LWT - Food Science and Technology 148 (2021) 111703

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LWT
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Enhancement of fructan extraction from garlic and fructooligosaccharide

purification using an activated charcoal column
Shalini R, Jayachandran Krishna, Meenakshisundaram Sankaranarayanan, Usha Antony *
Centre for Food Technology, Department of Biotechnology, Anna University, Chennai, 600025, India

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

Keywords: Conventional fructan extraction from most plant sources involves water combined with methanol or ethanol in
Enzyme assisted extraction different solvent ratios, time, and temperature combinations to increase extraction efficiency. Fructan recovery
Rapid solvent extractor step is usually very laborious using such conventional methods. Three different extraction methods from liter­
Combination
ature giving optimum fructan yield were compared. Methanol (62.5%) at 55 ◦ C, 15 min was effective and
Crude garlic extract
Shake flask and column purification
efficient to extract fructan from garlic (18.71 g/100 g) compared to the other two methods. Further, in this study,
extraction of fructan from garlic was optimized in a rapid solvent extractor system at room temperature (25 ◦ C)
with methanol (70%), which increased the fructan yield by 32.20% (20.73 g/100 g), with minimal loss in garlic
powder recovery. Hemicellulase treatment followed by solvent extraction increased the yield to 21.23 g/100 g
(total increase of 35.4%) compared to the conventional hot water method. Purification of fructooligosaccharides
(FOS) from a mixture containing 15.68 g/100 g of fructan in garlic extract using activated charcoal gave 94% 1-
kestose in shake flask and 82.4% in a column.

1. Introduction
Fructans comprise heterogeneous mixtures present in plant sources
with different degrees of polymerization (DP), and the presence of
specific type of fructans is species-dependent (Apolinario et al., 2014).
Occurrence of fructans with low DP values (DP < 10) are considered as
oligofructose or fructooligosaccharides (FOS) and high (DP > 10 and
<60) are described as inulin (Roberfroid et al., 2010). Fructans occur
naturally in many foods, including banana, onion, garlic, wheat, barley,
asparagus, and Jerusalem artichoke (Campbell, Fahey, & Wolf, 1997).
Natural plant fructans are more advantageous than chemically synthe­
sized ones since there is wide diversity in the occurrence of carbohy­
drates. Fructans are known to have prebiotic properties, giving several
health benefits (Roberfroid et al., 2010), including increased beneficial
microorganisms in the gut, increased mineral absorption, increased
bowel movements, decreased constipation, and decreased obesity and
diabetes. In humans, as there are no digestive enzymes to break these
fructan polymers, they reach the intestinal colon undigested, where the
microbes utilize them (Roberfroid et al., 2010).
Today, industrial production of fructan all over the world is from
chicory and Jerusalem artichoke (Lingyun et al., 2007), and appropriate
attempts to enhance extraction from other crops need to be addressed.
Jeruselum artichoke was reported as more sensitive to cold and seasonal
changes when compared to chicory tubers (Zhu et al., 2016). In India,
chicory cultivation is bi-yearly, confined to the North-western region,
and is used mainly as an ingredient in filter coffee powder both by
large-scale manufacturers and retail coffee grinding shops. Composi­
tional analysis of Jerusalem artichoke revealed 65% moisture, 15% free
sugars, 60% total sugars, and 80–90% inulin among its carbohydrate
content (Barta & Patkai, 2007). Chicory consists of 75% moisture, 4%
protein, 11% total soluble sugars, 89% carbohydrate, 31% total dietary
fiber and 45% inulin (Nwofer et al., 2017). Sugar structure in chicory
was found similar to Jerusalem artichoke (Bhagia et al., 2018). Both
Jerusalem artichoke and chicory tubers are rich sources of inulin. On the
other hand, garlic consists of a heterogeneous mixture of fructans from
<1000 Da to ~6800 Da molecular mass. Fructan constitutes about 84%
of the carbohydrate content in garlic (Lawson, 1995).
Per capita, dietary consumption of garlic in India is about 7.83 kg per
year (Gopal, 2013). At present, several value-added products like di­
etary supplements, garlic oil, garlic powder, garlic oil macerate, and
garlic extracts are produced to meet the consumer demand for
health-promoting supplements. However, garlic is not suitable for
extracting fructans large scale due to its lower fructan content
(16–18%), high cost, pungency, and difficulties in peeling the skin.

* Corresponding author.
E-mail address: usha.antony@gmail.com (U. Antony).

https://doi.org/10.1016/j.lwt.2021.111703
Received 6 October 2020; Received in revised form 29 April 2021; Accepted 11 May 2021
Available online 13 May 2021
0023-6438/© 2021 Elsevier Ltd. All rights reserved.
S. R et al.
Garlic is selected for this study because it is used commonly in almost all
households in India. Further, in our preliminary study (data not shown),
screening 48 foods consisting of commonly consumed fruits and vege­
tables, we recorded a viable fructan content in garlic cultivars. A market
research report in Asia and Southeast Asia shows that the production of
roots and tubers was only 6%, whereas post-harvest and handling loss of
commodity was comparatively higher at 19%. So, commonly used garlic
varieties were investigated to understand and optimize suitable extrac­
tion methods to enhance the fructan yield and purify it.
Optimum conditions for fructan extraction from plants were previ­
ously reviewed by different researchers (Pontis, 1990, pp. 353–369;
Davis, Tterry, Chope, & Faul, 2007; Sanz & Martínez-Castro, 2007:;
Benkeblia, 2013; Magwaza & Opara, 2015; Matros, Peukert, Lahnstein,
Seiffert, & Burton, 2019) concerning pH, temperature, time, solid and
solvent ratio. Almost all conventional extractions of fructan described in
the literature make use of water and solvents like ethanol or methanol in
different combinations with temperature and extraction time (Darby­
shire & Henry, 1978; Suzuki & Cutliffe, 1989; Prosky & Hoebregs, 1999;
Jaimne et al., 2000; Jaime, Martin-Cabrejas, Molla, Lopez-Andreu, &
Esteban, 2001; O’donogue et al., 2004; Benkeblia, Onodera, & Shiomi,
2004; Benkeblia, Onodera, Yoshihira, Kosaka, & Shiomi, 2004; Benke­
blia, Onodera, & Shiomi, 2005; Benkeblia, Ueno, Onodera, & Shiomi,
2005; Chope, Terry, & White, 2006; Chope, Terry, & White, 2007a;
Chope, Terry, & White, 2007b; Paseephol, Small, & Sherkat, 2007;
Vagen & Slimestad, 2008; Toneli, Park, Ramalho, Murr, & Fabbro, 2008;
Abozed, Abdelrashid, El-Kalyoubi, & Hamad, 2009; Muir et al., 2009;
Downes & Terry, 2010; Saengkanuk, Nuchadomrong, Jogloy, Patano­
thai, & Srijaranai, 2011; Abou-Arab, Talaat, & Abu-Salem, 2011; Apo­
linario et al., 2014). In all conventional methods mentioned above for
fructan extraction, there involved multiple extractions at high temper­
atures, using water and solvent mixtures. Conventional method uses hot
water for fructan extraction from food substances (Mccleary, Murphy, &
Mugford, 2000).
The industrial extraction of inulin from chicory includes many steps
in clarification like liming, pre-liming, and carbonation (Shoaib et al.,
2016). The purification steps required are very complex in the conven­
tional method due to the impurities from the process. Inulin extraction
by non-conventional methods like enzyme-assisted extraction,
ultrasound-assisted extraction, microwave-assisted extraction, super­
critical fluid extraction, and pulsed-electric field assisted extraction
were reviewed by Zhu et al. (2016). The authors concluded that pulse
electric field which involved lower temperature was most effective in
extracting inulin. Lingyun et al. (2007) suggest that a suitable method to
natural inulin from Jerusalem artichoke would be indirect sonication.
Inulin yield from Burdock root was increased by 24.31% using
ultrasound-assisted extraction (Milani, Koocheki, & Golimovahhed,
2011). The use of three stage homogenizer increased the fructan
extraction yield by 14% in artichoke compared with conventional hot
water extraction (Li, Meng, & Sun, 2012).
Enzymatic treatment was performed for enhancement of cap­
saicinoids and carotenoids extraction from Capsicum annuum (Santa­
maria et al., 2000), oil extraction from fruits and seeds (Domínguez,
Núñez, & Lema, 1995), and carotenoid extraction from tomato tissue
(Choudhari & Ananthanarayan, 2007). Limited studies are documented
for the enzyme-assisted extraction of inulin using protease from burdock
roots (Cao et al., 2010); cellulase, hemicellulase, and pectinase from
Nendran banana (Shalini & Antony, 2015); commercial preparation
pectinex treatment optimized in response surface methodology using
wild stool plant (Sánchez-Madrigal et al., 2018); and combination of
ultrasound and cellulase treatment using parsnip (Tecucianu & Oancea,
2019).
Few studies have reported the purification of extracted fructan.
Anion-exchange resins such as diethyl aminoethyl (DEAE) cellulose,
DEAE Sepharose, and DEAE Sephacel are used for the purification of
fructan (Apolinario et al., 2014). FOS produced from sugarcane molasses
using fructosyl transferase enzyme was purified by fixed-bed columns
2
LWT 148 (2021) 111703
packed with zeolite (Kuhn & MaugeriFilho, 2010; Kuhn, Mazutti, &
MaugeriFilho, 2014) and activated charcoal (Kuhn, Mazutti, Albertini,
& Filho, 2014; Nobre, Teixeira, & Rodrigues, 2012). FOS with 97%
purity, from the fermentative broth, was achieved using an activated
charcoal column, which is suited for large-scale applications (Nobre
et al., 2012). To date, purification of fructans particularly FOS was not
performed from a natural source like garlic.
This work aimed to enhance fructan extraction from garlic with the
combination of conventional and non-conventional methods. The
enzyme pre-treatment was followed by the use of a pressure-based sol­
vent extractor. Further, the adsorption kinetics of crude garlic fructan
onto activated charcoal using a shake flask and a column was also
investigated. FOS purification process involving three steps: adsorption,
column washing, and desorption as described by Nobre et al., 2012.
2. Materials and methods
2.1. Food selection and preparation
Two cultivars of garlic selected for the study were country garlic
(Allium oleraceum) and hill garlic (Allium ampeloprasum). The selection
was based on a previous screening of 48 commonly consumed foods in
South India (Preliminary work – data not shown). Samples of garlic
cultivars were procured from ten different locations of Chennai city
(Shalini & Antony, 2015). Composite samples were prepared by
measuring 50 g peeled garlic from each location and pooling the peeled
garlic to get half a kilogram (500 g). The garlic was pulped in a food
processor and the paste obtained was stored in air-tight glass containers
at − 20 ◦ C till further analysis. Fresh samples were used for moisture
content analysis on the same day of sampling. The garlic paste was
brought down to room temperature (25 ± 2 ◦ C) and used for extraction.
2.2. Chemicals
FOS like 1-kestose (GF2) and nystose (GF3) used for quantification
assays and enzymes cellulase and hemicellulase were acquired from
Sigma-Aldrich, St. Louis, USA. Cellulase with 1.45 Units/mg activity at
pH 4.5 and 40 ◦ C and hemicellulase with 1.5 Units/mg activity was
originally from Aspergillus niger. A commercial FOS mixture (DP: 2–5)
and inulin mixture (DP: 10–60) were gifted from Brenntag India Private
Limited, New Delhi, India. Activated charcoal extra pure (with a mean
particle diameter of 1.5 mm) was purchased from Merck (Darmstad,
Germany). All other reagents of analytical grade were purchased from
Merck (Mumbai, India). Kits for fructan analysis and measurement were
purchased from Megazyme International Ireland Ltd., Wicklow, Ireland.
2.3. Enzyme assisted extraction
The concentration of enzyme required for fructan and inulin
extraction was optimized based on preliminary experiments with vary­
ing enzyme concentrations. Garlic pulp was treated individually as well
as with an enzymatic cocktail of cellulase and hemicellulase, incubated
at 40 ◦ C, pH 4.5. The enzyme concentrations used were 1.0, 2.5, 5.0, and
7.5 Units/g of garlic pulp in 10 mL of buffer. Representative aliquots
were drawn at 0, 30, 60 and 90 min for total fructan and inulin esti­
mations in the control and enzyme-treated samples. The best yield of
fructan and inulin was obtained with hemicellulase at 5.0 Units/g garlic.
Based on this, 10 g of garlic pulp was treated with 5 Units/g of hemi­
cellulase at pH 4.5 at 40 ◦ C for 1 h. Enzyme action was inactivated by
heating the mixture to 80 ◦ C for 10 min.
2.4. Optimization of solvent for fructan extraction
Enzyme-treated garlic pulp fructan was extracted using three
different methods: 1) hot water, 2) ethanol, and 3) methanol. Many
different combinations of solvent ratio, time, and temperature were
S. R et al.
optimized by distinct researchers for fructan extraction (Darbyshire &
Henry, 1978; Suzuki & Cutliffe, 1989; Pontis, 1990, pp. 353–369; Jaime
et al., 2000; Jaime et al., 2001; O’donogue et al., 2004; Benkeblia,
Onodera, & Shiomi, 2004; Benkeblia, Onodera, Yoshihira, et al., 2004;
Benkeblia, Onodera, & Shiomi, 2005; Benkeblia, Ueno, et al., 2005;
Chope et al., 2006; Chope et al., 2007a; Chope et al., 2007b; Sanz &
Martínez-Castro, 2007; Davis et al., 2007; Vagen & Slimestad, 2008;
Downes & Terry, 2010; Benkeblia, 2013; Magwaza & Opara, 2015;
Matros et al., 2019). The particular three methods of extraction adopted
in this study were selected from those in literature based on their choice
of solvents, temperature, and time, taking into consideration employed
the maximum yield of fructan in the different extraction procedures. The
procedures used are as follows.
2.4.1. Extraction with hot water
Garlic pulp (2 g) was extracted with 80 mL hot distilled water (pH 6
and 80 ◦ C) for 15 min on a hot plate with a magnetic stirrer. The solution
was kept cool at room temperature (25 ± 2 ◦ C), and the volume adjusted
to 100 mL with distilled water. The extract was filtered using Whatman
No. 1 filter paper, and the filtrate was used for the determination, of total
fructan (Mccleary et al., 2000) and inulin (Ashwell, 1957, p. 75) content.
For identification and quantification of sugars, HPLC analysis carried
out with the extract which was filtered using a 0.2 μm syringe-driven
filter (Vagen & Slimestad, 2008).
2.4.2. Extraction with ethanol
Garlic pulp (2 g) was extracted in a water bath set at 75 ◦ C using 5 mL
80% (v/v) ethanol for 30 min. After decanting the supernatant, the
residue was again re-extracted twice under the same conditions. The
extract was removed and the pulp residue re-extracted twice in 2 mL
HPLC grade water for 10 min at 75 ◦ C. The pooled extract (19 mL) was
filtered using Whatman No. 1 filter paper. The filtrate was used for total
fructan (Mccleary et al., 2000) and inulin (Ashwell, 1957, p. 75) anal­
ysis. For identification and quantification of sugars, HPLC analysis was
carried out with 2 mL of the extract which was filtered using a 0.2 μm
syringe driven filter and the filtrate was analyzed for sugars as described
by Vagen and Slimestad (2008).
2.4.3. Extraction with methanol
Garlic pulp (2 g) was extracted for 10 min at 75 ◦ C with distilled
water (4.5 mL) in a water bath. To the slurry, 6.5 mL methanol was
added to provide a final concentration of 62.5% methanol (v/v) and re-
extracted for 15 min at 55 ◦ C (O’Donoghue et al., 2004). The extract was
filtered and analyzed as described in the section above 2.4.2.
Analysis of the yield obtained by the three methods described above
revealed that the one using methanol gave the best result. Hence, this
solvent was applied in the pressure-based system at room temperature
(25 ± 2 ◦ C) and the garlic samples were extracted with 70% methanol in
a benchtop rapid solvent extractor (Armfield, UK). Effect of the extrac­
tion parameters for fructans was programmed using pressure (4–7 MPa),
the number of cycles, and time and optimized for best extraction con­
ditions. The extraction conditions were optimized by varying the weight
of garlic pulp from 50 g to 400 g with 1500 mL of 70% methanol at
constant compression, decompression cycles at (25 ± 2 ◦ C).
2.5. Spray drying of fructan extract
The garlic extract obtained as given above was spray-dried in a
model mini spray dryer Laboratory (Jai Instruments Private Limited,
Mumbai, India) with 120 ± 2 ◦ C inlet and 80 ± 3 ◦ C outlet temperatures;
a peristaltic pump was (25 ± 2 ◦ C) set with 12–15 rotations per minute
to pump the extract, and the compressor maintained air pressure at 0.7
MPa. The feed volume used was 3 L, and the initial brix of garlic extract
was 13 ± 1%. The spray-dried crude garlic fructan powder was
collected, weighed and stored in sterile plastic bottles at room temper­
ature (25 ± 2 ◦ C). The samples were analyzed for total fructan (Mccleary
3
LWT 148 (2021) 111703
et al., 2000), inulin (Ashwell, 1957, p -75), and FOS (Vagen & Slimestad,
2008) by HPLC.
2.6. Purification of FOS
2.6.1. Kinetic studies in shake flask
Purification of FOS was performed using adsorption kinetics with
minor modifications in the method described by Nobre et al. (2012).
Spray-dried crude garlic fructan powder (2.5 g) was mixed with 20 mL
distilled water and 5 g of activated charcoal at room temperature (25 ±
2 ◦ C) and held for adsorption for 6 h in a shake flask at 150 rpm. During
the 6 h adsorption, a 2 mL aliquot was collected every 30 min and
centrifuged at 8000 rpm for 20 min at ambient temperature (25 ± 2 ◦ C).
Sugars in the supernatant were analyzed by HPLC (Vagen & Slimestad,
2008). The concentration of sugar adsorbed onto activated charcoal was
called loading (q) and determined by the following equation:
(C0 − C)⋅V
q=
m
C0, is the initial concentration, C, is the concentration at time t or at
the equilibrium, V is the volume of solution, M is the mass of dried
activated charcoal.
Washing was carried out using distilled water. Desorption was per­
formed using 100 mL each of different ethanol concentrations (5%, 10%,
15%). For each ethanol concentration, 10 mL aliquots were collected for
2 h every half an hour.
2.6.2. FOS purification in column
The column was set, with some modifications similar to the method
of Nobre et al. (2012). Before experimenting, activated charcoal was
washed and autoclaved to remove the air from the pores. Adsorbent
(activated charcoal, 10 g) was mixed with 20 mL water and loaded into a
column (10 cm × 1 cm diameter). For equilibrating the column, a
peristaltic pump set with 6 rpm and 0.6 mL/min flow rate was connected
at the bottom. The water was re-circulated till the activated charcoal bed
had set. After discarding the water, the activated charcoal column was
loaded with the mixture of 2.5 g of spray-dried crude garlic powder and
20 mL distilled water with a flow rate of 1 mL/min. The extract was
re-circulated for 3 h till equilibrium was attained between the adsorbent
and the moving phase. The charcoal was washed using 100 mL distilled
water. Aliquots of 200 μL were collected every 30 min and analyzed for
non-adsorbed sugars by HPLC. Desorption was performed by elution
with 10 mL ethanol (5%, 10%, and 15%). For each concentration of
ethanol (Nobre et al., 2012), 1 mL fractions were collected and analyzed
by HPLC to estimate the sugars recovered (Vagen & Slimestad, 2008).
2.7. Moisture analysis
The moisture content of garlic was determined by oven drying at
70 ◦ C for 6 h. Fresh garlic (5 g) was cut into pieces and spread equally in
sample holder made of stainless steel. The initial and final weights of the
sample were determined and corrected to 3 decimals. The sample holder
was removed and cooled to room temperature every 1 h and the weight
recorded. The final weight was measured until two or three consecutive
readings did not vary. Three determinations on the same test sample
were done and moisture content expressed as percentage. The bone-dry
weight was calculated using the final constant weight of the dried garlic.
2.8. Analysis of fructan and sugars
In the case of enzyme pre-treated garlic pulp, the fructans from
samples collected during the enzyme treatment was estimated by Meg­
azyme Fructan HK Assay Procedure (Mccleary et al., 2000). Garlic
extract (0.5 mL) was transferred for extraction with 80 mL of hot
distilled water (pH 6) at 80 ◦ C using a magnetic stirrer for 15 min. The
cooled solution was adjusted to 100 mL volume with distilled water in a
S. R et al. LWT 148 (2021) 111703

volumetric flask. Samples were filtered through a Whatman No. 1 filter

circle and immediately analyzed for fructans.
In the case of hot water, ethanol and methanol extraction of fructans,
the extracts were analyzed directly for total fructans, inulin and sugars
as described below.

2.8.1. Measurement of total fructans

For measuring total fructans, sample A was treated with fructanase,
while sample B was treated by buffer. The concentration of glucose plus
fructose was analyzed in samples A and B using mixed enzyme systems
by Megazyme assay kit (Mccleary et al., 2000). The fructan content was
calculated and expressed as g/100 g of the garlic sample.

2.8.2. Inulin
Inulin estimation was carried out as described by Ashwell (1957, p.
75). Garlic pulp (10 g) was extracted with 100 mL of 80% ethanol at
80 ◦ C for 6 h. The residue was passed through Whatman No.1 filter paper
and re-extracted again in 80% ethanol for 10 min. The residue was dried
using a hot air oven (60 ◦ C) and again hot water extracted (80 ◦ C and 10
min) using a magnetic stirrer. Inulin content was analyzed using
Resorcinol reagent. The standard graph was drawn using fructose
standard and inulin content expressed in terms of g fructose/100 g of
garlic.

2.8.3. Measurement of sugars

Sugars like glucose, fructose, sucrose, 1-kestose, and nystose were
determined using the HPLC method described by Vagen and Slimestad
(2008) with a slight modification. A high-performance liquid chroma­
tography (Shimadzu Corporation, Japan) with evaporative light scat­
tering detector (ELSD) and a Shodex Asahipak NH2P-50 4 E column was
used. The solvent used in binary gradient consisted of acetonitrile in
water: 0–14 m, 20–45%; 14–18 m, 45–20%; 18–25 m, 20% with flow
rate 0.75 mL/min. Injections of 10 μL of filtered extract were made. To
prepare a mixture of stock standard solution, 5 mg glucose, 5 mg fruc­
tose, 5 mg sucrose, 2.5 mg 1-kestose, 2.5 mg nystose are added to 5 mL
HPLC grade water. The working standard was prepared by diluting 100
μL, 200 μL, 300 μL, 400 μL, and 500 μL up to 1 mL. Injections of 10 μL of
mixed standard and individual standards were made. The mixed stan­
dard graph was compared with sample peaks for the quantification of
the sugars. Nitrogen gas (1.6 bar) was used as nebulizer with 90 ◦ C tube
temperature and 50 ◦ C nebulizer temperature in the ELSD.

2.9. Statistical analysis

All analysis of samples was performed in triplicate, to minimize the

variation and Graph Pad Prism 5.0, used for statistical analysis. The
correlations were analyzed by a two-way ANOVA test, with Bonferroni
post-tests for enzymatic assisted extraction.

3. Results and discussion

3.1. Moisture content

The moisture content of the country garlic and hill garlic was 59.02
Fig. 1. Effect of enzyme concentration and time on fructan extraction of
± 0.14% and 64.60 ± 0.02%, respectively. The bone-dry weight of country garlic. Results of fructan content varies significantly *(P < 0.05) and **
country garlic and hill garlic was 40.98 ± 0.17% and 35.4 ± 0.11%, (P < 0.01) from its respective control (0 min).
respectively. Hill garlic contained more moisture and had lower bone-
dry weight compared to country garlic.
which was followed by a slight decrease at 90 min. The decrease could
be due to the degradation of cell wall constituents. The yields with the
3.2. Enzyme assisted extraction
enzyme cocktail were slightly less compared to the effect of hemi­
cellulase when used alone, and may probably be due to the lower effect
The effect of different concentrations of cellulase, hemicellulase and
of cellulase. The best yield was obtained with 5 Units/g of hemicellulase
enzyme cocktail (cellulase and hemicellulase) for different periods of the
at 60 min (4.5 pH and 40 ◦ C). Similarly, 5 Units/g of hemicellulase at 60
extraction of total fructan (g/100 g), from country garlic cultivar are
min treatment with hill garlic was found to yield high inulin and total
represented in Fig. 1A, B, 1C, and 1D. It was clear that there was a
fructans compared with 1.0, 2.5, and 7.5 Units/g of cellulase and
constant increment in yield of fructans till the treatment time of 60 min,

4
S. R et al. LWT 148 (2021) 111703

enzyme cocktail (Figures not shown). mL) were obtained in parsnip using ultrasonic extracts and cellulase
Since the inulin content of garlic was higher compared to FOS, the pre-treatment compared to the conventional method (Tecucianu &
effect of enzyme treatment must be studied separately, to analyze the Oancea, 2019).
yield of both fructan and inulin. Both cellulase and hemicellulase
improved inulin and fructan yield when used at 5 Units/g of garlic in the 3.3. Solvent extraction
two cultivars (Table 1). Inulin yield was significantly higher in both the
varieties by hemicellulase treatment (enhanced by 54.32% in hill garlic Extraction of fructan utilizing different solvents like hot water
and 57.82% in country garlic). The increase in yield of total fructan was (80 ◦ C), 80% ethanol, and 62.5% methanol are compared in Table 2. The
relatively lower, 27.37% and 31.18% in hill and country garlic respec­ use of methanol was most effective in enhancing yield (50.80%), almost
tively. This could be explained by the hydrolytic action of the enzymes double that of ethanol (24.06%) compared to water extraction. Downes
on the components of the cell wall and membrane, which facilitate the and Terry (2010) reported methanol (62.5%) to be a more suitable
release of bound inulin and total fructans. solvent for fructan extraction from onions as compared to ethanol. Davis
Enzyme assisted extraction of inulin was employed previously in et al. (2007) on assessing different fructan extraction methods in onion,
limited cases. Work done by Cao et al. (2010), demonstrated increased concluded that methanol extraction was superior, compared with 80%
inulin yield (around 14.57%) in Burdock root using plant protease. In ethanol. The reason behind this could be the more polar solvent
one of our previous studies, pectinase assisted extraction in Nendran employing shorter extraction time (15 min) and low temperature
banana increased total fructan yield from 1.4 to 6.5 g/100 g, an increase (55 ◦ C). As methanol is known to be toxic, it was evaporated by spray
of 370% (Shalini & Antony, 2015). Commercial preparation pectinex drying to remove the adverse effect. The final spray dried product
application in wild stool plant at a concentration of 11.6–45.6 U/mL received in the cyclone separator was free of methanol. Thus, methanol
enhanced fructans from 37.58 to 38.58 g/100 g dry matter toxicity is removed from the extracted fructan and can therefore be used
(Sánchez-Madrigal et al., 2018). High levels of inulin (156.674 mg/100 as a functional food ingredient. Besides, methanol is reported to be the
best solvent used extensively for phytochemical extraction from plant
sources, but is removed in the end product before being used as a food
Table 1 additive (Altemimi, Lakhssassi, Baharlouei, Watson, & Lightfoot, 2017).
Effect of different enzyme concentrations on fructan yield in garlic cultivars. Therefore, the extraction of garlic fructan using 70% methanol was
Exp. No. Enzyme Enzyme Inulin and fructan Change in optimized in a rapid solvent extractor, a pressure based system.
(40 ◦ C and concentration yield (g/100 g of inulin/
60 min) (U/g garlic) garlic) fructan
3.3.1. Pressure-based solvent extraction of fructan
yield
Inulin Fructan
(%) Fructan extraction from garlic was optimized using 400 g of garlic in
1500 mL of 70% methanol. From Table 3, it is evident that as the weight
Hill Garlic
Control No enzymatic treatment 9.02 ± 14.54 ±
of garlic increased, the fructan yield was also enhanced. The highest
1 0.41 0.74 fructan yield was obtained at the weight of 200 g garlic. Further increase
Exp 1 C 1.0 10.19 15.93 ± 12.97; 9.55 in the quantity of garlic decreased the fructan yield. This may be due to
± 0.35 0.29 the solid/solvent ratio which interferes with the leaching of fructan from
Exp 2 C 2.5 11.16 16.23 23.72;
±
the substrate. For the next set of examinations, 200 g of garlic was
± 0.45 0.43 11.62
Exp 3 C 5.0 12.98 17.52 ± 43.93; considered to be optimum. Compression and decompression (4–7 MPa)
± 0.24a 0.28a 20.49 cycles of 3 min duration and 30 cycles were selected, as it gave the
Exp 4 C 7.5 12.52 16.98 ± 38.80; maximum efficiency. Fructan extraction was 32.2% more efficient using
± 0.19a 0.15a 16.78 the rapid solvent extractor at ambient temperature compared to regular
Exp 5 HC 1.0 11.48 16.37 27.27;
water extraction at high temperature (Table 2). FOS and inulin yield was
±
± 0.52a 0.62a 12.58
Exp 6 HC 2.5 13.14 17.93 ± 45.68; 73.51% and 64.09% respectively. The reason behind this could be
± 0.47a 0.28a 23.31 application of pressure during the compression process compress garlic
Exp 7 HC 5.0 13.92 18.52 ± 54.32; paste in sample bag and fructans get leached out from garlic during
± 0.36a 0.93a 27.37
decompression process. Application of pressure also protects a wide
Exp 8 HC 7.5 13.14 18.07 ± 45.67;
± 0.15a 0.93a 24.27 variety of other bioactive compounds because of the low temperature
Country Garlic exposure.
Control No enzymatic treatment 9.58 ± 15.68 ± Hemicellulase treatment followed by the pressure-based rapid sol­
2 0.23 0.37 vent system with methanol, brought about maximum extraction of garlic
Exp 9 C 1.0 10.93 16.46 14.09; 4.97
fructan, where the yield was 34.5% more efficient than regular high-
±
± 0.29 0.35
Exp 10 C 2.5 11.83 17.91 ± 23.49; temperature water extraction (Fig. 2). Both FOS and inulin increased
± 0.43 0.32 14.22 significantly by about 75.21% and 66.54%, respectively. This
Exp 11 C 5.0 14.11 18.82 ± 47.28;
± 0.36a 0.29a 20.02
Exp 12 C 7.5 13.48 17.52 ± 40.70; Table 2
± 0.62a 0.19a 11.73 Comparison of fructan yield in country garlic from different extraction methods.
Exp 13 HC 1.0 12.18 17.14 ± 27.14; 8.51 Extraction Fructan yield (mg/100 g garlic)
± 0.82a 0.71a method
Exp 14 HC 2.5 14.03 18.06 ± 46.45; Kestose Nystose Inulin Total fructans
± 0.84a 0.68a 15.17 Hot water 179.54 ± 621.34 ± 9580.16 ± 15680.23 ±
Exp 15 HC 5.0 15.12 20.57 ± 57.82; 5.6 10.2 23.4 37.4
± 0.16a 0.79a 31.18 80% Ethanol 254.94 ± 738.68 ± 12340.23 ± 17720.42 ±
Exp 16 HC 7.5 14.95 19.31 ± 56.05; 7.3a 17.3a 53.2a 63.2a
± 0.57a 0.62a 23.15 62.5% Methanol 352.32 ± 855.42 ± 13620.05 ± 18710.31 ±
C, cellulase enzyme; HC, Hemicellulase enzyme. 11.8a 19.6a 43.2a 82.4a
Rapid solvent 472.68 916.94 15720.21 ± 20730.23 ±
% change in inulin/fructan = (inulin/fructan at concentration C/HC – Inulin/ ± ±
extractor 15.3a 22.3a 52.7a 42.6a
fructan at no enzymatic treatment)/inulin/fructan at concentration C/HC.
a
Results in the same vertical column with superscript ‘a’ represent significant - Fructan yield was significant (P < 0.001) by two way ANOVA using
difference (P < 0.05) with their respective controls (No enzymatic treatments). different extraction methods compared with hot water extraction.

5
S. R et al. LWT 148 (2021) 111703

Table 3 extractor under pressure, operated at room temperature in this study

Optimization of fructan extraction yield in rapid solvent extractor. minimized the degradation and loss of fructan, preserving the bioactive
Weight of Fructan (g/100 g Inulin (g/100 g Efficiency of components and also enhanced significantly (p < 0.001) total fructans
country garlic garlic) garlic) extraction (%) extraction of 32.2%.
(g) (Fructan;
Inulin)
Extract Residue Extract Residue 3.4. Purification of FOS
50 g 15.13 ± 0.92 ± 8.32 ± 0.89 ± 93.91; 89.30
0.4 0.03 1.7 0.06 3.4.1. Kinetic study in shake flask
100 g 15.74 ± 0.83 ± 9.06 ± 0.38 ± 94.72; 95.80 From the adsorption curves (Fig. 3), it is clear that sugars were
0.7 0.04 1.2 0.03
selectively adsorbed depending on their molecular weights. The C/Co
150 g 16.15 ± 0.94 ± 9.62 ± 0.48 ± 94.17; 95.01
0.9 0.01 0.6 0.06 ratio is 0.11 ± 0.01 for nystose (GF3), 0.14 ± 0.02 for 1-kestose (GF2),
200 g 16.29 ± 0.21 ± 9.74 ± 0.14 ± 98.71; 98.56 0.24 ± 0.02 for sucrose (S), 0.26 ± 0.01 for glucose (G) and 0.35 ± 0.03
1.2 0.02 0.9 0.01 for fructose (F). The equilibrium was established after 120 min, and the
250 g 15.81 ± 0.52 ± 9.37 ± 0.92 ± 96.58; 90.18 calculated equilibrium loading (q) for FOS adsorbed was 212 mg/g of
0.4 0.05 1.1 0.02
300 g 15.52 ± 0.82 ± 9.14 ± 0.74 ± 94.71; 91.90
activated charcoal. It is very well known, that fructose and glucose have
0.5 0.05 1.2 0.02 the same molecular weight, but from the data, it was seen that glucose
350 g 15.16 ± 0.87 ± 8.74 ± 1.03 ± 94.26; 88.21 was adsorbed more onto activated charcoal than fructose. The activated
0.6 0.31 1.7 0.05 charcoal demonstrated good adsorption affinity for FOS, yet less for
400 g 14.95 1.37 ± 8.27 ± 1.69 ± 90.83; 79.56
glucose, fructose and sucrose. Comparable results were reported by two
±
0.9 0.14 1.1 0.01
different authors (Kuhn, Mazutti, Albertini, & Filho, 2014; Nobre et al.,
2012) despite use of crude extracts from different sources.
The desorption ratio of each sugar is represented in Fig. 4. The
centrifugation was carried out 5 times (each 25 mL) at all ethanol
concentrations to purify sugars (data not shown). The ethanol concen­
tration of 5% desorbed fructose and glucose, while sucrose desorbed
with 10% ethanol. FOS desorption was best at 15% ethanol concentra­
tion with a purity of 94%, and FOS recovery of 88.99%. This is the first
report on the purification of fructans from crude garlic extract, using
shake flask experiments.

3.4.2. FOS purification using column

The crude garlic extract was kept in recirculation until the concen­
tration of sugars reached equilibrium between liquid and solid phases.
Stabilization of the C/Co ratio took place after 2 h of contact time, which
was almost similar to the ratio observed in the shake flask experiment.
However, to ensure maximum sugar adsorption, the garlic extract was
re-circulated for 3 h. The C/Co ratio determined in the adsorbed stage at
equilibrium was 0.19 ± 0.02, 0.24 ± 0.01, 0.37 ± 0.01, 0.43 ± 0.02 and
0.49 ± 0.04 for nystose, 1-kestose, sucrose, glucose and fructose
respectively (Fig. 5). As the C/Co ratio increased, the molecular weight
Fig. 2. Comparison of different methods of extraction. Expansion: pET – pre- of sugar decreased, similar to the results observed by Nobre et al. (2012).
enzymatic treatment; RS – Rapid solvent extraction. Fructose and glucose were the least adsorbed sugars from crude extract
(Table 4). A washing step evacuated all non-absorbed sugars. Finally,
combination method represents a significant alternative that may be there was no sugar detected after 2 h of washing the column.
applied to the extraction of fructan from such plant sources.
The extracted garlic fructan was spray dried. For a single batch (200
g garlic), the final weight of the spray-dried fructan powder was 26.2 ±
0.46 g. The final concentration of fructan, inulin and FOS in spray-dried
powder was 95.3 ± 1.42%, 65.32 ± 0.94%, and 25.63 ± 0.52%,
respectively. The spray-dried fructan powder was stored at 4 ◦ C, in air-
tight containers for further analysis.
Inulin extracted from Jerusalem artichoke tubers by boiling water
treatment for 10–15 min (Paseephol et al., 2007); from the globe arti­
choke by hot distilled water (80 ◦ C) at pH 6.8 (Ronkart et al., 2007); and
from chicory roots by hot water diffusion technique using continuous
mixing at the temperature of 80 ± 2 ◦ C for 1 h (Toneli, Park, Ramalho.
Murr & Fabbro, 2008) have been documented. Ultrasound extraction of
inulin from Burdock root (Arctium lappa) enhanced yield by 24.31% and
the optimum conditions were demonstrated to be a 25 min sonication
time, 83.22% sonication amplitude, and 37 ◦ C temperature (Milani
et al., 2011). Lingyun et al. (2007) reported that direct sonication was
not suitable for inulin extraction because it degraded inulin partly, but
increased oligosaccharide yield. Many authors had reported the
extraction of fructan using different time-temperature combinations for
hot water (Apolinario et al., 2014). The usage of a rapid solvent
Fig. 3. Adsorption profile of sugars on activated charcoal in a shake flask

6
S. R et al. LWT 148 (2021) 111703

Fig. 6. Desorption profile of sugars on activated charcoal column

Fig. 4. Desorption profile of sugars on activated charcoal in a shake flask
4. Conclusion

Treatment with hemicellulase at 5 Units/g of garlic (country and hill

varieties) is effective in increasing fructan yield by 23.15% in country
garlic and 24.27% in hill garlic. The use of rapid solvent extractor with
pressure at ambient temperature reported for the first time here, effec­
tively enhanced the yield of fructan by 32.21% in country garlic and
29.34% in the hill variety. A combination of hemicellulase pre-
treatment and rapid solvent extraction with 70% methanol was more
efficient and increased fructan yield by 34.5% compared to the con­
ventional hot water method. Therefore, this combination method may
be scaled-up for an industrial process with suitable optimization. The
activated charcoal column is effective in separating FOS from crude
garlic extract. Increasing concentration of ethanol could likely assist in
eluting the polysaccharide inulin, which needs to be affirmed by further
investigations.
Garlic being pungent, is not preferred by all consumers. Further, its
strong flavour may not be suitable in many types of foods. So, by
extracting fructan and providing it in a relatively pure form will improve
fructan consumption. The extracted fructan after spray drying was
cream-colored and odourless. The ingredient could be utilized, in the
production of ready-to-eat foods providing functional prebiotic effects.
Fig. 5. Adsorption profile of sugars on activated charcoal column
The residue after extraction of fructan could serve as a source of insol­
uble fiber and other functional ingredients.
Table 4
Adsorption of sugars from country garlic extract in activated charcoal column. CRediT authorship contribution statement
Sugars Initial Concentration at Amount Adsorption
from Concentration time t (C) μg/mL adsorbed ratio (C/Co) Shalini R: Funding acquisition, Data curation, Formal analysis, Data
garlic (Co) μg/mL μg/mL (charcoal curation, Writing – original draft, revising the manuscript critically for
extract column) important intellectual content, Approval of the version of the manu­
Fructose 425.1 115.3 309.8 0.72 script to be published. Jayachandran Krishna: Funding acquisition,
Glucose 246.3 60.94 185.36 0.75 Data curation, Formal analysis, Data curation, Writing – original draft,
Sucrose 1935.6 424.13 1511.47 0.78 revising the manuscript critically for important intellectual content,
Kestose 552.6 71.27 481.33 0.87
Approval of the version of the manuscript to be published. Meenak­
(GF2)
Nystose 873.2 92.94 780.26 0.89 shisundaram Sankaranarayanan: Conceptualization, Formal analysis,
(GF3) Data curation, Writing – original draft, revising the manuscript critically
for important intellectual content, Approval of the version of the
manuscript to be published. Usha Antony: Conceptualization, Formal
The desorption profile (Fig. 6) shows that 72.71% fructose and analysis, Data curation, Writing – original draft, revising the manuscript
69.32% glucose eluted with 5% ethanol, 72.41% sucrose eluted with critically for important intellectual content, Approval of the version of
10% ethanol, 82.4% 1-kestose eluted with 15% ethanol, and 56.12% the manuscript to be published.
nystose, eluted with 20% ethanol. This outcome is similar to results from
Nobre Teixeira & Rodrigues (2012) where the 1-kestose and nystose Acknowledgement
eluted at 20% ethanol concentrations. The equilibrium loading (q)
calculated was 225 mg of FOS per gram of activated charcoal. The purity During the period of doctoral study, this work was completed by the
of sugar was 92% using the column. author, Ms. Shalini R., all the other authors contributed equally to the

7
S. R et al.
manuscript. This work was supported - by the Senior Research Fellow­
ship (SRF) from the Council of Scientific & Industrial Research, New
Delhi, India. Sanction grant number: 09/468(0470)/2013 and 09/468/
(0487)/2015.
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