 1.38 4.49 2.33 0.29 1.57 1.96 2.14
D3 butyric acid 1628 1637 1.37 3.48 0.88 0.36 2.03 1.03 1.03
D4 hexanoic acid 1854 1851 0.73 1.45 0.33 2.47 1.96 3.13 1.23

Esters
E1 ethyl acetate 891 0.40 n.d. 0.08 0.87 n.d. n.d. n.d.
E2 methyl acetate 827 n.d. 0.79 n.d. 0.70 n.d. 0.48 0.49
E3 heptyl formate 1366 1357 n.d. n.d. n.d. n.d. n.d. 0.41 n.d.
E4 butyl hexanoate 1407 1404 n.d. n.d. n.d. n.d. n.d. 0.32 n.d.
E5 ethyl caprylate 1450 1463 n.d. n.d. n.d. 0.51 n.d. 0.32 0.18
E6 butyrolactone 1624 1617 0.33 0.28 0.12 0.17 0.25 0.23 0.30
E7 ethyl decanoate 1641 1648 n.d. 0.22 0.06 0.80 0.14 0.31 0.18

Ethers
F1 4-hydroxyl-3-tert-butyl-benzaldehyde 2498 1.28 1.98 0.29 3.09 1.46 0.46 0.52
F2 anisole 1811 1817 n.d. n.d. n.d. 0.39 n.d. 0.18 0.12

Heterocycle compounds
G1 methyl pyrazine 1264 1264 n.d. 0.15 n.d. 0.20 0.29 0.15 0.11
G2 2-pentyl furan 1239 1249 n.d. 0.32 n.d. 0.32 0.89 0.37 0.32
G3 2,5-dimethyl pyrazine 1320 1323 n.d. n.d. n.d. n.d. 0.21 0.18 n.d.
G4 2,6- dimethyl pyrazine 1326 1328 n.d. n.d. n.d. 0.44 n.d. 0.09 0.20
G5 2,4-dimerhyl pyrimidine 1438 n.d. 0.37 n.d. n.d. n.d. n.d. n.d.
G6 furfural 1464 1456 n.d. 0.27 n.d. 0.16 0.10 n.d. 0.15
G7 furfuralcohol 1665 1669 0.88 1.24 0.32 0.58 0.64 0.49 0.46
G8 ethyl maltol 2049 2050 0.28 0.23 0.14 15.51 n.d. 0.17 0.14
G9 2-pyrrole cauboxaldehyde 2032 2030 n.d. 0.25 n.d. n.d. 0.10 n.d. 0.13
G10 4,5,7-trimethoxy-3-(4-methoxy phenyl)-1-benzopyran-2-one 1380 2.06 2.06 0.41 1.20 1.09 0.99 0.55

Phenols
H1 phenol 2006 2006 n.d. 0.23 n.d. n.d. 0.12 n.d. 0.12
H2 p-methoxyphenol 1844 0.14 n.d. 0.32 n.d. 0.23 n.d. 0.44
H3 butylated hydroxytoluene 1903 1910 0.33 n.d. n.d. n.d. n.d. 3.78 4.81
H4 Tert-butylhydroquinone 2899 n.d. n.d. n.d. n.d. 3.83 4.11 n.d.

Terpenes
I1 limonene 1197 n.d. n.d. 0.06 0.13 n.d. n.d. 0.09
I2 styrene 1263 1255 n.d. n.d. n.d. 0.16 n.d. n.d. 0.13

Polycyclic aromatic hydrocarbons

J1 naphthalene 1595 1791 0.14 0.12 0.05 0.12 n.d. 0.09 0.12
J2 2,3-diphenyl quinoxaline 1535 n.d. n.d. 0.85 n.d. n.d. 0.36 1.41

n.d.: not detected. RI: Retention index. All samples were tested in triplicate.
 M. Chen et al. / Animal Feed Science and Technology 225 (2017) 173181 177

Table 2
The preference test results of dry dog foods in the high level, medium level and low level (n = 8).

Sample First-approach CR (%)

High DF6 34 61.9 3.7a

level DF4 6 38.1 2.6b
Medium DF5 37 71.7 3.4a
level DF2 3 28.3 2.5b
Low DF1 28 56.8 3.1a
level DF3 12 43.2 2.4b

CR: Consume ratio. Different letters in the same level indicate signicant difference (p < 0.05).

Table 3
The preference test results of dog food between two different levels (n = 8).

Sample First-approach CR (%)

First DF4 35 63.1 3.8a

group DF5 5 36.9 2.5b
Second DF2 33 61.2 3.7a
group DF1 7 38.8 2.6b
Third DF3 36 74.5 3.5a
group DF7 4 25.5 2.3b

CR: Consume ratio. Different letters in the same group indicate signicant difference (p < 0.05).

palatability assessment is based on the preference ranking of two or more foods (Araujo and Milgram, 2004). Palatability is
often determined by using palatability preference test (two-pan method) or acceptance test (one-pan test) (Grifn, 2003).
The most common method of assessing palatability in dogs is the two-pan test, which involves comparing the consumption
of two different foods (Grifn et al., 1984).
The seven dry dog foods were rst classied by means of acceptance test (one-pan test). The intake ratios (IR) of the seven
basal dry dog foods coated with 7 DFAs were calculated. The IR of DF4 and DF6 were 73.5 and 74.6 respectively, indicating
that dogs consumed most of samples DF4 and DF6. In the cases of samples DF5 (68.8) and DF2 (66.0), the consumptions
were signicantly higher than those of DF1 (53.5) and DF3 (51.6). Diet DF7 (43.8) was the least consumed among all the
foods. Thus, from the results of IR, all the dry dog foods except for DF7 were roughly classied into three levels: high level
(DF6 and DF4), medium level (DF5 and DF2) and low level (DF1 and DF3).
The preference test (two-pan test) was used to rank the foods in each level. The results of preference test in the same level
are presented in Table 2. According to the results, DF6 was better than DF4, while DF5 was better than DF2, and DF1 was
better than DF3. Finally, the sample with the lower preference in the higher-level was compared with the higher preference
in the lower-level. Thus, all the dry dog foods except for DF6 were classied into three groups: rst group (DF4 and DF5),
second group (DF2 and DF1) and third group (DF3 and DF7). The results are shown in Table 3. The results have shown that
the sample DF4 was better than DF5, the sample DF2 was better than DF1, and the sample DF3 was better than DF7. Thus,
the seven DFAs could be ranked based on palatability values. In order to analyze the relationship between palatability and
aromatic compounds, the palatability ranking was assigned a value of 17 with 7 as the most palatable and 1 as the least.
The palatability values of DFA7, DFA3, DFA1, DFA2, DFA5, DFA4 and DFA6 were 1, 2, 3, 4, 5, 6 and 7, respectively.

3.3. Partial least squares regression analysis

The PLSR model with two latent variables (PC1 and PC2) was employed to predict the palatability of DFAs with high
degree of accuracy based on the contents of volatile compounds determined by GCMS (R2 = 0.98), as shown in Fig. 1. The
PLSR correlation loadings (PC1 vs. PC2) on the perceptions of DFAs attributes are presented in Fig. 2. The palatability value
(p.v.) was placed between the ellipses of 50% and 100%, particularly close to 100%, indicating that it was well explained by
the PLSR model (Martens and Martens, 2000; Song et al., 2010). It could be noticed from the variance in PC1 that palatability
value variables of DFA4, DFA6 and DFA5 were located on the right side. The compounds in the left anchors were implicit as
their negative correlations (Song et al., 2010), such as H2, J2, A5 and B8.
The inuences of aroma compounds on the palatability value are shown in Fig. 3. The 22 compounds, p-methoxyphenol
(H2), hexanoic acid (D4), acetaldehyde (B1), heptanone (C2), butyl hexanoate (E4), 2,3-diphenyl quinoxaline (J2), heptyl
formate (E3), methyl pyrazine (G1), 2,5-dimethyl pyrazine (G3), 2-heptanone (C3), pentanal (B2), ethyl decanoate (E7),
heptanal (B5), octanal (B6), pentanol (A2), acetone (C1), ethyl caprylate (E5), 3-methyl butanal (B3), anisole (F2), 2-ethyl
hexanol (A3) and 2-pentyl furan (G2) had VIP (variable importance for the projection) > 1. Eight aroma compounds of ethanol
(A1), trans-2,4-decadienal (B8), 2,3-butanediol (A6), 4-hydroxyl-3-tert-butyl-benzaldehyde (F1), benzaldehyde (B9), ethyl
vanillin (B10), vanillin (B11) and ethyl maltol (G8) had VIP between 0.8 and 1.0.
178 M. Chen et al. / Animal Feed Science and Technology 225 (2017) 173181

Fig. 1. Relationship between observed and estimated scores based on PLSR model.

Fig. 2. Correlation loading plot of the PLSR model between palatability value (p.v.) and volatile compounds. Ellipses represent r2 = 0.25, 0.50 and 1.00,
respectively.

3.4. Validation experiment

The amount of attractants added in each dry dog food was based on the difference between the maximum and minimum
values among the 7 DFAs. Thus, the amounts of benzaldehyde, vanillin and 2, 5-dimethyl pyrazine added to the DFA were
1.82 g g1 , 8.90 g g1 and 0.21 g g1 , respectively. The effects of benzaldehyde, vanillin and 2,5-dimethyl pyrazine on the
preference of dry dog foods are presented in Table 4. The increase in the palatability of all the dry dog foods was signicant
 M. Chen et al. / Animal Feed Science and Technology 225 (2017) 173181 179

Fig. 3. The inuence of aroma compounds on palatability value.

Table 4
The preference tests of dog food without and with the addition of the three aroma compounds (n = 8).

benzaldehyde 2,5-dimethyl pyrazine vanillin

control treatment control treatment control treatment

rst-approach 5 27 3 29 2 30
CR (%) 34.2 4.5b 65.8 5.2a 23.6 3.1b 76.4 6.5a 20.8 4.6b 79.2 5.5a

CR: Consume ratio. Different letters indicate signicant difference (p < 0.05).
Control: without the addition of the aroma compound.
Treatment: with the addition of the aroma compound.

upon the addition of these three aroma compounds. The consumption ratios (CR) of samples treated by 2, 5-dimethyl pyrazine
and vanillin were greater than 75%.

4. Discussion

Since olfactory sense is known to play an important role in dogs avor perception, the determination of aromatic
composition of the seven DFAs was inevitable as far as the understanding the functional effects of the attractants were
concerned. Food avor volatiles are often extracted from the headspace and analyzed using SPME-GCMS (Brunner, 2001).
Apart from its relatively low cost and simplicity of extraction, solid-phase microextraction (SPME) is also a rapid solvent-
free extraction technique, usually combined with GCMS for easy data analysis (Snchez-Palomo et al., 2005). The avor
compounds adsorbed on the SPME coating are greatly inuenced by the diffusion of the compounds through the sample into
the headspace. Dogs also perceive avor molecules in their food in a similar mechanism by which the volatiles are adsorbed
onto the SPME.
Alcohols were generally formed by the decomposition of the secondary hydroperoxides of fatty acids (Grosch, 1982)
characterized by green, fruity oral aroma (Yang et al., 2007; Liu et al., 2012). Aldehydes are also one of the most common
products resulting from lipid/fat oxidation reactions. These compounds could be used as an indicator of oxidized avor (Li
et al., 2012). In a recent study, aldehydes were shown to be the most abundant group of volatiles in grain products (Liu
et al., 2012). Since aldehydes generally have a low odor threshold value, their presence in cereal products could have a
considerable impact on the aroma (Koppel et al., 2013). Hexanal and nonanal are well known contributors of green and rose
odor notes, respectively in foods. Benzaldehyde is commonly known as almond oil. Benzaldehyde and hexanal have been
reported to be the most prevalent aldehydes in dry dog foods in a recent study (Koppel et al., 2013). These aldehydes have
also been reported in other foods, such as fruits (Jordan et al., 2002; Qiao et al., 2008), meat (Wettasinghe et al., 2001) and
dairy products (Li et al., 2012).
Esters are thought to have a little impact on uncured meat aroma. They are generally associated with fruity aroma
as well as cured pork avor (Wettasinghe et al., 2001). Furfural is formed by the Maillard reaction and has caramel-like
characteristic fruity avor. Furfuralcohol was detected in all the samples and is known to play an important role in the
180 M. Chen et al. / Animal Feed Science and Technology 225 (2017) 173181

avor of dry dog foods. Pyrazine compounds are often characterized by nutty and roasted aromatics (Piggoit and Paterson,
1994). Although two hydrocarbon compounds were detected in this study, the contribution of hydrocarbons to aroma is
insignicant (Vejaphan et al., 1988; Chung and Cadwallader, 1993).
Data analysis methods are able to provide both cognitive relationships and statistical tools. The soft multivariate bilinear
modeling method of PLSR combined with interactive computer graphics produces relevant and reliable information from
the data. Statistical assessment of the reliability of the results can be determined by cross-validation or jackkning a subset
of data (Wettasinghe et al., 2001). In this study, PLSR was used to analyze the relationship between volatile compounds
found by GC/MS and palatability value of seven DFAs. The X variable was designated as 53 volatiles and the Y variable was
designated as palatability value. The PLSR model included two signicant PCs explaining 98% of the cross-validate variance.
PC1 could reproduce 82% and PC2 reproduced only 16%. In Fig. 2, the big ellipses indicated 25%, 50% and 100% explained
variance, respectively. The palatability value (p.v.) was placed between the ellipses of 50% and 100%, particularly close to
100%, indicating that it was well explained by the PLSR model (Song et al., 2010).
The VIP above 1 indicates a very important role in palatability value. As shown in Fig. 2, the compounds of H2 and
J2 were in the left anchors and were implicit of their negative correlations. Thus, hexanoic acid (D4), acetaldehyde (B1),
heptanone (C2), butyl hexanoate (E4), heptyl formate (E3), methyl pyrazine (G1), 2,5-dimethyl pyrazine (G3), 2-heptanone
(C3), pentanal (B2), ethyl decanoate (E7), heptanal (B5), octanal (B6), pentanol (A2), acetone (C1), ethyl caprylate (E5), 3-
methyl butanal (B3), anisole (F2), 2-ethyl hexanol (A3) and 2-pentyl furan (G2) had signicant effects on palatability. Eight
aroma compounds had VIP between 0.8 and 1.0. The concentrations of 2,3-butanediol (A6), benzaldehyde (B9), ethyl vanillin
(B10) and vanillin (B11) in samples of DFA4, DFA6 and DFA5 were much higher than in the other four samples. Therefore,
these four compounds appeared to have signicant inuence on the palatability of DFAs.
The key compounds that are important to the palatability of DFAs were identied through PLSR analysis. The avor
thresholds were not determined and it was not possible to validate the impact of all of them independently. Benzaldehyde,
vanillin and 2,5-dimethyl pyrazine were chosen for palatability tests based on the availability of the concentration data to
validate the PLSR results. The improved CR scores showed that these three compounds had signicant effects on the prefer-
ence of dry dog food. This validation study suggests that the compounds selected by the PLSR model are avor compounds
important for palatability manipulation dry dog foods.

5. Conclusion

In this study, the volatile aroma compounds of seven self-made DFAs were extracted by HS-SPME and analyzed by
GCMS. A total of 53 aromatic compounds were identied, including 7 alcohols, 11 aldehydes, 4 ketones, 4 organic acids,
7 esters, 2 ethers, 10 heterocycle compounds, 4 phenols, 2 terpenes and 2 polycyclic aromatic hydrocarbons. The pref-
erence of these DFAs was investigated by preference tests (two-pan method) and acceptance tests (one-pan test). Using
the Partial Least Square regression analysis, the relationship between volatile aroma compounds and palatability results
was established. Twenty-three aroma compounds of hexanoic acid, acetaldehyde, heptanone, butyl hexanoate, heptyl for-
mate, methyl pyrazine, 2,5-dimethyl pyrazine, 2-heptanone, pentanal, ethyl decanoate, heptanal, octanal, pentanol, acetone,
ethyl caprylate, 3-methyl butanal, anisole, 2-ethyl hexanol, 2-pentyl furan, 2,3-butanediol, benzaldehyde, ethyl vanillin and
vanillin were related to the palatability of dry dog foods. Three aroma compounds (benzaldehyde, vanillin and 2, 5-dimethyl
pyrazine) were selected and added to dry dog food to validate the PLSR results. The addition of these three compounds
positively impacted on the avor of the dry dog foods and their presence signicantly increased the palatability of the all
the samples. This validation study suggests that the compounds selected by the PLSR model are avor compounds necessary
and sufcient for palatability development and that the manipulation of these compounds can signicantly improve the
palatability of dry dog foods.

Acknowledgment

This project was supported by the Self-determined Research Program of Jiangnan University (JUSRP 115A22) and by
program of Collaborative innovation center of food safety and quality control in Jiangsu Province.

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