J Food Sci Technol (March 2022) 59(3):990–1000

https://doi.org/10.1007/s13197-021-05103-7

ORIGINAL ARTICLE

Evaluation of microbiological safety, physicochemical

and aromatic qualities of shiikuwasha (Citrus depressa Hayata)
juice after high pressure processing
Yen-Ying Lai1 • Jian-Hua Chen1 • Yao-Chia Liu1 • Yun-Ting Hsiao1 •

Chung-Yi Wang1

Revised: 5 April 2021 / Accepted: 9 April 2021 / Published online: 17 April 2021
Ó Association of Food Scientists & Technologists (India) 2021

Abstract This study evaluated high pressure processing Abbreviations

(HPP) for achieving greater than 5-log reduction of HPP High pressure processing
Escherichia coli O157:H7 in shiikuwasha (Citrus depressa TPC Total phenolic content
Hayata) juices and compare quality parameters, including TFC Total flavanones
microbiological safety, total phenolic content (TPC), total PMF Polymethoxylated flavones
flavanones (TFC), and polymethoxylated flavones, HTST High-temperature short-time
browning, volatile aromatic, and physicochemical proper- TA Titratable acidity
ties of HPP-treated juice with those of high-temperature HMF Hydroxymethylfurfural
short-time pasteurized juice. A HPP of 600 MPa for 150 s BI Browning index
was identified capable of achieving greater than 5.15-log Y&M Yeast and mold
reductions of E. coli O157:H7 in shiikuwasha juice. The TSS Total soluble solids
microbiological shelf life of the juices were at least 28 days
when processed at HPP for 600 MPa/150 s or HTST for
90 °C/60 s. The color, aromatic, and antioxidant contents Introduction
(TPC, TFC, Tangeletin, Nobiletin) were well preserved
after HPP, however, HTST resulted in a significant Shiikuwasha (Citrus depressa) is a member of the citrus
(p \ 0.05) loss of antioxidant content (TPC (8.8%), Tan- family native to Taiwan and Japan (Okinawa and Kyushu).
geletin (6.8%)), and negatively impacted the juice color. It’s often used to prepare drinks, and its juice is commer-
By the end of storage, the amount of these aroma relevant cially available from a few vendors in Taiwan and Japan.
volatiles appears to still be higher in HPP pasteurized jui- Shiikuwasha contains numerous flavonoids, including
ces compared to their conventional counterparts. This study nobiletin, whose medicinal properties have been exten-
demonstrated that under optimal conditions of HPP can sively studied (Lee et al. 2010), which is widely recognized
attain the same level of microbiological safety as thermal as a therapeutic food, and its rind has been traditionally
pasteurization and preserved the acceptable quality of used in Chinese medicine (Lin 2006). In addition to fla-
shiikuwasha juice. vonoids, shiikuwasha contains many acridone alkaloids,
which are antispasmodics that can help relieve intestinal
Keywords Shiikuwasha  Juice  Pasteurization  High- spasms and reduce the incidence of digestive problems.
pressure processing  Aroma Akachi et al. (2010) reported that nobiletin extracted from
shiikuwasha can alleviate D-galactosamine-induced liver
damage and reduce the concentration of pro-inflammatory
mediators, such as tumor necrosis factor alpha and nitric
& Chung-Yi Wang oxide. Lee et al. (2011) studied the effects of shiikuwasha
cywang@nfu.edu.tw
extract on high-fat diet-induced obese mice and determined
1
Department of Biotechnology, National Formosa University, that the treatment group gained significantly less body
Huwei Township, No. 64, Wenhua Rd, Yunlin 632, Taiwan weight than the control group. Furthermore, histological

123
J Food Sci Technol (March 2022) 59(3):990–1000 991

examinations revealed that the white adipose tissue weight Materials and methods
and adipocyte size in the treatment group were significantly
lower and smaller, respectively, than those in the control Shiikuwasha juice
group.
High-pressure processing (HPP) is a non-thermal food Fresh shiikuwasha fruits were obtained from a farm in
processing technique that involves subjecting sealed food Pingtung County (Taiwan). After the fruit juice was
items to high pressure (100–600 MPa) via a liquid medium squeezed and filtered, it was immediately vacuum packed
(typically water) at room temperature. This process inac- and pasteurized. The E. coli O157:H7 (BCRC No. 14824)
tivates microbes and enzymes, and therefore, prolongs the strain was purchased from the Bioresource Collection and
storage life of food items while preserving their original Research Center (Taiwan). The strain was cultured in a
flavor and nutritional value. HPP can achieve the same nutrient broth, which contained 0.5% NaCl, at 37 °C until
level of food safety as conventional thermal pasteurization its population reached stationary phase. The bacterial cells
techniques (Huang et al. 2017). In a study on the use of were collected via centrifugation at 3000 9 g for 10 min
HPP for inactivating Escherichia coli and Listeria innocua and were subsequently washed once with phosphate-buf-
in kiwi and pineapple juice, Buzrul et al. (2020) determined fered saline (pH 7.3). The washed bacrerial cells were
that these pathogens were not significantly inactivated after resuspended in fruit juice at a concentration of
HPP at 300 MPa (20 °C). However, after HPP at 400 MPa, 1 9 108 CFU/mL. Fresh bacterial cultures (prepared using
the microorganisms were almost fully inactivated. For low- the aforementioned procedure) were used for all HPP
acid fruits and vegetables (pH C 4.6), ‘‘hurdle technolo- experiments.
gies,’’ which combine HPP with other treatments (at low
intensity), can be used to minimize processing intensity; High-pressure and thermal pasteurization
reduce the use of chemical additives, salt, or sugar;
decrease the temperature and duration of thermal inacti- A 6.2 L HPP experimental unit utilized water as the
vation treatment; and attain the same level of food safety pressure-transmitting medium. The vacuum-packed juices
that conventional thermal pasteurization confers (Huang were subjected to HPP at 600 MPa and 25 °C for 0.5, 1,
et al. 2014). Many studies have suggested that HPP does 1.5, 2, and 2.5 min. All subsequent pasteurization experi-
not alter the nutritional content and taste of fruit juices or ments were performed using the HPP conditions that led to
damage their functional components. Therefore, HPP pas- a 5-log reduction in E. coli O157:H7 counts. HTST was
teurized can be used to extend the storage life of heat- performed by heating the juice to 93 °C for 3 min. During
sensitive fruit juices (e.g., mango, pineapple, watermelon, the storage experiment, the sterilized juice samples were
grape, strawberry, and coconut juice) while maintaining a maintained at 4 °C, and the changes in juice quality were
fresh-like taste (Wang et al. 2016). examined by sampling the juices every 7 d.
Based on the microbiological safety standards for high-
pressure food pasteurization, this study will investigated Microbiological analysis
the effects of HPP on the overall quality of shiikuwasha
juice and experimentally determined the conditions The fruit juice samples were serially diluted with 0.1%
required to attain a 5-log reduction in E. coli O157:H7 buffered peptone water, and the diluted samples were
counts. Moreover, the quality parameters of HPP-treated immediately inoculated onto PetrifilmTM plates for total
juice were compared to those of juices subjected to high- aerobic bacteria (TAB), psychrophile, and yeast and mold
temperature short-time (HTST) treatment during a 28 d (Y&M) count analysis. The TAB count plates were incu-
cold-storage period. The analyzed quality parameters bated at 30 °C for 24 h, the psychrophile count plates were
included antioxidant contents (TPC, TFC, Tangeletin, and incubated at 10 °C for 5 d, and the Y&M count plates were
Nobiletin), physicochemical properties (pH, titrat- incubated at 25 °C for 5 d. Subsequently, the number of
able acidity (TA), and color), degree of browning colonies on the plates with 25–250 colonies were counted
(browning index (BI) and hydroxymethylfurfural (HMF) and were recorded in units of CFU/mL.
concentration), and aromatic compounds. The results of the
aforementioned analyses were subsequently used to deter- Total phenols and flavonoids
mine the optimal expiration date of cold-stored shi-
ikuwasha juice. A 0.5 mL juice sample was reacted with 0.5 mL of the
Folin–Ciocalteu reagent for 5 min. Next, 2 mL of 200 mg/
mL Na2CO3 was added to the mixture followed by thor-
ough homogenization. The resulting mixture was allowed

123
992 J Food Sci Technol (March 2022) 59(3):990–1000

to react for 15 min at room temperature. When the reaction HMF content analysis
was completed, 10 mL of ultrapure water was added to the
mixture, which was subsequently centrifuged at 4000 9 g Two mL of fruit juice was pipetted into a 50 mL plastic
for 5 min to remove all precipitates. The absorbance of the centrifuge tube, to which 10 mL of double-distilled water
solution was measured at 725 nm, and the result was was added, followed by vortexing for 1 min. Thereafter,
compared against a calibration curve for gallic acid. The 10 mL of methanol/1% formic acid was added to the
TFC of the sample was expressed in units of lg of gallic mixture followed by 1 min of vortexing and centrifugation
acid/mL. For flavonoid content analysis, a 1.5 mL of at 3500 rpm for 10.5 min. Subsequently, 500 lL–1.5 mL
deionized water (or ethanol), 0.1 mL of 10% Al(NO3)3, of the supernatant was pipetted into an Eppendorf tube, a
0.1 mL of 1 M CH3COOK, and 2.8 mL of H2O were mixture of 1000 lL of methanol/1% formic acid and
added to a 0.5 mL juice sample After the reactants were ultrapure water (1:1, v/v) was added to the tube, and the
mixed and were allowed to settle for 40 min, the absorption tube was vortexed to homogenize the solution. After 200
of the solution was measured at 415 nm using a spec- lL of the obtained solution was filtered through a 0.22 lm
trophotometer. The TFC of the sample was subsequently filter, the filtrate was subjected to high-performance liquid
determined by comparing the recorded data to a calibration chromatography–photometric diode array analysis using a
curve for quercetin. Waters 2695 HPLC separation module with a Luna C18
(5 lm 9 100 mm 9 4.60 mm) column and a Waters 2998
Polymethoxyflavonoids photodiode array detector. The analysis parameters were as
follows: the chromatogram was monitored at 283 nm, the
The contents of polymethoxyflavonoids (PMFs) of the fruit flow rate was 1.0 mL/min, and the injection volume was
juice samples were determined using the method of Tak- 10.0 lL. Mobile phases A and B consisted of a 1% acetic
enaka et al. (2007). A 20–85 ATNNNS (Takara ATM, acid solution and methanol, respectively. Thereafter, 2 mL
Tokyo, Japan) freeze dryer was used to freeze-dry 10 mL juice aliquots were pipetted into 50 mL plastic centrifuge
juice samples. The freeze-dried juice samples were tubes and HMF standards of different concentrations were
extracted twice with 10 mL of 70% ethanol, and the extract added to the tubes. After the samples were preprocessed,
was made up to 25 mL with 70% ethanol. The dried resi- solutions with concentrations of 100, 20, 10, 2, 1, and
due samples were milled into uniform powders using an 0.5 lg/g were prepared to create a calibration curve. Next,
IDS-50 (Sunbeam Products, Boca Raton, FL, USA) blen- the relationship between the instrument signals of the
der. The dried residue powder (100 mg) was extracted analytes with known concentrations and those of the
three times with 70% ethanol (2 mL), and the extracted samples was used to determine the HMF concentrations of
solution was made up to 10 mL with 70% ethanol. Each the samples.
extract was analyzed using a high-performance liquid
chromatography (HPLC) 600E (Waters, Milford, MA, Physicochemical properties
USA) multi-solvent flow system equipped with a Cosmosil
5C18-MS (4.6 mm i.d. 9 150 mm; Nacalai Tesque, The pH of the juice samples was determined using a
Kyoto, Japan) column. The mobile phase, which consisted JENCO 6173 (JENCO Quality Instruments) pH meter at
of 10 mM phosphoric acid and acetonitrile with a ratio of 25 °C. The total soluble solids (TSS, (°Brix)) content of the
90:10 (solvent A) and acetonitrile (solvent B), was pro- juice samples was determined at 20 °C using an ATAGO
grammed to mix 20% to 100% solvent B to solvent A for Master (ATAGO Co., LTD., Tokyo, Japan) handheld
15 min at a flow rate of 1 mL/min. Nobiletin and tangeletin refractometer. Color measurements were performed using
were detected at 280 nm. an LC100 (LovibondÒ) handheld spectrocolorimeter. The
TA of the juices was determined using the AOAC method
Brown index (1990).

To determine the BI of juice, a 5 mL juice sample was Aroma

centrifuged at 10 000 9 g for 10 min, and the supernatant
was collected and diluted with double-distilled water. The The volatile aromatics of the fruit juice samples were
mixture was filtered using a Whatman no. 1 filter, and the analyzed using the liquid–liquid continuous extraction–
absorbance of the filtrate was measured at 420 nm. solvent-assisted flavor evaporation (LLCE/SAFE) tech-
nique reported by Asikin et al. (2014). First, 200 mL of
sample, 0.5 mg of n-hexanol (the internal standard), and
200 mL of diethyl ether were placed in an LLCE system.
An extract containing the volatile aroma components was

123
J Food Sci Technol (March 2022) 59(3):990–1000 993

obtained after a 3 h continuous extraction process. The

volatile aroma components were separated from the non-
volatile fraction using the SAFE procedure under vacuum,
at 50 °C. The volatile aroma extract was dried with 10 g of
anhydrous sodium sulfate for 12 h at 10 °C, and its volume
was reduced to 2 mL using a Vigreux column. The isolated
volatile aroma extract was subsequently analyzed using gas
chromatography–flame ionization detection–mass spec-
trophotometry (GC–FID–MS). The composition of the
volatile aroma components was examined using an Agilent
6890 N GC instrument with a bonded-phase fused silica
capillary column (DB-Wax, 60 m 9 0.25 mm i.d., film
thickness 0.25 lm, Agilent J and W, Santa Clara, CA,
USA) and a FID apparatus. The temperature of the GC and Fig. 1 Table 1 Effects of HPP (600 MPa) on E. coli O157H7 in
FID instruments was set to 250 °C. Samples (1 lL) were Shiikuwasha juice. Data are means of three determinations ± stan-
dard deviation
injected in split mode injection (1:50) under a helium
atmosphere. The linear velocity of the helium carrier gas
poisoning and is highly resistant to pressure. The changes
was 32 cm/s. The chemical compositions were expressed
in the microorganism counts of fruit juices subjected to
as milligrams of volatile compound per liter of juice. The
600 MPa/1.5 min HPP treatment or 95 °C/3 min HTST
conditions were as follows: electron impact ion source and
treatment during storage at 7 °C are summarized in
interface temperatures of 230 °C, mass range (m/z) of
Table 1. The initial TAB of fresh shiikuwasha juice varied
29–450 amu, scan rate of 1.77 scans/s, and ionization
between 0.87–1.05 log CFU/mL, and TAB increased to
energy of 70 eV. The volatile aroma components were
2.11 log CFU/mL after 28 d of cold storage. The TAB of
identified by calculating their linear retention indices (RIs)
the HPP-treated fruit juice could not be measured during
using a homologous series of n-alkanes (C7–C30) and
the first 28 d of cold storage and only reached 0.69 log
comparing their fragmentation patterns with the data of
CFU/mL after 28 d. Furthermore, the TAB of the HTST-
authentic volatile aroma compounds from the library of the
treated juice could not be measured during the first 28 d.
National Institute of Standards and Technology and peak
The psychrotrophs of the HPP- and HTST-treated fruit
enrichment on co-injection with authentic standards.
juices remained below the countable limit even after 28 d
of cold storage. Coliforms could not be detected in the
Statistical analysis
control, HPP-treated and HTST-treated juices. The Y&M
count of untreated shiikuwasha juice was 0.29 log CFU/
The aforementioned measurements were performed in
mL, and HPP fully suppressed Y&M growth. The Y&M
triplicate. Data processing and statistical analysis were
counts of the HPP- and HTST-treated groups were identical
performed using the Statistical Analysis System (SAS)
throughout the 28 d cold-storage period.
software suite (Statistica, 1999 edition, Statsoft Co., Tulsa,
HPP treatments must be optimized to guarantee micro-
OK, USA). Analysis of variance and the Duncan’s multiple
biological safety. Hence, it is important to understand the
range test were performed to measure the differences
pressure resistance of the indicator microorganism in dif-
between groups.
ferent types of food (Huang et al., 2017). In a study
regarding the high-pressure pasteurization of wheatgrass
juice, it was determined that L. innocua was more pressure-
Result and discussion
resistant than E. coli and Salmonela typhimurium. To attain
a 5-log reduction in L. innocua counts, the juice had to be
Microbial analysis
subjected to 500 or 600 MPa for more than 60 s. The
400 MPa/180 s treatment only reduced L. innocua counts
The decrease in bacterial counts increased proportionally
by 3 logs (Ali et al., 2019). HPP pasteurization experiments
with holding time (Fig. 1). HPP at 600 MPa for 1.5 min
on açaı́ juice with different TSS values at different pH
was sufficient to reduce the E. coli O157:H7 count in
levels revealed that 400 MPa/3 min HPP was sufficient to
shiikuwasha juice by 5.67 log CFU/mL. The USDA (2012)
attain an 8-log reduction in the Salmonella spp. population
recommends that the pressure resistance of the pathogen
of açaı́ juice with a TSS of 2.9°Brix at pH 4.0. Gouvea
must be tested to find the conditions that will achieve a
et al. (2020) reported that the Salmonella spp. counts of a
5-log reduction in its population. E. coli O157:H7 is a
sample with a TSS of 14.9°Brix at pH 5.5 were only
common foodborne pathogen, which easily induces food

123
994 J Food Sci Technol (March 2022) 59(3):990–1000

Table 1 Total aerobic bacterial,

Treatment Time (d)
psychrotrophs, and Yeast and
Molds counts in Shiikuwasha 0 0à 7 14 21 28
juice storage at 7 °C
TAB
Control 1.05 ± 0.10 – 1.22 ± 0.15 1.29 ± 0.19 1.35 ± 0.14 2.11 ± 0.17
HPP 0.87 ± 0.09 ND ND ND ND 0.69 ± 0.08
HTST 0.98 ± 0.11 ND ND ND ND 0.53 ± 0.13
Psychrotrophs
Control 0.51 ± 0.07 – 0.81 ± 0.04 0.69 ± 0.06 0.94 ± 0.07 0.89 ± 0.15
HPP 0.38 ± 0.05 ND ND ND ND ND
HTST 0.29 ± 0.08 ND ND ND ND ND
Y&M
Control 0.29 ± 0.06 – 0.30 ± 0.04 0.48 ± 0.09 0.42 ± 0.11 0.74 ± 0.25
HPP 0.33 ± 0.07 ND ND ND ND ND
HTST 0.46 ± 0.05 ND ND ND ND ND
All data were the means ± SD, n = 3. 0 , day 0 results before treatment; 0à, day 0 results after treatment.
TAB total aerobic bacteria; Y&M yeast and mold. -, not analysed; ND not detectable (\ 1 CFU/mL)

decreased to 2 logs after 400 MPa/3 min HPP. Because juice; however, the TPCs of juices treated at 300, 400, and
microbial pressure resistance depends on the food envi- 500 MPa were not significantly different (Hasni et al.
ronment, Buzrul (2020) developed a probabilistic model 2020). Yuan et al. (2018) reported that HPP slightly
for HPP target microorganism log-reductions in a variety increased the TPC and anthocyanidin content of aronia
of food matrices, which assumed that the conditions berry puree. This can be attributed to the cellular structure
defined by experimental data yielded the same degree of of the treated fruits and vegetables being destroyed at high
microbial inactivation during commercial applications. pressures, which alters cell permeability and induces the
release of intracellular phenolics, and therefore, increases
Antioxidant content the antioxidant content of the HPP-treated juices. Ali et al.
(2019) determined that the TPC of wheatgrass juice heated
HPP and HTST affected the TPC, TFC, and PMF content to 75 °C for 15 s was only 64% of the TPC of fresh
of shiikuwasha juice to different degrees (Table 2). Both wheatgrass juice. By contrast, the TPC of the HPP-treated
HPP and HTST decreased TPC. However, the TPC of the wheatgrass juice was only 7.5% lower than that of fresh
HPP-treated juice did not change throughout a 28 d cold- juice. Hence, HPP sterilization maintained relatively high
storage period, whereas the TPC of the control group juice TPCs. Fernandez et al. (2019) reported that the pectin-
decreased gradually, and by the 28th d, the TPC of the methylestearase, peroxidase and polyphenoloxidase activ-
control group juice was lower than that of the HPP-treated ities of fruit and vegetable smoothies treated at 630 MPa
juice. Similar trends were observed for TFC. Although the for 6 min at 20 °C were significantly lower than those of
TFCs of the HPP- and HTST-treated juices were not sig- untreated smoothies. Furthermore, TPC increased signifi-
nificantly different after pasteurization, by the 28th d of cantly after HPP but gradually decreased during storage.
cold storage, the THC of the HPP-treated juice was higher These results were consistent with the TPC trends observed
than that of the HTST-treated juice. Although HTST for shiikuwasha juice during cold storage.
slightly reduced TFC, the difference was not statistically
significant. The tangeletin and nobiletin contents of fresh Browning
shiikuwasha juice were 53.64 and 82.14 lg/mL, respec-
tively, and changed to 53.91 and 82.59 lg/mL, respec- HTST increases fruit juice browning, which darkens juice
tively, after HPP. Therefore, HPP did not affect tangeletin color. The BI of fresh juice was 0.1 and increased to 1.0
and nobiletin levels. Conversely, the post-HTST levels of and 0.6 after HTST and HPP, respectively (Table 3). The
tangeletin and nobiletin were significantly decreased (50.27 BI of all juices increased during cold storage and was the
and 79.57 lg/mL, respectively). No significant changes highest for the HTST-treated juice, which presented a BI of
occurred in the tangeletin and nobiletin levels of the con- 0.52 on the 28th d of cold storage. This BI was significantly
trol, HPP-, and HTST-treated groups during cold storage. higher than those of the control group (0.43) and HPP-
HPP significantly increased the TPC of Sabah snake grass treated juice (0.44). Analysis of HMF, the primary

123
J Food Sci Technol (March 2022) 59(3):990–1000 995

Table 2 The contents of phenolic content (TPC), total flavanones (TFC), and polymethoxylated flavones (PMF) in Shiikuwasha juice storage at
7 °C
Treatment Time (d)
0 0à 7 14 21 28

TPC (mg/ml)
Control 251.42 ± 2.34Ab – 249.67 ± 3.87Bb 242.37 ± 4.91Ba 237.48 ± 6.10Bab 223.71 ± 3.89Bb
HPP 253.70 ± 3.66Aa 243.81 ± 3.57Ab 239.11 ± 4.15Ab 235.68 ± 7.28Ab 236.62 ± 4.59Ab 231.33 ± 5.27Ab
HTST 250.39 ± 5.52Aa 228.37 ± 5.41Bb 228.75 ± 6.81Bb 231.93 ± 6.47Cb 229.38 ± 7.69Cb 224.18 ± 5.94Cb
TFC (mg/ml)
Control 20.24 ± 1.12Aa – 20.79 ± 1.13Aa 21.42 ± 0.89Aa 20.81 ± 0.92Ba 20.33 ± 1.03Aa
HPP 20.97 ± 0.85Aa 22.21 ± 1.11Aa 21.25 ± 0.88Aa 22.54 ± 0.67Aa 22.24 ± 0.98Aa 21.65 ± 1.17Aa
HTST 20.37 ± 0.80Aa 19.29 ± 0.94Aa 19.24 ± 1.14Aa 19.67 ± 0.64Aa 19.37 ± 0.76Ba 19.34 ± 0.82Aa
Tangeletin (lg/ml)
Control 53.64 ± 0.57Aa – 54.47 ± 0.83Aa 54.10 ± 0.47Aa 55.39 ± 0.37Aa 53.57 ± 0.81Aa
HPP 54.11 ± 0.73Aa 53.91 ± 0.62Aa 54.29 ± 0.65Aa 55.88 ± 0.49Aa 54.61 ± 0.67Aa 54.07 ± 0.65Aa
HTST 53.97 ± 0.64Aa 50.27 ± 0.48Bb 51.29 ± 0.48Bb 50.89 ± 0.77Bb 50.23 ± 0.54Bb 51.22 ± 0.62Bb
Nobiletin (lg/ml)
Control 82.14 ± 1.17Aa – 83.57 ± 0.85Aa 84.67 ± 0.48Aa 82.16 ± 0.91Ba 83.57 ± 0.37Aa
HPP 82.33 ± 1.08Aa 82.59 ± 0.67Aa 81.92 ± 0.47Aa 83.25 ± 0.68Aa 84.49 ± 0.64Aa 83.24 ± 0.80Aa
HTST 82.58 ± 0.98Aa 79.57 ± 0.34Bb 80.11 ± 0.66Bb 79.57 ± 0.52Bb 81.74 ± 0.38Bb 80.64 ± 0.59Bb
All data were the means ± SD, n = 3. 0 , day 0 results before treatment; 0à, day 0 results after treatment. Different capital letters in the same
column indicate significant statistical differences, Different lower case letters in the same row indicate significant statistical differences
(p \ 0.05)

browning reaction product demonstrated that the HMF changes in pH, and the pH values of the control, HTST-
concentrations of the HPP- and HTST-treated juices were treated, and HPP-treated groups did not change signifi-
0.5 and 0.9 lg/100 mL, respectively. Furthermore, HMF cantly throughout the 28 d cold-storage period. The same
could not be detected in fresh (untreated) juice. The HMF trend was observed for TA. During color analysis, it was
content of the HTST-treated juice decreased with time revealed that HPP and HTST caused small changes in color
during cold storage but it was still significantly higher than (4E = 1.2 and 1.47, respectively). By the 28th d of cold
that of the HPP-treated juice by the 28th d of cold storage. storage, the 4E values of the control, HPP-treated, and
Thermal pasteurization promotes browning reactions (e.g., HTST-treated groups were 5.23, 5.4, and 4.37, respec-
sugar oligomerization), which generate products, such as tively. These results were consistent with the changes in
indole-5,6-quinone, and HMF. Gooseberry juice steriliza- BI. Therefore, HPP helped to maintain the color of shi-
tion experiments revealed that browning increased with ikuwasha juice, as it reduced darkening over time. Previous
temperature during thermally-assisted HPP sterilization. studies have also revealed that the pH, TSS, and TA values
However, given the same sterilization time, the BI of the of HPP-treated juices did not change significantly during
juice that did not undergo HPP was significantly higher cold storage. Pei et al. (2018) demonstrated that HPP-
than that of the HPP-treated juice; hence, HPP reduced treated melon juice presented a small change in color
browning (Raj et al. 2019). A study on HPP-treated throughout treatment and thermal inactivation at 85 °C for
watermelon juice determined that the increase in pressure 10 min significantly increased the 4E and TA of melon
or holding time significantly decreased the browning of juice. However, the BI, pH, and TSS of HPP- and HTST-
watermelon juice (Liu et al. 2013). treated melon juice samples were not significantly differ-
ent. Hasni et al. (2020) reported that 400 or 500 MPa HPP
Physicochemical properties treatment was sufficient to suppress microbial growth in
Sabah snake grass juice, and the juice maintained its pH
The changes in physicochemical properties of shiikuwasha and TA. The microbial growth in the untreated and
juice after HPP and HTST treatments are summarized in 300 MPa-treated juice samples caused the pH and TA to
Table 3. The pH of fresh shiikuwasha juice was approxi- decrease and increase, respectively. These findings were
mately 2.3. Neither HTST nor HPP led to significant similar to our results for shiikuwasha juice, as the TA and

123
996 J Food Sci Technol (March 2022) 59(3):990–1000

Table 3 Brown pigment formation, HMF content and Physicochemical properties of Shiikuwasha juice storage at 7 °C
Treatment Time (d)
0 0à 7 14 21 28

BI
Control 0.01 ± 0.01Ad 0.01 ± 0.01Bd 0.12 ± 0.05Bc 0.24 ± 0.06Bb 0.26 ± 0.07Bb 0.43 ± 0.04Ba
HPP 0.01 ± 0.02Ae 0.06 ± 0.02Ad 0.11 ± 0.04Bc 0.26 ± 0.04Bb 0.29 ± 0.04Bb 0.44 ± 0.06Ba
HTST 0.01 ± 0.01Ae 0.10 ± 0.01Ad 0.19 ± 0.04Ac 0.33 ± 0.04Ab 0.37 ± 0.07Ab 0.52 ± 0.09Aa
HMF (lg/100 mL)
Control ND – ND ND ND ND
HPP ND 0.5 ± 0.1A 0.5 ± 0.1Aa 0.5 ± 0.1Aa 0.5 ± 0.1Aa 0.5 ± 0.1Aa
HTST ND 0.9 ± 0.1A 0.9 ± 0.02Ab 0.8 ± 0.1Ab 0.8 ± 0.1Ab 0.7 ± 0.1Ab
pH
Control 2.33 ± 0.01Aa – 2.35 ± 0.01Aa 2.32 ± 0.01Aa 2.30 ± 0.03Aa 2.32 ± 0.02Aa
HPP 2.31 ± 0.01Ab 2.24 ± 0.02Bc 2.36 ± 0.01Aa 2.27 ± 0.01Ac 2.27 ± 0.01Ac 2.31 ± 0.01Ab
HTST 2.32 ± 0.01Aa 2.31 ± 0.01Aa 2.31 ± 0.01Aa 2.30 ± 0.01Aa 2.26 ± 0.02Aa 2.32 ± 0.02Aa
TA (%)
Control 6.26 ± 0.09Aa – 6.16 ± 0.07Ab 6.10 ± 0.09Ab 6.33 ± 0.03Aa 6.30 ± 0.06Aa
HPP 6.25 ± 0.09Aa 6.17 ± 0.12Aa 6.49 ± 0.21Aa 6.21 ± 0.13Aa 6.34 ± 0.04Aa 6.13 ± 0.15Aa
HTST 6.22 ± 0.05Aa 6.20 ± 0.07Ab 6.20 ± 0.07Ab 6.26 ± 0.06Ab 6.40 ± 0.08Ab 6.39 ± 0.03Ab
TSS
Control 7.33 ± 0.06Ab – 7.87 ± 0.06Aa 7.80 ± 0.01Aa 7.87 ± 0.06Aa 7.73 ± 0.06Aa
HPP 7.26 ± 0.06Ab 7.33 ± 0.06Ab 7.83 ± 0.06Aa 7.77 ± 0.06Aa 7.63 ± 0.06Ba 7.80 ± 0.01Aa
HTST 7.45 ± 0.15Ab 7.37 ± 0.14Ab 7.83 ± 0.06Aa 7.83 ± 0.06Aa 7.90 ± 0.10Aa 7.70 ± 0.10Aa
Color(D)
Control - – 3.83 ± 0.49Ab 4.63 ± 0.06Aa 4.83 ± 0.47Aa 5.23 ± 0.55Aa
HPP 0.25 ± 0.13Ad 1.20 ± 0.10Ac 3.50 ± 0.30Aa 3.60 ± 0.26Bb 4.40 ± 0.10Ba 4.37 ± 0.32Ba
HTST 0.27 ± 0.11Bd 1.47 ± 0.15Ac 3.97 ± 0.38Ab 4.60 ± 0.78Ab 4.90 ± 0.36Ab 5.40 ± 0.17Aa
All data were the means ± SD, n = 3. 0 , day 0 results before treatment; 0à, day 0 results after treatment. Different capital letters in the same
column indicate significant statistical differences, Different lower case letters in the same row indicate significant statistical differences
(p \ 0.05)

pH of HPP-treated shiikuwasha juice did not change sig- physicochemical properties of the juice. Hence, the color
nificantly throughout the 28 d cold-storage experiment, and flavor of HPP-treated juice were more similar to those
owing to a lack of microbial growth. 4E and TA of the of fresh juice than to those of HTST-treated juice.
wheatgrass juice treated at 75 °C for 15 s increased 7.75-
and 1.7-fold, respectively; conversely, 4E of the wheat- Aroma components
grass juice treated at 500 MPa for 60 s only increased 1.06-
fold and TA remained unchanged (Ali et al., 2019). Nayak Aroma is an important quality indicator of shiikuwasha
et al. (2017) compared the quality of apple juice samples juice, and aroma analysis indicated that its primary aroma
subjected to HTST and HPP pasteurization and revealed components are a-methylmaleic anhydride, butylated
that the pH, TSS, and TA of the samples were not signif- hydroxytoluene, linoelaidic acid, cis-butenedioic anhy-
icantly different throughout 60 d of storage. Furthermore, dride, limonene and palmitic acid (Fig. 2 and Table 4).
the increase in total bacteria count of the HPP-treated juice HPP or HTST treatment affected some aroma components;
was lower than 1.0 log over 60 d, and its 4E was smaller moreover, HTST (but not HPP) significantly decreased the
than that of the HTST-treated juice. Therefore, HPP was concentrations of acetic acid and diethyl acetal and both
effective at preserving the quality and prolonging the shelf HPP and HTST significantly decreased the concentrations
life of apple juice. These findings were in agreement with of a-methylmaleic anhydride, cis-butenedioic anhydride,
our results, as we also demonstrated that HPP outper- and butylated hydroxytoluene. In addition, both HPP and
formed HTST for maintaining the original HTST increased the concentrations of limonene, c-

123
J Food Sci Technol (March 2022) 59(3):990–1000 997

Fig. 2 Chromatograms of volatile compounds from C. depressa juice a Fresh juice (control); b HTST juice; and c HPP juice

terpinene, palmitic acid, linoelaidic acid, and linolenic terms of retention of aroma components. Aroma compo-
acid. Furthermore, the limonene and c-terpinene concen- nents are important quality indicators for fruit juice prod-
trations of HPP-treated juice were higher than those of ucts. Many studies have demonstrated that room-
HTST-treated juice. Hence, HPP outperformed HTST in temperature HPP pasteurization affected the aroma

123
998 J Food Sci Technol (March 2022) 59(3):990–1000

Table 4 Volatile compounds of of Shiikuwasha juice storage at 7 °C

Peak no. Retention time Volatile aroma component Fresh juice (ppm) TP juice (ppm) HPP juice (ppm)

1 5.842 b-Pinene 0.104 ± 0.018 0.128 ± 0.009 0.157 ± 0.010

2 6.167 3-Penten-2-one (Ethylidene acetone) 0.304 ± 0.021 0.376 ± 0.020 0.412 ± 0.021
3 7.405 b-Myrcene 0.233 ± 0.017 0.286 ± 0.010 0.330 ± 0.017
4 7.735 a-Terpinene 0.138 ± 0.011 0.102 ± 0.009 0.122 ± 0.015
5 8.279 Limonene 6.039 ± 0.048 7.398 ± 0.064 8.663 ± 0.072
6 8.490 b-phellandrene 0.111 ± 0.007 0.072 ± 0.007 0.084 ± 0.015
7 9.656 c-Terpinene 2.898 ± 0.039 3.545 ± 0.026 4.133 ± 0.029
8 10.331 p-Cymene 0.168 ± 0.010 0.181 ± 0.007 0.197 ± 0.004
9 15.549 Acetic acid 3.201 ± 0.061 2.885 ± 0.028 3.105 ± 0.031
10 18.302 Diethyl acetal 4.903 ± 0.085 3.858 ± 0.042 4.537 ± 0.027
11 19.017 Linalool 1.145 ± 0.029 1.302 ± 0.014 1.324 ± 0.011
12 20.342 Caryophyllene 0.400 ± 0.022 1.498 ± 0.017 0.576 ± 0.024
13 20.424 Terpinen-4-ol 0.275 ± 0.013 0.282 ± 0.014 0.287 ± 0.008
14 22.404 a-Caryophyllene 0.087 ± 0.008 0.106 ± 0.015 0.124 ± 0.006
15 23.190 a-Methylmaleic anhydride 12.442 ± 0.102 11.160 ± 0.094 11.755 ± 0.092
16 23.538 cis-Butenedioic anhydride 6.211 ± 0.073 5.989 ± 0.062 5.554 ± 0.084
17 27.028 2-Pentenoic acid 0.297 ± 0.011 0.243 ± 0.014 0.247 ± 0.013
18 29.171 Butylated Hydroxytoluene 8.882 ± 0.093 7.544 ± 0.051 7.802 ± 0.089
19 30.557 6-Ethoxy-2,6-dihydropyran-3-one 0.876 ± 0.014 0.767 ± 0.015 0.811 ± 0.012
20 34.943 Eugenol 0.122 ± 0.016 0.095 ± 0.008 0.107 ± 0.007
21 35.110 c-Eudesmole 0.142 ± 0.007 0.117 ± 0.007 0.177 ± 0.010
22 35.538 2-Methoxy-4-vinylphenol 2.959 ± 0.009 2.101 ± 0.039 2.088 ± 0.021
23 36.540 a-Cadinol 0.242 ± 0.012 0.318 ± 0.005 0.344 ± 0.006
24 37.902 2-methyloctacosane 0.336 ± 0.019 0.375 ± 0.006 0.396 ± 0.017
25 28.920 Eicosane 0.744 ± 0.023 0.820 ± 0.019 0.858 ± 0.028
26 39.819 Coumaran 0.574 ± 0.029 0.412 ± 0.008 0.401 ± 0.016
27 41.063 Eicosane 0.470 ± 0.018 0.507 ± 0.014 0.525 ± 0.027
28 42.045 Dodecanoic acid 0.262 ± 0.016 0.287 ± 0.006 0.320 ± 0.019
29 42.180 2-methyloctacosane 0.653 ± 0.031 0.738 ± 0.016 0.777 ± 0.025
30 46.243 Tridecanoic acid 0.421 ± 0.015 0.479 ± 0.025 0.523 ± 0.026
31 50.203 Palmitic acid 5.812 ± 0.041 7.253 ± 0.038 7.452 ± 0.070
32 50.849 9-Hexadecenoic acid 0.925 ± 0.016 1.129 ± 0.017 1.088 ± 0.008
33 54.362 Squalene 1.297 ± 0.021 1.556 ± 0.025 1.726 ± 0.038
34 55.271 Octadecanoic acid 0.949 ± 0.028 0.966 ± 0.012 1.014 ± 0.007
35 56.120 9-Octadecenoic acid 2.185 ± 0.014 2.275 ± 0.040 2.492 ± 0.063
36 56.322 cis-Vaccenic acid 2.733 ± 0.032 3.572 ± 0.027 3.462 ± 0.048
37 57.887 Linoelaidic acid 7.763 ± 0.057 10.526 ± 0.065 10.315 ± 0.011
38 60.484 Linolenic acid 2.464 ± 0.032 3.459 ± 0.025 3.214 ± 0.017
All data were the means ± SD, n = 3

components of juices less than HTST pasteurization. Chen effects of HPP and HTST treatments on the aroma of
et al. (2015) demonstrated that the aldehyde, alcohol, and mango juice. Twelve aroma-active compounds were iden-
ketone concentrations of HPP-treated asparagus juice were tified in fresh mango juice, and it was determined that both
higher than those of HTST-treated counterpart, and the HPP and HTST caused the loss of a few aroma compounds.
difference was attributed to the thermal sensitivity of Nonetheless, the HPP-treated juice retained more aroma
aroma components. Zhang et al. (2019) used odor activity compounds than the HTST-treated juice and its aroma was
values and detection frequency analysis to compare the more similar to that of fresh mango juice. Similar results

123
J Food Sci Technol (March 2022) 59(3):990–1000 999

were reported when the pasteurization of Thai green-chili Funding This research work was supported by the Ministry of Sci-
pastes was investigated. Aroma compounds gradually dis- ence and Technology, MOST 109–2622-E-150 -010 -CC2, Taiwan,
Republic of China.
sipated and disappeared during an 8-week storage experi-
ment; however, the pressurized pastes retained greater Data availability Yes.
ester, aldehyde, and alcohol concentrations thermally
treated pastes (Apichartsrangkoon et al. 2014). Kebede Declarations
et al. (2018) compared the effects of pulsed electric field
Conflict of interest The authors declare that they have no conflict of
(PEF) processing, HPP, and HTST on the volatile aroma interest to declare.
compounds of apple juice and reported that mild thermal
pasteurization (30 °C) followed by PEF treatment led to
the best retention of odor-active volatiles (e.g., (E)-2-hex- References
enal and hexyl acetate), followed by HPP, and lastly,
HTST. During cold storage, the concentrations of the Akachi T, Shiina Y, Ohishi Y, Kawaguchi T, Kawagishi H, Morita T,
aroma components of the PEF- and HPP-treated juices Mori M, Sugiyama K (2010) Hepatoprotective effects of
flavonoids from shekwasha (Citrus depressa) against D-galac-
were always higher than those of the HTST-treated juice. tosamine-induced liver injury in rats. J Nutr Sci Vitaminol
Hence, nonthermal pasteurization methods preserve the 56:60–67
aroma compounds of fruit juices better than thermal pas- Ali N, Popovi V, Koutchma T, Warriner K, Zhu Y (2019) Effect of
teurization methods. thermal, high hydrostatic pressure, and ultraviolet-C processing
on the microbial inactivation, vitamins, chlorophyll, antioxi-
dants, enzyme activity, and color of wheatgrass juice. J Food
Process Eng 43:13036
Conclusion Asikin Y, Fukunaga H, Yamano Y, Hou DX, Maedae G, Wada K
(2014) Effect of cultivation line and peeling on food composi-
tion, taste characteristic, aroma profile, and antioxidant activity
This study demonstrated that a 600 MPa/1.5 min HPP of Shiikuwasha (Citrus depressa Hayata) juice. J Sci Food Agri
treatment reduced the E. coli O157:H7 counts of shi- 94:2384–2392
ikuwasha juice by 5 logs. Because HPP preserved the AOAC, Official Methods of Analysis (15th edn). Association of
quality of shiikuwasha juice and provided the same Official Analytical Chemists, Arlington, VA. 1990.
Apichartsrangkoon A, Chaikham P, Srisajjalertwaja S (2014) Storage
microbiological safety as conventional thermal pasteur- stability of aroma volatiles in ultra-high pressure and thermally
ization methods, it can be used as a nonthermal replace- treated Thai green-chili pastes (Nam PrigNhum). Food Bio
ment for thermal pasteurization. The physicochemical and 5:91–100
browning parameters of 28 d cold-stored HPP-treated juice Buzrul S (2020) High hydrostatic pressure inactivation of microor-
ganisms: a probabilistic model for target log-reductions. Int J
samples revealed that HPP maintained the microbiological Food Microbiol 309:108330
safety of the juice; preserved the fresh juice color, acidity, Fernandez MV, Denoya GI, Jagusa RJ, Vaudagnab SR, Agüero MV
and TSS; reduced browning, and decreased HMF concen- (2019) Microbiological, antioxidant and physicochemical stabil-
tration. Hence, the quality of the HPP-pasteurized juice ity of a fruit and vegetable smoothie treated by high pressure
processing and stored at room temperature. LWT - Food Sci
was more similar to that of fresh juice than to that of the Technol 105:206–210
HTST- pasteurized juice. Analysis of the antioxidant con- Gouvea FS, Padilla-Zakour OI, Worobo RW, Xavier BM, Walter
tents and aroma components of the juices revealed that EHM, Rosenthal A (2020) Effect of high-pressure processing on
HPP increased the retention of aroma components, reduced bacterial inactivation in açaı́ juices with varying pH and soluble
solids content. Innov Food Sci Emerg Technol 66:102490
heat damage of aroma components, and caused antioxidant Hasni HN, Koh PC, Noranizan MA, Tahir PNFMM, Mohamad A,
concentrations (TPC, TFC, and PMFs) to increase more Limpot N, Hamid N, Aadil RM (2020) High-pressure processing
than HTST did. Hence, we concluded that HPP is suit- treatment for ready-to-drink Sabah snake grass juice. J Food
able for the pasteurization of shiikuwasha juice. Process Prese 44:14508
Huang HW, Lung HS, Yang BB, Wang CY (2014) Responses of
microorganisms to high hydrostatic pressure processing. Food
Acknowledgements This research work was supported by the Min- Control 40:250–259
istry of Science and Technology, MOST 109-2622-E-150 -010 -CC2, Huang HW, Wu SJ, Lu JK, Shyu YT, Wang CY (2017) Current status
Taiwan, Republic of China. and future trends of high-pressure processing in food industry.
Food Control 72:1–8
Authors’ contributions Y-YL performed the analysis. J-HC per- Kebede B, Lee PY, Leong SY, Kethireddy V, Ma Q, Aganovic K,
formed the analysis. Y-CL performed the analysis. Y-TH collected Eyres GT, Hamid N, Oey IA (2018) Chemometrics approach
the data, and contributed data or analysis tools. C-YW conceived and comparing volatile changes during the shelf life of apple juice
designed the analysis, contributed data or analysis tools, and wrote the processed by pulsed electric fields, high pressure and thermal
paper. pasteurization. Foods 7:169
Lin SY, Chen IZ (2006) Native Citrus in Taiwan and current status of
research and utilization. Seed Nursery 8:1–12

123
1000 J Food Sci Technol (March 2022) 59(3):990–1000

Liu Y, Zhao XY, Zou L, Hu XS (2013) Effect of high hydrostatic Takenaka M, Nanayama K, Isobe S, Ozaki K, Miyagi K, Sumi H,
pressure on overall quality parameters of watermelon juice. Food Toume Y, Morine S, Ohta H (2007) Effect of extraction method
Sci Technol Int 19:197–207 on yield and quality of citrus depressa juice. Food Sci Technol
Lee YH, Charles AL, Kung HK, Ho CT, Huang TC (2010) Extraction Res 13:281–285
of nobiletin and tangeretin from Citrus depressa Hayata by United States Department of Agriculture (USDA) (2012) High
supercritical carbon dioxide with ethanol as modifier. Ind Crop pressure processing (HPP) and inspection program personnel
Prod 31:59–64 (IPP) verification responsibilities. FSIS Directive 6120.1
Lee YS, Cha BY, Saito K, Choi SS, Wang XX, Choi BK, Yonezawa Wang CY, Huang HW, Hsu CP, Yang BB (2016) Recent advances in
T, Teruya T, Nagai K, Woo JT (2011) Effects of a Citrus food processing using high hydrostatic pressure technology. Crit
depressa Hayata (shiikuwasa) extract on obesity in high-fat diet- Rev Food Sci Nutr 56:527–540
induced obese mice. Phytomedicine 18:648–654 Yuan B, Danao MGC, Stratton JE, Weier SA, Weller CL, Lu M
Nayak PK, Rayaguru K, Krishnan KR (2017) Quality comparison of (2018) High pressure processing (HPP) of aronia berry purée:
elephant apple juices after high-pressure processing and thermal effects on physicochemical properties, microbial counts, bioac-
treatment. J Sci Food Agri 97:1404–1411 tive compounds, and antioxidant capacities. Innov Food Sci
High pressure processing (HPP) and inspection program personnel Emerg Technol 47:249–255
(IPP) verification responsibilities. FSIS Directive 6120.1 Zhang W, Dong P, Lao F, Liu J, Liao X, Wu J (2019) Character-
Raj AS, Chakraborty S, Rao PS (2019) Thermal assisted high- ization of the major aroma-active compounds in Keitt mango
pressure processing of Indian gooseberry (Embilica officinalis juice: comparison among fresh, pasteurization and high hydro-
L.) juice – Impact on colour and nutritional attributes. LWT - static pressure processing juices. Food Chem 289:215–222
Food Sci Technol 99:119–127
Pei L, Hou S, Wang L, Chen J (2018) Effects of high hydrostatic
Publisher’s Note Springer Nature remains neutral with regard to
pressure, dense phase carbon dioxide, and thermal processing on
jurisdictional claims in published maps and institutional affiliations.
the quality of Hami melon Juice. J Food Process Eng 41:12828

123