International Food Research Journal 26(5): 1505-1513 (October 2019)
Journal homepage: http://www.ifrj.upm.edu.my

Effects of acidic treatment on the physicochemical, microstructural, andmicrobiological properties of fresh-cut lotus root during storage
Lara, G. R., 1,2Uemura, K., 3Khalid, N., 1,2Kobayashi, I., 2Takahashi, C.,
1,2,4
Nakajima, M. and 1,2,4*Neves, M. A.

1,2

Tsukuba Life Science Innovation Program, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba,
Ibaraki 305-8572, Japan
2
Food Research Institute, NARO, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan3
School of Food and Agricultural Sciences, University of Management and Technology, Lahore
54000, Pakistan
4
Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba,
Ibaraki 305-8572, Japan
1

Article history

Abstract

Received: 29 March, 2019
Received in revised form:
28 June, 2019
Accepted: 6 August, 2019Freshly cut slices of lotus root are a popular food item in various countries; however, problems
such as enzymatic browning affect the storage stability of these products. In the present work,
we evaluated for the first time the effect of low pH treatments on the overall quality of fresh-cut
lotus root (physicochemical, microbiological, and morphological properties) during storage.
The effect of acidic treatment, using citric acid solution at pH 2 or pH 4 at 2% w/w and 0.002%
w/w, respectively, will help to improve our understanding of the mechanism of browning and therole of polyphenol oxidase in catalysing browning reactions. Treatments were prepared using
citric acid and were applied to 5 mm thick fresh-cut lotus for 2 min. Samples were then stored
for 16 days at 5°C. We found that the whiteness values (L*) of pH 2 (68.76) and pH 4 (65.77)
treated samples were significantly higher than the control (57.37), suggesting an enhancementof colour quality during storage. We also observed reduced polyphenol oxidase (PPO) enzyme
activity for the treated samples as compared to the control (12.5, 16.5, and 17.13 U/min-mL,
respectively), as well as reduced total phenolic content (46.5, 44.3, and 57.8 mg/100 g sample)
and microbial counts. Despite these promising results, we also observed undesirable effects asa result of the pH treatments 2 and 4, including tissue softening and microstructural changes.
However, we believe that our findings will be significant in the development of an optimised
formulation for dipping treatments of fresh-cut lotus root products.

Keywords
Lotus root
Enzymatic browning
Microstructure
Storage stability
pH reduction© All Rights Reserved

Introduction
An expansion of the market for fresh-cut fruits
and vegetables has been reported in recent years(Qadri et al., 2015). Convenience and freshness are
the two demands associated with freshly cut products(Francis et al., 2012). In addition, the International
Fresh-cut Produce Association (Garrett, 1998) has
emphasised that the recent increase in fresh-cut
produce is related to the increased availability ofhealth information and the typically busier lifestylesof consumers, thus putting a higher value on fresh,
nutritious and convenient food products. However,
fresh-cut products are prone to increased perishability*Corresponding author.
Email: marcos.neves.ga@u.tsukuba.ac.jp

and a shorter shelf life as compared to whole products
due to the physical stress such as peeling, cutting,slicing, shredding, trimming and/or coring, and theremoval of protective epidermal cells (Watada et al.,1996).
Elevated respiration rates and ethylene
production, as well as oxidative browning reactions,lipid oxidation or enhanced water loss are some of
the undesirable changes that occur in these products(Facundes et al., 2013). One of the most perceivabledamages in fresh-cut fruits and vegetables is
enzymatic browning (Roura et al., 2008). Thisphenomenon involves the oxidation of phenols intoo-quinones, which are further induced by the action of

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polyphenol oxidase (PPO) (Altunkaya and Gokmen,
2008). The final products of oxidation are brown, redor black pigmentation (Garcia and Barrett, 2002).
Factors involved in enzymatic browning include
oxygen, PPO activity, and phenolic substrates(Shinoda, 2005; Holderbaum et al., 2010).
Recently, fresh-cut lotus (Nelumbo nucifera) root
slices have drawn the attention of researchers and theindustry as a novel minimally-processed vegetable(Zhang et al., 2013). However, fresh-cut lotus root isprone to enzymatic browning, thus deteriorating its
quality and shortening its shelf-life. Xing et al. (2010)
found that browning and oxidation occur in untreatedfresh-cut lotus root slices after two days of storage,
making this product undesirable in the market, while
Hu et al. (2014) reported that PPO and phenylamine
ammonia lyase (PAL) activity increased after one
day of storage in fresh-cut lotus root products, which
resulted in increased browning indices. However, tothe best of our knowledge, there are few available
studies on the control of fresh-cut lotus root
enzymatic browning. Xing et al. (2012) studied the
combined effects of anti-browning agents, cinnamon
oil fumigation, and moderate vacuum packaging.
Zhang et al. (2013) considered carbon monoxide on
browning of fresh-cut lotus root. Son et al. (2015)
evaluated the effects of ascorbic acid, heat treatment,
and modified-atmosphere packaging. However, no
studies have reported the effects of low pH treatmentson the overall quality of fresh-cut lotus root duringstorage. Chiabrando and Giacalone (2012) foundthe use of 1% ascorbic acid, 1% citric acid and 1%
calcium chloride was effective in reducing enzymaticbrowning in fresh-cut apples. Similar results
were also reported by Sanchís et al. (2017), whoemphasised the combined use of 1% ascorbic acid,
1% citric acid, and controlled atmosphere packaging
for decreasing flesh colour browning and loss offirmness in fresh-cut persimmons during storage. The
present work thus investigated the effect of acidic
treatments on the physicochemical, microstructural,and microbiological properties of fresh-cut lotus root
during 16 days of storage.
Materials and methods
Chemicals
The chemical reagents anhydrous citric acid,polyvinylpyrrolidone, catechol, sodium phosphate,
Folin-Ciocalteu, gallic acid, and standard method
agar were purchased from Wako Pure Chemical
Industries, Ltd. (Osaka, Japan). Anhydrous citric acidwas used to adjust the pH for the acidic treatments.

Preparation of acidic solutions
For the preparation of the pH 2 and pH 4
treatments, 2 g and 0.002 g of anhydrous citric acid,respectively, were weighed and added to Milli-Q
water to obtain a final weight (w/w basis) of 100 g.
The citric acid solutions were magnetically stirred(hot stirrer, HS 360 H; As One, Tokyo, Japan) for
1 min at room temperature (25°C) to assure thecomplete dissolution of the citric acid. The resulting
pH of each citric acid solution was verified (827 pHlab; Metrohm, Herisau, Switzerland) before use as
the acidic treatment for the fresh-cut lotus root.
Sample preparation
Three experimental treatments (control, pH 2, andpH 4) were prepared. The control consisted of freshly
sliced lotus root without any pH adjustment. The lotus
root samples were obtained from a local supermarket
in Tsukuba, Japan, and were confirmed to be freshand of good quality by evaluating their whiteness andthe length of duration on the supermarket shelf. The
samples were thoroughly washed under tap water,then peeled and sliced into 5-mm thick sections. The
dipping technique was used for the acidic treatmentof the fresh-cut lotus root. Five slices (5-mm thick)of fresh-cut lotus root per treatment were immersed
for 2 min. Preliminary studies were performed prior
to this experiment with regards to dipping time; only
slight differences in the storage of fresh-cut lotus root
were found with a longer dipping time. The freshcut lotus root samples were then quickly dried byallowing the excess citric acid to drip from the surfaceof the samples. The fresh-cut lotus root slices were
packed into polyethylene bags (60 μm × 160 mm ×
260 mm; GT-1626, Sigma Tube, Tokyo, Japan) andwere stored for 16 days at 5°C to evaluate the storage
stability parameters of colour, texture, pH, enzymatic
activity, total phenolic content, and microbiologicalproperties.
Evaluation of physicochemical changes duringstorageColourColour changes during the 16 days of storage
were monitored using a spectrophotometer (CM-5;
Konica Minolta, Tokyo, Japan). The 16-day storage
time was found to be sufficient to observe the colour,
textural, enzymatic, and microstructural changes andto determine the stage at which enzymatic browning
occurs after the acidic treatments. The L* values wereused to express lightness or darkness of the treatedand untreated samples.

Lara et al./IFRJ 26(5) : 1505-1513

TextureThe textural changes were evaluated using the
Texture Profile Unit (TPU 2DL; Yamaden, Tokyo,
Japan). The 5-mm thick samples were longitudinallycompressed with a probe at a deformation rate of
2.5 mm/s. The hardness value, which is defined as
the peak force at the first compression cycle, wasexpressed in Newton (N).
pH measurement
The pH measurement was adapted from Son etal. (2015) with a few modifications. Twenty gramsof each fresh-cut lotus root sample was homogenised
in 40 mL Milli-Q water for 30 s using a multipurpose
blender (Vitaprep 3; Vitamix Corp., USA). The pHof the blended samples was determined using a
digital pH meter (827 pH lab; Metrohm, Herisau,
Switzerland).
Weight loss
The weight loss of the samples during storage
was also evaluated. The values were expressed aspercentage losses from the initial weight of thesample using Equation 1, as follows:% WL =

w0-wd

× 100
w0		

(Equation 1)

where w0 = initial weight (g) at day 0 and wd =final weight (g) at each storage day interval.
Polyphenol oxidase activity
The method for the determination of the
enzymatic activity of PPO was based on the standard
procedures reported by Son et al. (2015) with a few
modifications. Ten grams of each fresh-cut lotus
root sample per treatment was homogenised in
sodium phosphate buffer (0.2 mol/L, pH 7.0 with 2%
polyvinylpyrrolidone) at a ratio of 2.0 mL of buffer
per 1 g of lotus root in an ice bath. The homogenates
were centrifuged using a high-speed refrigerated
centrifuge (Tomy MX -307; Tomy Seiko, Tokyo,
Japan) at 12,000 g for 10 min at 4°C. The supernatant
was separated from the filtrate and was reserved
for analysis. Enzymatic analysis was performed byevaluating the increase of absorbance at 410 nm forcatechol at 25°C using a spectrophotometer (UVVis, V-530; JASCO Inc., Tokyo, Japan). The 0.2
mL supernatant was then added to 2.8 mL of thecatechol substrate solution (0.02 mol/L catechol in
0.05 mol/L sodium phosphate buffer, pH 7). The
catechol substrate solution alone (catechol in sodium
phosphate buffer) was used as a reference. The
enzymatic activity (units/(min-mL) enzyme) was

1507

determined using the linear section of the activity
curve. A change in 0.001 in the absorbance value per
min was defined as 1 unit of PPO enzyme activity.
Total phenolic content
The method used to evaluate the total phenoliccontent of the fresh-cut samples was based onprocedures reported by Xing et al. (2012) with a few
modifications. Ten grams of lotus root sample pertreatment was added to 25 mL Milli-Q water aftercrushing with a mortar and pestle. Centrifugation was
performed for 15 min at 5,000 g. One millilitre of thesupernatant was mixed with 5 mL Folin-Ciocalteu
solution (1 mL of FC reagent in 10 mL Milli Q
water). Between 0 and 8 min of reaction time, 4 mL
(7.5% w/v sodium carbonate solution) was slowly
added. The samples were incubated for 2 h at 30°C
in a controlled incubator (Panasonic cool incubator;
ICI-200, Tokyo, Japan). The spectrophotometer wasused to measure the absorbance of the solution at
760 nm. Gallic acid was used as a standard for the
analysis. Acid solutions at concentrations ranging
from 0.01 - 0.1 mg/mL dissolved in Milli-Q water
were prepared prior to plotting the standard curve.
The results were expressed as mL of gallic acid per
100 grams of fresh-cut lotus root.
Evaluation of microbiological count during storage
Prior to the microbiological analysis, a slice ofeach of the fresh-cut lotus root samples (control, pH
2 treatment, and pH 4 treatment) was cut into three
portions to ensure the same flora of microorganisms
were present. For the analyses, one slice of thelotus root (10 g) was aseptically removed fromthe polyethylene bags and transferred into a sterile
stomacher bag. Ten millilitres of Milli-Q water wereadded to the sample prior to homogenisation. Thesesamples were then homogenised in the stomacher
for 1 min. One millilitre of the sample was pipetted
for serial dilutions. One millilitre per dilution (101to 10-6) was placed onto petri dishes, onto whichplate count agar (PCA) was poured before incubatingfor 24-48 h at 37°C. The resulting microorganism
colonies were manually counted. The results wereexpressed as log CFU/g (colony forming units per gof lotus root sample). All microbiological tests were
performed at least in duplicate on a clean bench atroom temperature.
Evaluation of morphological changes duringstorageMorphological analyses were performed using ascanning electron microscope (SEM Miniscope 1000;
Hitachi, Tokyo, Japan). Prior to microscopy, 2 g of

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samples per treatment were sliced into 1 mm3 cubes
and oven-dried at 100°C for 5 h to achieve 16% ± 2.0
total solids content (w/w). After drying, the samples
were mounted onto an aluminium sample probe formagnification at 500×. The samples were observed
after 16 days storage at 5°C.
Statistical analysis
All experiments were performed at least in
triplicate, and the results were reported as the mean
± standard deviation (SD) of the measurements.
SPSS Statistics (IBM Statistics 22; New York, USA)was used to perform one-way analysis of variance(ANOVA). The Duncan multiple range test was used
as a post hoc test at a significance of 95% probability.
The significant difference among the treatments (p <0.05) was indicated using different superscript letters.
Results and discussion
Changes in colour of acid-treated lotus root
samples during storage
Colour is one of the most important quality
attributes that consumers take into consideration
when purchasing a food product. Table 1(a) showsthe effect of pH 2 and pH 4 on the whiteness (L*values) of the lotus root samples. L* values wereused to indicate the lightness of the samples. A higher
whiteness (L* value) indicates a brighter sample
surface (Du et al., 2009). A decreasing whiteness (L*values) was observed in all samples, which can be
attributed to enzymatic browning. After four daysof storage, the control samples showed significant
differences in terms of colour as compared to thesamples treated with pH 2 and pH 4. This suggeststhat the control samples exhibited the highest rateof enzymatic browning as compared to the pH 2and pH 4-treated samples. However, no significant
difference (p > 0.05) in whiteness (L* values) was
observed after 16 days of storage in any of thesamples, indicating that browning occurred in all thesamples.
Altunkaya and Gokmen (2008) found that citricacid functions via two inhibitory mechanisms: (1) bylowering the pH below that required for optimal PPO
activity at pH 4.0-8.0 and (2) by chelating copper. In
the present work, citric acid was used to lower the
pH to determine whether two pH values influenced
the storage quality of fresh-cut lotus root. We foundthat a lower pH helped to reduce PPO activity, thus
reducing enzymatic browning, observed in the pH 2
and pH 4 samples. However, after 12 days of storage,
enzymatic browning also occurred in the pH 2 andpH 4 samples, which can be attributed to the partial

inactivation of PPO during acidic treatments. Li et al.
(2018) reported that at pH 2, the PPO activity in freshcut potato only decreased to 46.10%, suggesting only
the partial inactivation of the enzyme. Ali et al. (2015)
confirmed the same results in their study, where
1% citric acid was found to reduce the browningindices of lettuce heads during eight days of storage
in comparison to the untreated samples. However,despite the reduction of browning indices in the citricacid treated samples, browning was observed after
only two days of storage. Ali et al. (2015) proposedthat citric acid does not completely avoid browning,
but delays the development of browning, indicating
its action as a PPO inhibitor. Based on our results,
pH 2 treatment showed superior results than the pH
4 treatment in terms of enhancing the colour qualityof the samples.
Changes in texture of acid-treated lotus root
samples during storage
Toivonen and Brummell (2008) demonstrated thatthe disruption of sub-cellular compartmentalisation
occurs at the surfaces of the sliced lotus roots, which
results in undesirable reactions. In addition, changes
in turgor pressure and the structure and compositionof cell walls, particularly cell wall polysaccharides,
results in tissue softening (Pinheiro and Almeida,2008; Krall and McFeeters, 1998). Table 1(b) showsthe effect of pH on the texture, indicated as hardness,of the fresh-cut lotus root samples during 16 days ofstorage.
A decreasing hardness in all samples (Table
1(b)) indicates tissue softening. Based on theseresults, the pH 2-treated samples showed the lowest
hardness values after 16 days of storage, followedby the pH 4-treated samples and the control. Son etal. (2015) found similar decreasing hardness values
during storage, which can be attributed to weight loss
associated with loss of water (Bico et al., 2009).
Changes in pH of acid-treated lotus root samples
during storage
Changes in the pH values of the treated and thecontrol fresh-cut lotus roots were also evaluated in
the present work, as shown in Table 1(c). Our results
showed an increase in the pH of the untreated, pH
2-treated, and pH 4-treated samples during the 16
days of refrigerated storage; however, significant
differences (p < 0.05) were observed for the pH 2- andpH 4-treated samples. Gil et al. (2006) and Facundes
et al. (2013) compared the changes in quality andnutrient retention in fresh-cut apples during storage.
Their results showed an increase in pH for fresh-cut
apple slices during storage, which was attributed to a

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Table 1. Physicochemical changes of fresh-cut lotus root treated at different pH’s during storage at 5°C.
Treatment

Storage time (day)
0

4

8

12

16

a) Colour (L*)
Control

68.26 ± 0.16a

64.84 ± 0.67b

57.37 ± 1.53c

53.04 ± 0.84b

52.72 ± 1.45a

pH 2.0a69.96 ± 0.95

a69.26 ± 1.23

a68.76 ± 1.09

a58.87 ± 1.09

54.7 ± 2.22a

pH 4.070.14 ± 0.73a

69.44 ± 0.16a

65.77 ± 1.40b

60.26 ± 2.56a

54.5 ± 1.24aControl

44.19 ± 2.33a

35.7 ± 3.34b

21.66 ± 1.37a

17.52 ± 1.92a

16.08 ± 1.31a

pH 2.0a46.01 ± 3.15

a37.01 ± 1.48

a21.13 ± 2.42

a18.55 ± 2.87

12.24 ± 0.66c

pH 4.044.14 ± 0.40a

30.65 ± 0.87c

24.34 ± 0.77a

18.82 ± 2.39a

13.48 ± 0.66b

Control

6.42 ± 0.07a

6.45 ± 0.12a

6.48 ± 0.01a

6.49 ± 0.03b6.57 ± 0.17a

pH 2.04.81 ± 0.03b

4.9 ± 0.11b5.12 ± 0.08b5.15 ± 0.06c

5.43 ± 0.00b

pH 4.0a6.33 ± 0.02

a6.40 ± 0.03

a6.55 ± 0.03

a6.74 ± 0.03

6.76 ± 0.23a

Control

0.00 ± 0.00

0.92 ± 0.43a

2.57 ± 0.35a

2.93 ± 0.86a

4.81 ± 0.71a

pH 2.00.00 ± 0.00

2.03 ± 0.23a

3.55 ± 0.67ab

5.33 ± 0.98ab

6.75 ± 1.75a

pH 4.00.00 ± 0.00

0.66 ± 0.76

1.28 ± 0.91b1.99 ± 0.37

4.09 ± 2.19ab) Texture (N)c) pHd) Weight Loss (%)abe) PPO Enzymatic Activity (U/min-mL)
Control

19.25 ± 1.06a

18.5 ± 0.35a

17.25 ± 1.06a

17.13 ± 0.71a

17.13 ± 0.35a

pH 2.017.25 ± 3.89b13.5 ± 1.41

b13.25 ± 1.06

b

12.5 ± 1.41

12.5 ± 0.00b

pH 4.016.25 ± 1.77a

18.75 ± 0.35a

18.25 ± 0.35a

16.5 ± 0.71a

16.5 ± 0.71aaf) Total Phenolic Content (mg/100 g fresh weight)
Control

57.8 ± 0.64a

71.78 ± 1.24a

73.53 ± 0.54a

76.77 ± 1.68a

71.45 ± 0.21a

pH 2.0b46.5 ± 2.83

a69.5 ± 0.75b72.0 ± 0.63a72.9 ± 0.71

68.3 ± 2.06a

pH 4.044.3 ± 1.41b

69.4 ± 2.39a

74.2 ± 0.76a

75.6 ± 1.51a

71.3 ± 0.21a

Different superscript letters indicate significant differences with 95% probability.

deterioration of characteristics such as colour, texture
and nutritional components.
Generally, lotus root cultivars consist of 13%
protein, 2.3% crude fat, 66% carbohydrates and2.8% fibre (Park et al., 2009). A possible breakdownof these components could have caused the increasein pH of the samples during storage. Initially, the pHof the samples treated with pH 2 increased to 4.81,
then increased further with storage time, while thepH of samples treated with pH 4 had an initial pHof 6.33. This could also be a possible explanationfor enzymatic browning during storage, in which
the monitored pH increased towards the optimal
pH range for PPO that is pH 4.0 - 8.0 (Yoruk andMarshall, 2003).
Changes in weight loss of acid-treated lotus root
samples during storage
The removal of the peel during post-harvest
processing results in agricultural commodities thatare more susceptible to being perishable (Garcia
and Barrett, 2002). The weight loss of freshly cut
lotus root samples (control, pH 2, and pH 4) during

16 days of storage is presented in Table 1(d). As
observed, the weight loss of the control, pH 2-treated,and pH 4-treated samples increased during storage.
Son et al. (2015) reported similar results of increased
weight loss during storage, which could be attributed
to water loss or dehydration. However, we observed
a higher weight loss in the samples treated with pH 2
in comparison with the control and pH 4 samples. We
hypothesised that at a low pH, such as pH 2, cell wall
disruption and cells shrinkage can occur, thereby
causing water loss and further affecting the product’s
weight loss.
Changes in enzymatic activity of polyphenol oxidase(PPO) in acid-treated lotus root samples duringstorage
Wang et al. (2009) found that the optimum
pH and temperature of PPO activity of lotus roots
were 7.5 and 35°C, respectively. However, theeffects of pH on storage stability parameters such as
colour, texture, microbiological, and morphologicalproperties have not been reported. Table 1(e) shows
the variations in PPO enzymatic activity between the1510

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untreated, pH 2-treated, and pH 4-treated samples.
As observed in Table 1(e), a smoother decreasing
trend of enzymatic activity was visibly observedin the control and pH 2 samples throughout the 16
days of storage, while more marked increases anddecreases were observed for the pH 4 sample. The
same PPO enzymatic activity trend was reported by
Xing et al. (2010) in fresh-cut lotus root, which canbe attributed to the stability of the PPO enzyme being
influenced by treatments, such anti-browning agents,
during storage.
The higher PPO activity at day 0 could be
attributed to the wounding and slicing processes(Xing et al., 2012). However, the lower PPO activityof pH 2 and pH 4 as compared to the control could be
due to the lowering of the pH resulting from the acidtreatments. The experimental results showed thatthe pH 2-treated samples had the lowest enzymatic
activity, as compared to other treatments, at days 0, 4,
8, 12 or 16 of storage. The lower enzymatic activity
could be related to the lower degree of browning,
previously reported as L* values. Our results also
showed that the enzymatic activity of the untreatedsamples as compared to pH 4-treated samples show
almost no difference at storage times of 0, 4, 8, 12,
and 16 days, which suggest that pH 4 treatment wasless effective in suppressing PPO activity.
Changes in the total phenolic content of acid-treatedlotus root samples during storage
Slicing is one of the abiotic stress processes
that occur during fresh-cut lotus preparation (Huet al., 2014), which could lead to the accumulationof phenolic compounds (Dixon and Paiva, 1995;
Chalker-Scott, 1999; Reyes and Cisneros-Zevallos,
2003). Table 1(f) shows the increasing phenoliccontent of untreated and treated fresh-cut lotus root
samples up until 12 days of storage and a decrease
after 16 days of storage. The increase in the phenoliccontents of the samples can be attributed to thewounding of the tissues during the cutting process(Hu et al., 2014). Our results show that, immediately
after acidic treatment, the total phenolic contents ofthe pH 2- and pH 4-treated samples were lower thanthat of the control. The pH 2-treated samples exhibiteda lower total phenolic content than the untreated andpH 4-treated samples. This suggests that the pH 2 andpH 4 treatments reduced the production of phenolics
resulting from slicing and wounding during postharvest processing, thus decreasing the amount of
substrate available for oxidation. However, after
12 days of storage, we found that there was no
significant difference between the control and the pH
2- and pH 4-treated samples, which suggested thatphenolics, which cause enzymatic browning, hadbeen produced; thus resulting in colour changes in all
the treatments during storage.
Changes in microbiological count of acid-treatedlotus root samples during storage
The presence of water, starch, and vitamins inlotus root make it an excellent source of nutrientsfor the proliferation of microorganisms during postharvest processing and storage (Du et al., 2009).
Thus, the microbiological quality of the samples
must be assessed for the effective preservationof fresh-cut produce (Bico et al., 2009).The total
microbial counts for the untreated, pH 2-treated, andpH 4-treated samples are presented in Figure 1. Our
results show that, on day 0, the untreated (control)
and pH 4-treated samples had higher microbial
counts (6 log CFU/g) than the pH 2-treated sample

Figure 1. Total microbial counts of fresh-cut lotus root treated at different pH’s, stored at 5°C. Different letters indicate
significant difference at 95% probability.

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Lara et al./IFRJ 26(5) : 1505-1513(4 log CFU/g). These results indicate that pH 2
treatment decreased the initial microbial count fromthe control by 2 log reductions. An increase of the
microbial counts was observed for both the control(7 log CFU/g to 8 log CFU/g) and pH 4-treated (6 log
CFU/g to 8 log CFU/g) samples from day 2 to 4, withno observable increase in the pH 2 samples (5 log
CFU/g). The storage experiment was terminated after
day 4 due to excessive microbial growth in the control
and pH 4-treated samples. These results suggest thatpH 2 treatment could reduce the microbial counts infresh-cut lotus root.
Morphological analyses using scanning electron
microscopy
Despite the importance of the microstructure andits degradation, its role in the maintenance of foodquality has been largely underestimated (Tu et al.,2015). To evaluate the effect of low pH treatments(pH 2 and pH 4) on the microstructure of the freshcut lotus root samples, morphological analyses were
performed. SEM micrographs, presented in Table

2, show the results of the microstructure analysisof the control, pH 2-treated, and pH 4-treatedsamples between days 0 and 16 of storage. From themicrographs, it can be observed that the cell walls of
the control samples remained intact and were visibly
defined during the 16 days of storage. Meanwhile, thepH 2-treated samples showed cell wall breakdownwith increasing storage time. Initially, the pH 2-treatedsamples exhibited distortion and less defined cell
walls and these were continuously observed untilday 16, which most likely indicated cell wall damage
caused by low pH conditions. The cell walls of pH
4-treated samples showed similar distortions to thoseobserved in the pH 2-treated samples, which alsosuggests partial cell wall damage. Tu et al. (2015)
found that raw lotus root samples were composedof defined and organised individual cells, which iscorrelated with the results obtained in the present
work. However, after freezing, the cells appeared
torn and were irregularly shaped with a partial lossof amorphous materials and tissue distortions (Tuet al., 2015). Our findings suggest that the acidic

Table 2. SEM micrographs of fresh-cut lotus root treated at different pH’s, at day 0 and after storage for 16 days at 5°C.
Treatment

Control

pH 2

pH 4

Storage time (day)
0

16

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treatments had a similar effect on cell wall damage
and disruption. Krall and McFeeters (1998) reported
that pectin, a major component of the cell wall,
rapidly degraded at pH 2.5 - 4.5. Fraeye et al. (2007)
also reported that the acid hydrolysis of pectin occurs
at pH values as low as 3.8. As such, we speculated
that, at pH 2, cell wall damage would occur, as canbe observed in Table 2. We propose that this cell walldamage resulted in aggressive tissue softening in the
pH 2-treated samples.
Conclusion
One of the major problems that fresh-cut lotus
root slices encounter is enzymatic browning. As far
as the authors were aware, there are currently noother studies focusing on the specific effect of low
pH treatments on the overall quality of fresh-cut lotus
root, in particular the impact on the microstructuralproperties of this food product. Based on our results,
we found out that lowering the pH of the immersion
treatments to pH 2 was the most effective method
for controlling enzymatic browning in fresh-cut
lotus root. However, this method may also result intissue softening and microstructural changes duringstorage. Our study elucidated the effect of low pH
on the storage stability of fresh-cut lotus root. Thesefindings provide a basis for the formulation of dipping
treatments for fresh-cut lotus root, as well as otheragricultural products.
Acknowledgement
The present work was financially supported by
JSPS Kakenhi (grant no. 15K07452), Japan.
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