Food Chemistry: X 19 (2023) 100790

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Food Chemistry: X
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Exploring the growth characteristics of Alicyclobacillus acidoterrestris for

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

Keywords: Fruit juice spoilage that caused by contaminated Alicyclobacillus has brought huge losses to beverage industry
Alicyclobacillus acidoterrestris worldwide. Thus, it is very essential to understand the growth and metabolism processing of Alicyclobacillus
Controlling juice spoilage acidoterrestris (A. acidoterrestris) in controlling juice spoilage caused by Alicyclobacillus. In this work, simulative
Vanillin
models for the growth and metabolism of A. acidoterrestris were systematically conducted in the medium and
Guaiacol
Volatile components
fruit juice. The results showed that low temperature (4 ℃) and strong acidic environment (pH 3.0–2.0) of
medium inhibited the growth and reproduction of A. acidoterrestris. In addition, with decreasing temperature, the
color, smell and turbidity of commercially available juice supplemented with A. acidoterrestris significantly
improved. This work provided a clear exploration of growth characteristics of A. acidoterrestris by applying
theory (medium) to reality (fruit juices), and pave fundamental for exploring the zero additives of controlling
juice spoilage.

Introduction not kill all spores, thus generating negative organoleptic effects on fruit
juice when used alone. Adding natural antibacterial products (e.g.,
Fruit juice is popular for consumers due to its ability to preserve most bacteriocins, lysozyme, chitosan, and some plant extracts etc.) com­
nutrients of fruits and good taste (Zhang et al., 2021). The flavor of fruit bined with or without physical methods is an effective alternative and
juices plays an important role in beverage industry, but once the un­ has become a research hotspot for controlling Alicyclobacillus contami­
pleasant flavors are present, it will dramatically reduce consumer nation in fruit juices (Roig-Sagués et al., 2015; Song et al., 2019).
acceptance and bring economic losses for juice industry (Bianchi et al., Despite the excellent antimicrobial properties of natural antibacterial
2010). Microbiological contamination is one of the possible pathways to products, the high cost of extraction and purification processing limits
produce off-flavors. Species of Alicyclobacillus, one of the common their utilization. Moreover, consumers prefer the natural and free of
sources of contamination, can multiply in fruit juice and lead to juice additive fruit juice (Pornpukdeewattana et al., 2020). Thus, it is
spoilage along with the production of medicinal and disinfectant-like imperative to have a clear understanding for the growth characteristics
off-flavor, phenolic, and visible sediment when cell concentration is of Alicyclobacillus in juice under different storage conditions, which is
high enough (Huang et al., 2014; Witthuhn et al., 2011). Due to its acid necessary in developing new methods for controlling Alicyclobacillus
tolerance and wide survival temperature, spores of Alicyclobacillus can contamination in the absence of additives and provide fundamental
survive pasteurization of fruit juices. The residual spores germinate and theory for exploring the zero additives for controlling juice spoilage.
produce guaiacol (off-flavor) when the bacterial counts reach 105 CFU/ In response to these demands, herein, the growth and metabolism
mL (Ju et al., 2020). Thus, it is very important to control and even conditions of Alicyclobacillus acidoterrestris (A. acidoterrestris) were
prevent Alicyclobacillus growth at early stage for juice manufacture. simulated in the medium and juice environment. As previous study has
Many approaches have been reported to prevent or control Alicy­ been reported, pH and temperature are important for growth and
clobacillus contamination including physical treatments, chemical metabolism of A. acidoterrestris due to its thermophilic and acidophilic
treatments, adding natural antibacterial products, and controlling bio­ properties (Bianchi et al., 2010), thus, the simulative models for
film (Ju et al., 2020). Nevertheless, traditional physical methods may contaminated A. acidoterrestris at different pH and temperature were

* Corresponding authors.
E-mail addresses: zhangwt@nwsuaf.edu.cn (W. Zhang), fanmt@nwsuaf.edu.cn (M. Fan).
1
Congdi Shang and Tong Zhang contributed equally to this work.

https://doi.org/10.1016/j.fochx.2023.100790
Received 17 May 2023; Received in revised form 5 July 2023; Accepted 8 July 2023
Available online 10 July 2023
2590-1575/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
C. Shang et al. Food Chemistry: X 19 (2023) 100790

Fig. 1. The schematic diagram of guaiacol production for A. acidoterrestris (a). Effect of different concentration of vanillin for the growth of A. acidoterrestris (CK:
normal culture; 100 mg /L: normal culture with 100 mg /L vanillin; 500 mg /L: normal culture with 500 mg /L vanillin; 1000 mg /L: normal culture with 1000 mg /L
vanillin) (b).

systematically investigated using guaiacol as a representative in fluid incubated for 24 h at 45 ◦ C and 150 r/min with shaking, and OD600 were
nutrient medium. Furthermore, the simulative conditions were verified measured every 2 h. The concentration of guaiacol was determined by
in juice to simulate actual contamination situations including viable high performance liquid chromatography (HPLC) based on Xu et al.
count and analysis of the volatile components in the study. (2019). Samples were taken at the following time points: 0, 4, 6, 8, 10,
12 and 16 h.
Materials and methods
Effect of different temperature on growth and metabolism of guaiacol
Materials production by A. Acidoterrestris
Activated A. acidoterrestris were cultured in liquid medium (pH 4) at
Vanillin and guaiacol were purchased from Shanghai Yuanye Co. Ltd. different temperature (45 ◦ C, 25 ◦ C and 4 ◦ C) for 7 d (Samples were
(Shanghai China). Both pure apple (pH 3.6) and orange (pH 3.6) juices taken daily). Samples were used as standard plate counting to determine
are pasteurized concentrate and were purchased from local supermar­ the number of viable bacteria. The samples were centrifuged and the
ket. Alicyclobacillus acidoterrestris (A. acidoterrestris) was stored at the supernatant was passed through a 0.22 μm membrane for guaiacol
Lab of Food Microbiology and Food Biotechnology in Northwest A&F concentration determination using HPLC.
University (Yangling, Shaanxi).
Effect of storage temperature on growth and metabolism of a.
Effect of vanillin on a. Acidoterrestris Acidoterrestris in fruit juice

Strain activation was referenced to the method of Zhao et al. (2021). The activated bacteria were inoculated into sterile apple and orange
Vanillin was selected as a direct precursor to guaiacol, and vanillin stock juice at a concentration of about 104 CFU/mL and stored at 45 ◦ C (op­
solution (10 mg/mL vanillin dissolved in 50% ethanol (v/v) and filtered timum growth temperature), 25 ◦ C (room temperature) and 4 ◦ C
through a 0.22 µm membrane to remove bacteria) was added to the (refrigerated temperature) for 30 d. The juice without A. acidoterrestris
liquid medium to achieve the following final concentrations of vanillin: was used as control, during which samples were taken at regular in­
100, 500 and 1000 mg/L. A. acidoterrestris was inoculated at 2% (v/v) tervals and the viable bacteria were counted by standard plate counting.
into the AAM medium containing different concentrations of vanillin. The metabolism of A. acidoterrestris was measured by analyzing the
The culture was incubated at 45 ◦ C for 24 h at 150 r/min. Samples were volatile components in fruit juice with the method of Xu et al. (2019) at
measured every 2 h at OD600. the end of storage. Samples were taken after 30 days respectively,
centrifuged at 5000 rpm for 10 min, and 5 mL of supernatant was added
to a 20 mL headspace flask with 6 μL of 2-Octanol as internal standard,
The establishment of growth and metabolism model at different condition
followed by 1.5 g of NaCl, and the volatile components in juice were
determined using SPME-GC–MS. The volatile compounds were identi­
Two common three-parameter mathematical models (Gompertz
fied with reference to the retention indices of the standards, character­
model and Logistic model) were chosen to fit the growth curve and
ized by comparing the full scan mass spectra with those stored at NIST
metabolic guaiacol production curve of A. acidoterrestris in this study.
and quantified using the internal standard method.
Non-linear curve fitting in Origin software was used to complete the
fitting of the A. acidoterrestris growth curve and the metabolic guaiacol
production curve (Zhao et al., 2021). Sensory analysis

Effect of different pH on growth and metabolism of guaiacol production by a. Sensory analysis was completed according to the literature with
Acidoterrestris some modifications (Piskernik et al., 2016). 10 experienced experts in
Activated A. acidoterrestris were inoculated in liquid medium at the field of juice made up the evaluation team and carried out the sen­
different pH (2.5, 2.7, 3.0, 3.2, 3.5, 3.8 and 4.0). The liquid medium was sory properties of labelled juices in a standard sensory laboratory. All

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C. Shang et al. Food Chemistry: X 19 (2023) 100790

Fig. 2. The growth curves of A.acidoterrestris in medium with different pH (a); the curves of guaiacol production metabolism by A.acidoterrestris in medium with
different pH (b); the growth curves of A.acidoterrestris in medium with different temperatures (c); Curves of guaiacol production metabolism by A.acidoterrestris in
medium with different temperatures (d). At 4℃, the growth and metabolism of A. acidoterrestris did not conform to the S-shaped curve, so only the changes in data
were shown in Fig. 2c and d.

samples were evaluated by the same team. For purpose of the evalua­ mg/L vanillin had not markedly inhibitory influence, but the addition of
tion, three parameters (color, smell, and turbidity) were introduced 500 mg/L and 1000 mg/L vanillin had remarkably negative effect on the
applied with analytical-descriptive test. In the test, sensory attributes of growth of A. acidoterrestris. Considering the produce of guaiacol requires
color and smell in juices were scored by non-structured scale from 1-4-7, certain concentration of A. acidoterrestris at least 4–5 Log CFU/mL
and point 4 was considered to be the best, while scores ≥4.5 indicated (Witthuhn et al., 2012), 100 mg/L vanillin addition kept
the greater description of the property and scores ≤3.5 stand for the A. acidoterrestris at low concentration with very weak ability to produce
lesser description of the property. For turbidity, the structured scale guaiacol.
score was 1–7, in which higher score means greater description of the
property. The establishment of growth and metabolism model in medium
The samples for sensory analysis were prepared by storage uncon­ The three-parameter models, and the estimated value having one
taminated and contaminated juice in 4, 25 and 45℃ respectively for 30 more degree of freedom, were used to fit the S-shaped curve which is
days. As presented in Table S7, S8, all samples labelled as A-G and necessary for curves with a small amount of test points. Therefore, the
offered to experts in separate glasses for analysis. growth and metabolism of A. acidoterrestrisit could be predicted by the
curve (Bahçeci and Acar, 2006). Two mathematical models, Gompertz
and Logistic, were used to fit the growth curves and guaiacol production
Statistical analysis metabolism of A. acidoterrestris at different pH (Table S1 and S2). The
criteria used to check the accuracy of the model are the coefficient of
The data were represented as the mean ± standard deviation (SD). determination of the fit (R2) and the standard error of prediction (S), and
All experiments were triplicated. The data was analyzed with ANOVA higher R2 and smaller standard error S indicate more accuracy of the
using IBM SPSS 22 statistical software. Significant differences (P < 0.05) model. The results showed that there were larger determination co­
were evaluated by Tukey’s significant difference test. efficients in Gompertz model and smaller standard error than that in
Logistic model. So Gompertz model was chosen to fit the growth curves
Result and discussion of A. acidoterrestris in medium at different conditions.
As is known to all, pH and temperature are important factors
The growth and metabolism of A. Acidoterrestris in medium affecting the growth of A. acidoterrestris comparing to other environ­
mental stresses (Zhao et al., 2021). As pH decreases, the maximum
Effect of vanillin on A. Acidoterrestris bacterial density (ODmax) and the maximum growth rate (μ) was
Guaiacol as a representative of metabolite for A. acidoterrestris re­ decreased, and the hysteresis period (λ) was also prolonged (Fig. 2a,
quires the substrate vanillin to synthesize (Bahçeci and Acar, 2006; Corli Table S3). Although the growth of A. acidoterrestris was inhibited, it still
Witthuhn et al., 2013). As a commonly used approach (Witthuhn et al., grew at extreme pH environment (e.g., ODmax = 0.323, pH 2.5), illus­
2012; Cai et al., 2015a; Wang et al., 2021a), vanillin-guaiacol mode has trating that as an acidophilic bacterium, A. acidoterrestris was not
been adopted in much research to explore the growth metabolism of completely inhibited by extreme acidic environments. The prolonged
A. acidoterrestris by adding vanillin to the studied system (Cai et al., hysteresis period indicated A. acidoterrestris took longer time to accli­
2015b; Cai et al., 2019; Wang et al., 2021b). To explore the ability of mate and resume growth when dealt with acid stress at low pH (Estilo
A. acidoterrestris to produce guaiacol, different concentration of sub­ and Nakano, 2019). At different temperature, it can be found that the
strate vanillin was added to the fluid nutrient medium, Fig. 1a, b shows maximum bacterial count at 45 ℃ was higher about 1 Log10 CFU/mL
the effect of different vanillin concentration on growth of than that at 25 ℃, while the bacterial count further decreased at 4 ℃,
A. acidoterrestris. Compared with the normal culture, the addition of 100

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Fig. 3. The schematic diagram of the growth of A. acidoterrestris at different temperature in fruit juice (a). Effect of different temperature on growth of
A. acidoterrestris in apple juice (b) and in orange juice (c). (For interpretation of the references to color in this figure legend, the reader is referred to the web version
of this article.)

indicating that low temperature inhibited the growth of A. acidoterrestris The growth and metabolism of a. Acidoterrestris in fruit juice
(Fig. 3c, Table S4). Moreover, the maximum growth rate of
A. acidoterrestris at 45 ℃ was significantly higher than that at 25 ℃. In the process of fruit juice production, juice was easily contaminated
Compared with A. acidoterrestris at 45 ℃, the hysteresis period at 25 ℃ with microorganisms, and A. acidoterrestris was the main source of
was significantly prolonged, indicating that A. acidoterrestris needed contamination in fruit juice spoilage (Kakagianni et al., 2020). As a heat-
longer time to adapt to new environment at lower temperature. It was tolerant bacterium, temperature plays an important role in affecting the
reported that A. acidoterrestris was classified as a mild to highly ther­ growth of A. acidoterrestris. This work aimed at exploring the effect of
mophilic bacterium and grew slowly at lower temperatures (Estilo and temperature on the growth and metabolism of A. acidoterrestris in juice,
Nakano, 2019). Corli Witthuhn et al. (2013) also found that the growth so as to provide fundamental theory for fruit juice manufacturers in
rate of A. acidoterrestris at 25 ℃ was slower than that at 45 ℃. Under the controlling A. acidoterrestris.
non-optimal growth temperature (25 ℃), the maximum number of
A. acidoterrestris was less than the maximum number under the optimal The growth of a. Acidoterrestris in fruit juice
growth temperature (45 ℃). At low temperature (4 ℃), the bacterial Based on our preliminary experiments and literatures reports
counts further decreased (Fig. 2c). (Bianchi et al., 2010; Wang et al., 2021), concentrate juice was used for
In order to determine the effect of different conditions on meta­ the latter experiment. The growth diagram of A. acidoterrestris in fruit
bolism of A. acidoterrestris, the metabolism of guaiacol production was juice at different temperature was shown in Fig. 3a. The results showed
chosen as representative indicator. Fig. 2b shows that low pH value has that, at optimum growth temperature (45 ◦ C), the number of bacterial
an inhibitory effect on guaiacol production by A. acidoterrestris, but not counts increased continuously during the first 8 days, with a maximum
suppressed completely. As pH decreased, the rate of guaiacol formation count reaching 7.21 Log10 CFU/mL in apple juice (Fig. 3a) and 7.38
(μ) and hysteresis period (λ) were also influenced (Table S5). The results Log10 CFU/mL in orange juice (Fig. 3b), and then leveled off after 15
also showed that low pH (pH < 3.2) retarded the growth of the bacteria days until the end of storage. At 25 ◦ C, the bacterial counts increased in
and also delayed the production of guaiacol, because A. acidoterrestris the first 2 days, then declined from 3 to 8 days and levelled off after 8
needed more time to adapt to the new environment. The lag time was days. At 4 ◦ C, the counts declined continuously from beginning to 8 days
about 2 h at pH 3.5 to 3.0, and about 4 h at pH lower than 3.0, the same and levelled off after 12 days. The counts in both apple and orange juice
as the results obtained from the growth curve in Fig. 2a. For example, at remained at around 1.10 Log10 CFU/mL until the end of the storage
pH 2.5, guaiacol was still produced by A. acidoterrestris, and the final period.
concentration was only 52.105 mg/L. A. acidoterrestris in pH 3.5 needed In contrast to the situation in the medium, room temperature (25 ◦ C)
longer time to produce guaiacol than in pH 4.0 and 4.5, which was was not suitable for growth of A. acidoterrestris in juice, with a gradual
consistent with the results in previous study (Chang et al., 2015). decrease of the count to 1.0–1.5 Log10 CFU/mL compared to the initial
Temperature also affected the formation rate of guaiacol，more guaia­ inoculation, it may be attributed that in nutrient-rich medium, room
col was produced at 45 ◦ C than at 25 ◦ C (Table S6). It has been reported temperature (25 ◦ C) storage only retarded bacterial growth, while in
that A. acidoterrestris could produce guaiacol at both 25 ◦ C and 45 ◦ C, but nutrient-limited and more acidic juice, the growth of bacteria was
the time required to produce guaiacol at 25 ◦ C was longer than at 45 ◦ C further inhibited. Moreover, refrigerated temperatures (4 ◦ C) has greater
(Hu et al., 2020). Fig. 2d shows that the final concentration of guaiacol inhibitory effect on bacterial growth, with bacterial counts decreased by
at 45 ◦ C was almost the same as at 25 ◦ C, suggesting that the potential 3.0–3.5 Log10 CFU/mL compared to the initial inoculation.
for spoilage of juice products stored at ambient temperatures (25 ◦ C)
should not be underestimated. Guaiacol was not detected at 4 ◦ C，it The metabolism of a. Acidoterrestris in fruit juice
may be due to low number of A. acidoterrestris. Since the aim of our work was to investigate the effect of
A. acidoterrestris for zero additives juice other than vanillin-guaiacol
mode, therefor, no additional vanillin added in juice system. Limited

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C. Shang et al. Food Chemistry: X 19 (2023) 100790

Fig. 4. The schematic diagram of the metabolism for A. acidoterrestris with different temperature in fruit juice (a). Total positive (b, d) and negative (c, e) contents of
volatile components in different treatments for A. acidoterrestris in apple juice (b, c) and orange juice (d, e) after storage (CK: normal juice, AA: normal juice with
A. acidoterrestris). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

by the used methods or the low content of guaiacol or vanillin (precursor 2022; Sauceda-Gálvez et al., 2021): volatile components with positive,
of guaiacol), guaiacol was not detected in fruit juice. Thus, the effect of negative effect on the flavor of fruit juice and other volatile components
temperature on the metabolism of A. acidoterrestris in fruit juice was with uncertain contributions. This work mainly focused on the first two
studied by sensory analysis and measuring the total volatile broad categories in detail. The research schematic diagram and effect of
components. storage temperature on metabolism of A. acidoterrestris in fruit juices
As shown in Table S7 and S8, the evaluated color, smell and turbidity was shown in Fig. 4a and the volatile components of the juices were
of contaminated juice (apple juice and orange juice) treated with 45 ◦ C determined. The total concentration of positive volatile flavor compo­
showed the darkest color, worst smell and the highest turbidity nents of the contaminated juice was higher than that of uncontaminated
compared with other treatments. At 4 ◦ C, compared with untreated juice (Fig. 4 b, d), and negative effects on the flavor of contaminated
juice, the appearance and smell of uncontaminated juice had no signif­ juice were lower than that of uncontaminated fruit juice under three
icant difference, while the contaminated juice had slight changes. It can temperatures tested (Fig. 4 c, e). Even at low temperatures, the meta­
be assumed that the sensory property of the juice gradually deteriorates bolic activity of the bacterium was still going, resulting in juice spoilage.
with the increase of temperature, which, combined with the previous For apple juice, a total of 54 volatile compounds was detected at
results of the effect of temperature on the growth of A. acidoterrestris, different temperatures, including 12 alcohols, 12 aldehydes, 14 ketones,
suggests that the growth and metabolic activity of A. acidoterrestris in the 5 acids, 9 esters and 2 phenols (Supplementary Material: Table S9), in
juice increased with raising temperature. which alcohols and esters are considered to contribute to the aroma.
In order to deeply investigate the effect of temperature on the Compared with esters, alcohol, a degradation product of unsaturated
metabolism of A. acidoterrestris in fruit juice, volatile components were fatty acids, was considered to be the second important contributor to
measured with different treatments. To clearly analyze the variations of aroma of apple juice (Han et al., 2021). As shown in Table 1, the alcohols
different volatiles, the volatile components into were divided into three that positively influenced the flavor of apple juice were significantly
categories according to literatures (Bianchi et al., 2010; Liu et al., 2019; higher in the uncontaminated juice than in the contaminated juice at 45
Schmutzer et al., 2014; Zhang et al., 2022; Reznik et al., 2021; Zhu et al., and 25 ◦ C, while at 4 ◦ C, the alcohols that positively affect the flavor in

Table 1
Volatile compound total contents of uncontaminated and contaminated apple juice treated at different temperatures.
Volatile compounds（μg/L) Indicators detected

45 CK 45AAT 25CK 25AAT 4 CK 4AAT

Alcohols
Positive effect alcohols 85.41c ± 3.14 31.48d ± 0.92 140.53a ± 4.55 93.89b ± 3.26 140.94a ± 4.78 140.47a ± 4.07
Other alcohols 6.41c ± 0.16 12.92b ± 0.21 14.56a ± 0.25 12.97b ± 0.31 7.14d ± 0.19 3.35e ± 0.11
Esters
Positive effect esters 0.75c ± 0.05 0.55c ± 0.04 4.14c ± 0.14 1.41c ± 0.12 369.98a ± 12.81 278.38b ± 8.14
Ketones 1.17b ± 0.05
Positive effect ketones 9.41d ± 0.14 12.30ab ± 0.36 8.52e ± 0.13 10.39c ± 0.27 11.99b ± 0.31 nd
Negative effect ketones nd 48.39a ± 1.54 nd 16.77b ± 0.42 nd nd
Other Ketones 2.17c ± 0.11 21.33a ± 0.45 1.11d ± 0.06 17.34b ± 0.44 1.02d ± 0.06 1.22d ± 0.06
Acids
Negative effect Acids 1.37c ± 0.07 214.17a ± 6.90 4.62c ± 0.16 16.83b ± 0.78 1.90c ± 0.06 1.76c ± 0.07

All data were expressed as mean ± standard deviation (n = 3). Different letters in the same line indicate significant differences at 95% confidence according to Tukey
test; CK: normal juice, AA: normal juice with A. acidoterrestris.

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Table 2
Volatile compound total contents of uncontaminated and contaminated orange juice treated at different temperatures.
Volatile compounds（μg/L) Indicators detected

45 CK 45AAT 25CK 25 AAT 4 CK 4 AAT

Alcohols
Positive effect alcohols 87.04d ± 2.13 19.25e ± 0.37 155.84b ± 3.15 97.92c ± 2.54 167.74a ± 4.22 164.37a ± 4.31
Other alcohols 18.47b ± 0.33 19.11a ± 0.34 17.02c ± 0.36 18.66ab ± 0.41 nd 2.54d ± 0.14
Esters nd
Positive effect esters 64.17c ± 0.94 15.22e ± 0.35 104.66b ± 2.40 21.81d ± 0.64 183.73a ± 5.39 107.35b ± 1.79
Terpenes
Positive effect terpenes 4791.53d ± 68.79 97.10f ± 3.23 5805.63c ± 83.56 2304.78e ± 41.15 6995.33a ± 92.75 6153.25b ± 89.47
Negative affect terpenes 195.24c ± 4.54 425.34a ± 9.73 151.28d ± 3.79 226.51b ± 6.94 140.63f ± 4.02 148.75e ± 4.23
Other Terpenes 427.80d ± 7.88 107.98f ± 2.54 583.96b ± 8.27 253.60e ± 4.36 644.45a ± 7.14 Other Terpenes
Aldehydes
Positive effect aldehydes 53.52c ± 1.28 nd 32.67d ± 0.66 4.27e ± 0.06 156.56a ± 4.21 101.67b ± 3.84
Other Aldehydes nd nd 31.09c ± 1.17 33.35a ± 1.23 31.98b ± 0.94 nd
Ketones
Positive effect ketones 7.68d ± 0.13 6.34e ± 0.14 8.33c ± 0.14 6.63e ± 0.12 13.01a ± 0.25 10.84b ± 0.23
Negative effect ketones nd 95.19a ± 2.89 nd 9.83b ± 0.14 nd nd
Other ketones 12.27a ± 0.17 7.01d ± 0.08 4.13e ± 0.02 10.56b ± 0.14 8.50c ± 0.19 8.23c ± 0.06
Acids
Negative effect acids nd 499.60a ± 9.49 1.40c ± 0.02 4.20b ± 0.09 nd nd

All data were expressed as mean ± standard deviation (n = 3). Different letters in the same line indicate significant differences at 95% confidence according to Tukey
test; CK: normal juice, AA: normal juice with A. acidoterrestris.

uncontaminated apple juice were not significantly different with that in investigated in orange juice (Li et al., 2022) and the result was presented
contaminated juice. The results indicated that the metabolism of alcohol in Table 2 and Table S10. The concentration of positive effect terpenes
production for A. acidoterrestris increased with temperature rising. In was increased rapidly with temperature decreased from 45 ℃ to 4 ℃ in
contrast, at 4 ◦ C, the activity of A. acidoterrestris was very low, so the both contaminated and uncontaminated juice. Moreover, the concen­
ability to produce alcohol was greatly diminished. Similar results were tration of positive effect terpenes in 45 CK was far higher than that in 45
also observed for esters which were important contributors to apple AAT. Meanwhile, we also detected off-odor for terpenes including
juice flavor due to their fruity and sweet flavor (Li et al., 2021), and α-terpineol and carvone which was the degradation product of limonene
compared with 4 ◦ C for normal juice having A. acidoterrestris, the total and linalool (Reznik et al., 2021). Considering that terpenes can be used
content of positive effective esters at 45 ◦ C were greatly decreased (from as carbon source (Bianchi et al., 2010), We believe that off-odors can be
278.38 to 0.55 μg/L). Similarly, ketone, also important contributors for produced not only through self-deterioration but also from
apple juice, was divided into positive effect ketones, negative effect A. acidoterrestris using the positive flavor. In addition, the variation of
ketones and other ketones, of which, β-Damascenone had a rose-like alcohols, esters, ketones and acids in orange juice were almost the same
aroma and was used as a representative of positive effect ketones. The as in apple juice.
results showed that the content of β-Damascenone was high overall at
4 ◦ C, and the content for uncontaminated juice was higher than Conclusion
contaminated juice. Meanwhile, as temperature increased, the content
of β-Damascenone decreased and was completely eliminated at 45 ◦ C In summary, we simulated two ways to probe the growth and
(Supplementary Material: Table S9), which the probable reasons were metabolism of A. acidoterrestris for providing fundamental theory in
volatilization of flavor placed at high temperature for long time (45 ◦ C, exploring the zero addition to control juice spoilage. The results showed
30 days) and metabolic exhaustion of A. acidoterrestris. Negative effect that guaiacol was not produced by A. acidoterrestris in absence of pre­
ketones including 2,3-Butandione, 3-Hydroxy-2-butanone, 2,3-Heptane­ cusor substance (vanillin). Undesirable flavors produced by
dione had cream flavor which is off-odor for fruit juice (Zhu et al., 2022), A. acidoterrestris in fruit juices not only originated from guaiacol, but
and were only detected at 45 AAT (normal juice with A. acidoterrestris at also through the metabolism of other substances such as ketones and
45 ◦ C) and 25 AAT (normal juice with A. acidoterrestris at 25 ◦ C), indi­ terpenes, which can be a direction for monitoring juice spoilage.
cating that negative effect ketones could be synthesized in the process of
growth and metabolism for A. acidoterrestris. Volatile acids, mainly fatty CRediT authorship contribution statement
and cheesy odor, were assigned to negative effect acids in this work,
which gave apple juice an unpleasant odor in high concentrations Congdi Shang: Conceptualization, Validation, Writing – original
(Sauceda-Gálvez et al., 2021). The content of volatile acids was signif­ draft. Tong Zhang: Conceptualization, Investigation, Writing – review
icantly higher in the contaminated juice than in the uncontaminated & editing, Writing – original draft. Junnan Xu: Visualization. Ning
juice at 45 ◦ C and 25 ◦ C, in which the highest content was 45 AAT Zhao: Software. Wentao Zhang: Validation, Supervision. Mingtao Fan:
(214.17 μg/L). However, the negative effect volatile acids in apple juice Writing – review & editing, Project administration, Resources.
were not significantly different from 45 CK (normal juice at 45 ◦ C), 25
CK (normal juice at 25 ◦ C), 4 CK (normal juice at 4 ◦ C)and 4 AAT
(normal juice with A. acidoterrestris at 4 ◦ C), the reason may be that Declaration of Competing Interest
A. acidoterrestris could produce the volatile acids during its growth and
metabolism. To sum up, it was mostly contaminated at 45 ◦ C, the The authors declare that they have no known competing financial
possible reason was that 45 ◦ C is the optimum temperature for interests or personal relationships that could have appeared to influence
A. acidoterrestris (Zhao et al., 2021). The result (Fig. 4b, c) showed that the work reported in this paper.
the negative volatile components of apple juice almost the same as
original at 4 ◦ C, which means the activity of A. acidoterrestris was Data availability
inhibited at 4 ◦ C.
As a typical volatile compound of citrus fruits, terpenes were No data was used for the research described in the article.

6
C. Shang et al. Food Chemistry: X 19 (2023) 100790

Acknowledgments and aroma release. Food Chemistry, 374, Article 131705. https://doi.org/10.1016/j.
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