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Characterization of spray dried probiotic Sohiong fruit powder with Lactobacillus plantarum
Journal Pre-proof Characterization of spray dried probiotic Sohiong fruit powder with Lactobacillus plantarum Kambhampati Vivek, Sabyasachi Mishra, Rama Chandra Pradhan PII:
S0023-6438(19)31041-2
DOI:
https://doi.org/10.1016/j.lwt.2019.108699
Reference:
YFSTL 108699
To appear in:
LWT - Food Science and Technology
Received Date: 29 May 2019 Revised Date:
28 September 2019
Accepted Date: 30 September 2019
Please cite this article as: Vivek, K., Mishra, S., Pradhan, R.C., Characterization of spray dried probiotic Sohiong fruit powder with Lactobacillus plantarum, LWT - Food Science and Technology (2019), doi: https://doi.org/10.1016/j.lwt.2019.108699. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
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Characterization of spray dried probiotic Sohiong fruit powder with
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Lactobacillus plantarum
3
Kambhampati Vivek, Sabyasachi Mishra*, Rama Chandra Pradhan
4
Department of Food Process Engineering, National Institute of Technology, Rourkela,
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Odisha-769008, India.
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*Corresponding author: Email: [email protected], Tel: +91-661-2462905.
7
ABSTRACT
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Non-dairy based probiotic foods have gained consumer interest due to lactose intolerance,
9
casein allergy, and cholesterol associated risks with consumption of dairy-based products.
10
Probiotic fruit juice powder can be a suitable alternative for dairy-based probiotic powders.
11
Enzymatically extracted Sohiong juice was fermented with Lactobacillus plantarum and
12
spray dried to obtain probiotic Sohiong fruit powder. The storage stability of the powder was
13
determined at 25oC and 50% RH. Acceptable probiotic viability of 6.12 log CFU/g was
14
obtained at 36 days of storage period. Changes in color value, density, porosity, flow
15
properties, hygroscopicity, and moisture content were observed in acceptable ranges. FESEM
16
images confirmed proper encapsulation of probiotics. Glass transition temperature of the
17
powder decreased from 57.04 to 45.81oC during storage. Moisture sorption isotherms of the
18
powder were modeled using various sorption equations at different temperatures ranging
19
between 25 to 45oC. The Peleg model showed the best fitting with highest R2adj. (0.998) and
20
lowest RMSE (0.005) at 35oC.
21
maintained for 36 days without refrigeration and packaging suggesting potential application
22
in the food industry.
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Keywords: Sohiong, spray drying, probiotic powder, moisture isotherm, storage stability.
Quality of probiotic Sohiong juice powder could be
1
24 25
1. Introduction
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Consumer interest in functional foods has increased in recent years due to various
27
health benefits. Probiotics are such functional foods with live microbial supplements
28
(Arepally & Goswami, 2019). Dairy-based probiotic food products are commercially
29
available all over the world in different forms. However, casein allergy, lactose intolerance,
30
and cholesterol associated risks are the major setbacks to dairy products. Therefore, there is a
31
need to develop non-dairy (cereals, fruits and vegetables, and meat) based probiotic
32
functional food products. Among the various non-dairy products, fruits have a wide variety of
33
phytochemicals and have ability to decreases the risk of various diseases (Vivek, Mishra, &
34
Pradhan, 2017). Hence fruit juice could be an alternative option for probiotic food
35
formulation. Probiotification of fruit juice also enhances the digestibility, nutritional, shelf
36
life, and sensory attributes.
37
Sohiong (Prunus nepalensis) is an important, underutilized fruit in the northeastern
38
part of India. It belongs to the Rosaceae family and is also known as Khasi cherry. It is a
39
seasonal fruit available from August to October. The fruit is a good source of total phenols,
40
antioxidants, β-carotenes, anthocyanins, vitamins, and minerals (Vivek, Mishra, & Pradhan,
41
2017). This makes Sohiong juice an excellent substrate for probiotic fermentation (Vivek,
42
Mishra, Pradhan, & Jayabalan, 2019). The poor probiotic survivability in fermented juices is
43
a significant drawback because of free probiotic cells (Mansouripour, Esfandiari, & Nateghi,
44
2013). Therefore, encapsulation is required to protect the probiotic cells from adverse
45
environment, storage, and intestinal conditions.
46
Spray drying is an innovative and economical method for encapsulating the probiotic
47
micro-organisms in fruit matrix. This process produces dried probiotic fruit powders with 2
48
good viable probiotic cells (Anekella & Orsat, 2014). The fruit powders produced by spray
49
drying have low water activity with long shelf life and easy handling (Bhusari, Muzaffar, &
50
Kumar, 2014). The viability of probiotic micro-organisms and changes in physicochemical
51
properties of the powder mainly depends on the storage conditions, i.e. temperature and
52
relative humidity. The moisture sorption isotherms are essential in identifying the optimal
53
storage conditions of the probiotic powder. It describe the relationship between equilibrium
54
moisture content and water activity of powder. It is mainly used for shelf life and packaging
55
predictions and modeling the moisture changes that occur during storage (Koç, Yilmazer,
56
Balkır, & Ertekin, 2010). This work is aimed at characterization and physical stability
57
evaluation of spray dried probiotic Sohiong powder during storage period and measurement
58
of moisture isotherms at various tempratures and relative humidities.
59
2. Materials and methods
60
2.1. Sample preparation
61
Ten kilograms of uniform size, shape and maturity (ripe) of Sohiong fruits without
62
bruises were procured from Meghalaya, India. The fruits were washed under running tap
63
water and deseeded to obtain fresh fruit flesh. The flesh was grounded using a blender for
64
homogenous pulp. The pulp was then subjected to ultrasound pre-treatment (90% ultrasound
65
amplitude, 9.95 min ultrasound treatment time, and 0.05% w/w pectinase) to extract Sohiong
66
juice (Vivek, Mishra, & Pradhan, 2019).
67
Analytics, India) with the frequency of 20 kHz was used for the experiments. Ultra-sonicator
68
had the maximum output power of 250 watts with a maximum amplitude of 240 µm. The
69
juice was then pasteurized with the probe sonicator at 80% amplitude for 15 minutes and
70
fermented with L. plantarum for 72 hours at 37oC to get a bacterial load of 10 log CFU/ml
71
before spray drying (Vivek, Mishra, Pradhan, & Jayabalan, 2019).
Probe type ultra-sonicator (PKS-250F, PCI
3
72 73 74
2.2. Spray drying of fermented Sohiong juice with L. plantarum
75
A tabletop laboratory-scale mini spray dryer (LSD-48, JISL, India) with a concurrent
76
flow regime and a two flow nozzle (0.7 mm diameter) was employed for the
77
microencapsulation process. Based on the preliminary experiments the drying chamber
78
pressure, aspiration rate, and feed temperature were set at 2.5 kg/cm2, 50% and 26±1oC,
79
respectively. The fermented juice (100 ml) was mixed with an anti-caking agent (0.5% w/w
80
magnesium carbonate), and spray dried under optimum conditions of 120oC inlet temperature
81
12% w/w maltodextrin and 201 ml/hr inlet feed rate to obtain probiotic Sohiong fruit powder.
82
The powder was collected from the cyclone separator, according to Muzaffar and Kumar
83
(2015). The spray dried powder had 7.08 Log CFU/g.
84
2.3. Determination of moisture sorption isotherms
85
The gravimetric method was adopted for determining the moisture sorption isotherms
86
of probiotic Sohiong juice powder (Fabra, Talens, Moraga, & Martínez-Navarrete, 2009).
87
Ten grams of powder samples with known moisture content was kept in the desiccators.
88
Relative humidity (10 to 85 %) in the desiccators were maintained with the saturated salt
89
solutions (Polachini, Betiol, Lopes-Filho, & Telis-Romero, 2016). The desiccators were
90
placed inside the incubators to sustain over different temperatures, i.e., 25, 30, 35, 40, and
91
45oC. The sample weight was recorded (± 0.001 g) until constant weight. Then the moisture
92
content was determined as equilibrium moisture content (EMC). Potassium sorbate (5 mg)
93
was placed inside the desiccators to prevent mold growth during the study (Amreen, Khojare,
94
& Jadhao, 2017). All the experiments were done at atmospheric pressure and repeated thrice.
95
The moisture isotherm data at different temperatures and relative humidity were analyzed, 4
96
according to (Muzaffar & Kumar, 2015). A plot was made between equilibrium moisture
97
content (EMC) and water activity which is in conversion from relative humidity (Shah,
98
Tawakkul, & Khan, 2008) for producing the moisture sorption isotherms. The experimental
99
data were fitted to the nine different empirical mathematical models (Brunauer-Emmett-
100
Teller (BET), Harsey, Henderson, Oswin, Iglesias and Chirife, Smith, Caurie, Guggenheim,
101
Anderson and De Boer (GAB) and Peleg) for obtaining a good fit (Chang, Karim,
102
Abdulkarim, Kong, & Ghazali, 2019). Curve fitting and regression analysis were performed
103
by MATLAB, 2015a. The best fit of the model was determined using the adjusted coefficient
104
of determination (R2adj.), and root mean square error (RMSE).
105
2.5. Storage properties of Sohiong powder
106
The freshly prepared probiotic Sohiong powder samples were placed in the Petri
107
plates with open lid and stored at 25oC and 50% RH. The sample was periodically analysed
108
for different physicochemical properties, i.e. color, bulk density, true density, porosity, flow
109
properties, moisture content, hygroscopicity and degree of caking. These properties were
110
determined every four days until the viable cell reaches 6.0 log CFU/g in the powder sample.
111
2.5.1. Probiotic viability
112
The viability of L. plantarum was determined by following according to Vivek,
113
Mishra, Pradhan, and Jayabalan, (2019). One gram of the powder sample was serially diluted
114
in accordance with Koç et al. (2010). Then the aliquots were taken and spread on Petri dish
115
containing De Man, Rogosa and Sharpe (MRS) agar then the petri dish was incubated at 37oC
116
for 72 hours in anaerobic conditions. The number of colonies formed on the surface of the
117
agar was counted and used for calculating the colony-forming unit (CFU) per gram.
118
2.5.2. Colour measurement
5
119
The color of the spray dried powder samples were determined using the colorimeter
120
(Hunter lab, Colorflex EZ, USA). The values were expressed in L* (light to dark), a* (red to
121
green) and b* (yellow to blue).
122
2.5.3. Flow properties
123
The flow properties include Carr’s index (CI, and Hausner ratio (HR) are used to
124
estimate cohesiveness and flowability of the spray dried Sohiong powder (Arepally &
125
Goswami, 2019). The CI and HR are calculated using the following expression.
Carr s index % =
Tapped bulk density − Loose bulk density x 100 tapped bulk density
Hausner Ratio =
126
Tapped bulk density Loose bulk density
2.5.4. Moisture content
127
The moisture content of the spray dried powder samples were determined according
128
to Arepally and Goswami (2019) and expressed in % w/w, wet basis.
129
2.5.5. Bulk and true densities
130
The bulk density i.e., loose and tapped bulk density and true density of the spray-dried
131
probiotic powder were calculated using the following expressions (Arepally & Goswami,
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2019).
Loose bulk density g/cm# =
Tapped bulk density g/cm# =
True density ml =
weight of powder bulk powdered volume
weight of powder Tapped powdered volume
1 Change in volume of toluene
6
133
2.5.7. Porosity
134
The porosity of the spray dried Sohiong powder is calculated using the following expression. Porosity = 1 −
)* )+
135
Where, ρb and ρt are loosed bulk density and true density, respectively.
136
2.5.8. Hygroscopicity and degree of caking
137
The hygroscopicity was determined by placing 1g of a powder sample in the
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desiccator at ambient temperature (25oC). Saturated sodium chloride solution was kept in the
139
desiccator to maintain a relative humidity of 73.36% (Bhusari et al., 2014). The weight
140
change of sample was observed after a week. All the measurements were analysed in
141
triplicates, and the results were expressed in grams of adsorbed moisture per 100 g of dry
142
solids. The degree of caking was measured in accordance with Molina, Clemente, Scapim,
143
and Vagula (2014). The samples after the hygroscopicity test were kept in the oven at 70oC
144
until it reaches the constant weight and allowed to cool in a desiccator. After cooling the
145
sample was manually agitated in 500 µm sieve for five minutes. The powder left over on the
146
screen was measured for calculating the degree of caking using the following equation.
147
148
Degree of caking =
,-./01 .2 3.4567 76189065 .:67 1;6 <=7660 ,-./01 .2 3.4567 /<65 2.7 <96:90>
2.5.9. Glass transition temperature
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Differential scanning calorimetry (DSC, Mettler Toledo, DSC 822, Switzerland) was
150
used for determining the glass transition temperature (Tg) of spray dried Sohiong powder.
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Ten milligrams of the powder sample was taken in an aluminium pan covered with a lid.
152
While the empty container was used as a reference. The temperature was set in the range of -
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20 to 250oC with a heating rate of 10oC/min (Koç et al., 2012). 7
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2.5.10. Powder morphology and Degree of crystallinity
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Morphology, surface and cross-sectional microstructures of spray dried Sohiong fruit
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powder was examined using Field emission scanning electron microscopy (FESEM, Nova
157
NanoSEM, FEI, USA). The powder was fixed on the carbon adhesive tape and plasma coated
158
before analysis. The microscope was operated at a voltage of 10 kV. The degree of
159
crystallinity of spray-dried probiotic Sohiong powders was determined using X-ray
160
diffractometer (XRD, D8 Advance A25, Brucker, USA). The radiation was generated at 40
161
mA and 40 kV. The scattering angle (2θ) was measured from 10o to 99o at a step size of 0.013
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(Papoutsis et al., 2018).
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2.5.11. Statistical analysis
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The analysis of variance (ANOVA) was carried out using SPSS version 20 (SPSS
165
Inc., Chicago, IL, USA) to determine significant differences (p<0.05) of properties among the
166
storage period. The Duncan multiple range test with a 95% confidence interval was used for
167
all the data analysis.
168
3. Results and discussion
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3.1. Moisture sorption isotherm
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Change in EMC at different water activity and temperatures are shown in Table 1.
171
The coefficients of all the sorption models were presented in Table 2. The adjusted
172
coefficient of determination (R2adj) and Relative Mean Square Error (RMSE) was used to
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evaluate the best fit of the model. The data obtained from the isotherm study can be useful to
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describe the energy requirements of a dehydration process. It can also be used to define
175
transport, storage, and process conditions, to predict the shelf life of the material and
176
adsorption or desorption behavior of the material. The Equilibrium moisture content
8
177
increased with the temperature at constant water activity. This indicates the characteristics of
178
a powder, which is amorphous and rich in hydrophilic components (Muzaffar and Kumar,
179
2015). The data of probiotic Sohiong powder followed a type III isotherm behavior,
180
indicating the formation of multilayer. This may be due to the presence of soluble
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components i.e. sugars in the powder (Brunauer, Deming, Deming, & Teller, 1940).
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Generally the isotherms are classified into three regions, monolayer moisture region,
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multilayer moisture region and free water region. The isotherm data of probiotic Sohiong
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powder showed no flattish portion in the curve indicating no monolayer formation (Polachini
185
et al., 2016). The equilibrium moisture of the powder was increased with water activity at all
186
the temperatures. This might be due to the increase in soluble compounds and porosity of the
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probiotic Sohiong powder (Molina et al., 2014). The adsorption behavior decreased when
188
water activity kept constant at high temperatures. At greater temperatures, the water-binding
189
potential is less compared to that at a lower temperature. Similar results were reported on
190
pineapple, yogurt and kiwi fruit powders (Gabas, Telis, Sobral, & Telis-Romero, 2007;
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Moraga, Martínez-Navarrete, & Chiralt, 2006; Seth, Dash, Mishra, & Deka, 2018). Among
192
all the models Peleg model showed the highest R2adj with less RMSE at all the temperatures
193
compared to other models (Table 2). This indicates the data fits exceptionally well to the
194
model. The goodness of fit parameters among pelage model was best at 35oC. The pelage
195
model is suitable to describe type II and III types of isotherm data. Therefore the Peleg model
196
is the best form to represent the moisture isotherms for probiotic Sohiong powder.
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3.2. Effect of storage on probiotic viability
198
The viability of L. plantarum in spray-dried Sohiong powder showed a decreasing
199
trend during the storage period (Fig. 1). This may be due to the stress involved during spray
200
drying, thermal shock (storage at ambient temperatures), and exposure of bacteria to an
201
oxygen-containing environment and water adsorption of powder (Anekella & Orsat, 2014). A 9
202
significant decrease in probiotic viability was observed from 16 days of storage period. The
203
probiotic viability reduced below 6 log CFU/g after 36 days of storage period. It is important
204
for any probiotic formulation to maintain the minimum required standard counts of >106
205
CFU/g throughout the shelf life of the product (Pinto et al., 2015). The survival of the
206
probiotic microorganism is a strain-specific and depends on storage temperature and relative
207
humidity. The results of this study agree with the findings of other similar study reported by
208
Koç et al. (2010); Anekella and Orsat (2014) on yogurt and probiotic raspberry powder,
209
respectively.
210
3.3. Effect of storage on color values of probiotic Sohiong powder
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The color values (L*, a* and b*) of spray dried probiotic Sohiong powder was
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presented in Table 3. The L* and b* values decreased, and a* value increased during the
213
storage period. A significant difference in L* values was observed between 20-24 days and
214
24-28 days of storage period. A similar result was observed for aloe vera powder, where
215
lightness decreased during the storage period (Ramachandra & Rao, 2011). A significant
216
difference in b* values was observed between 20-24 days of storage period. This might be
217
due to the increase in the a* values (Fig. 2). The a* values increased significantly from 16
218
days of storage period. This might be due to the increase in the moisture content of the
219
powder during storage. This phenomenon might be due to the release in water-soluble
220
pigments, i.e. anthocyanins, which increases a* values (Table 3). Therefore, encapsulated
221
anthocyanins in the powder can be protected until 16 days of storage period. The change in
222
color during storage may be due to the physical and chemical reactions during the storage
223
period (Chang, Karim, Abdulkarim, & Ghazali, 2018).
224
3.4. Effect of storage on flow properties of probiotic Sohiong powder
10
225
Carr’s index (CI) and Hausner ratio (HR) are important flow properties in the
226
handling and transportation of powders. The CI estimates powder stability and bridge
227
strength, while HR estimates the inter particulate friction (Shah et al., 2008). The CI and HR
228
values of spray dried Sohiong powder ranged from 16.07 to 23.67 and 1.19 to 1.31,
229
respectively, during the storage period. The CI and HR values increased with the storage
230
period. The significant difference for CI was observed from 32 days, while a significant
231
difference for HR was observed at 8 and 32 to 40 days (Table 4). The increase in values of CI
232
and HR during storage may be due to the increase in the amount of water adsorbed by the
233
powder. This increased the cohesiveness of the powder particles, which results in decreased
234
flowability during the storage period. The flow properties of Sohiong powder falls in the fair
235
and possible category.
236
properties of a powder (Carr, 1965; Hausner, 1967). The flow energy of powder increases
237
when the powder tends to compress readily by tapping (Shah et al., 2008). Similar results
238
were reported by Bhusari et al. (2014) and Arepally and Goswami (2019).
239
3.5. Effect of storage on the moisture content of probiotic Sohiong powder
A lower CI and HR values are recommended for better flow
240
The moisture content is a fundamental property for spray-dried powders for
241
determining its stability and storage. It also influences the probiotic viability during storage
242
(Arepally & Goswami, 2019). The moisture content of spray dried Sohiong powder increased
243
from 3.98 to 7.67 % during the storage period. It showed a non-linear increasing trend (Fig.
244
3). A Significant increase in moisture was observed from 12 days of storage period. This may
245
be due to the sugars present in fruit and carrier agent, i.e. maltodextrin, which increased the
246
water adsorption of powder (Molina et al., 2014). The degree of increase or decrease in the
247
moisture content of powder may also depend on the concentration of maltodextrin and
248
anticaking agent used in the feed. Similar results were reported for spray dried yogurt and
249
tamarind powders (Bhusari et al., 2014; Koç et al., 2010). 11
250
3.6. Effect of storage on density and porosity of probiotic Sohiong powder
251
Loose bulk density, tapped bulk density, and true density of spray dried probiotic
252
Sohiong powder was measured, and the results were shown in Table 5. These densities
253
influence storage space, packaging, and transportation cost of the product. It was observed
254
that all the three densities increased with storage period. A significant increase in loose
255
density and true density was observed from 16 of storage period. While a significant increase
256
in tapped density was observed from 12 days of storage period. This may be due to the
257
increase in powder moisture content during storage. Porosity is the measure of void space in
258
the powder sample. It plays an important role during the reconstitution of powder samples.
259
The porosity of probiotic Sohiong powder increased during the storage period. A significant
260
increase in porosity was observed from 28 days of storage period. This may be due to the
261
increase in the amount of water adsorbed by the powder during storage from the surrounding
262
environment through the packaging material. This results in the agglomeration (lumps) of
263
powder. The formation of lumps responsible for increases in density and porosity of the
264
powder. Similar results were reported by Bhusari et al. (2014) and Arepally and Goswami
265
(2019). The reconstitution of Low-density powders (lighter powders) is rapid compared to
266
high-density powders.
267
3.7. Effect of storage on hygroscopicity and degree of caking of probiotic Sohiong powder
268
The hygroscopicity of Sohiong powder describes the products ability to uptake water
269
from the ambient atmosphere. It ranges from 11.3 to 31.16 % during the storage period (Fig.
270
4). A significant increase in hygroscopicity was observed from 16 days of storage period. The
271
increase in hygroscopicity may be due to the wider contact surface of the powder particles
272
and the use of higher dextrose equivalent (DE-17 to 20) maltodextrin (Molina et al., 2014).
273
The fine particles with a wider contact surface have a large number of active sites for water
12
274
adsorption. A significant increase in the degree of caking was observed from 12 days of
275
storage period. The degree of caking mainly depends on the anticaking agent added in the
276
feed and water adsorption during storage. Lower hygroscopicity and degree of caking can be
277
achieved by using the high concentration of maltodextrin (low DE) and anticaking agents.
278
This is recommended to get non-sticky free-flowing powders. Similar results were observed
279
for spray dried probiotic lychee juice (Kingwatee et al., 2015).
280
3.8. Effect of storage on the glass transition temperature of probiotic Sohiong powder
281
The glass transition temperature (Tg) is the temperature where the material undergoes
282
a physical transition from glassy to a rubbery state. The Tg of spray dried Sohiong powder
283
was measured on 0, 20 and 40 days of storage period. The glass transition temperature was
284
decreased during the storage period. The 0, 20 and 40 days stored powder showed 57.04oC,
285
49.12oC and 45.81oC, respectively. The adsorption of moisture during storage period leads to
286
the mobilization of molecules to exhibit a higher tendency to undergo physical transition.
287
This phenomenon decreases the Tg of spray dried probiotic Sohiong powder. The plasticizing
288
effect of water leads to depressing the Tg of amorphous materials (Fabra et al., 2009). The
289
glass transition temperatures depend on the fruit solids, maltodextrin, and anticaking agents.
290
Anticaking agents increase the stability of spray dried fruit powders (Chang et al., 2018). The
291
results showed that the obtained Tg values are higher than the room temperature (Tg>25oC);
292
therefore, the powder is in the glassy state (Fig. 5). The addition of maltodextrin in feed
293
solution increased the Tg values of powder, which has a higher molecular weight (Jaya &
294
Das, 2004). Similar results were observed for spray dried sucrose and mango powder
295
(Adhikari, Howes, Bhandari, & Langrish, 2009).
296
3.9. Effect of storage on powder morphology and degree of crystallinity of probiotic Sohiong
297
powder
13
298
The morphology of spray dried Sohiong powder was determined at 0, 18 and 36 days
299
of storage period. The maltodextrin added in feed was used as a control (Fig.6). The
300
magnification level used for determining the microstructure for maltodextrin and spray dried
301
probiotic Sohiong powder were 2500x and 10000x, respectively. The spray dried powder
302
resulted in almost spherical with smaller size. While the maltodextrin particles were irregular,
303
hollow and shriveled with larger size (Fig.6). No free cells were visible in FESEM images,
304
confirming the formation of good microcapsules. Similar results were observed for spray
305
drying of mango (Caparino et al., 2012) and lychee (Kingwatee et al., 2015). The smoothness
306
and spherical shape decreased with increase in storage period. This may be due to the
307
formation of liquid bridges resulting in agglomeration of particles. The degree of crystallinity
308
of spray-dried probiotic Sohiong powder at 0, 20, and 40 days of storage was observed for the
309
crystalline-amorphous state. The x-ray diffraction patterns did not show a crystalline peak
310
formation. It showed a broad background pattern which indicates the amorphous
311
characteristics (Caparino et al., 2012). This may be due to the low molecular weight
312
compounds, i.e. organic acids and fruit sugars present in the Sohiong fruit. These compounds
313
have insufficient time to crystallize, thus produces an amorphous metastable state of dried
314
powders. Similar results were observed for spray dried mango powder (Caparino et al.,
315
2012).
316
4. Conclusion
317
The enzymatically extracted Sohiong juice was fermented with L. plantarum and
318
subjected to spray drying to get the probiotic Sohiong powder. The moisture sorption
319
isotherms of the powder were determined using various sorption models in the range of 25 to
320
45oC. The equilibrium moisture content of spray dried Sohiong powder increased with water
321
activity. Peleg model was recommended as the best model due to highest goodness of fit
322
parameters. The product stability at 25oC and 50% RH was determined until the probiotic 14
323
viability reduces beyond 6 Log CFU/g. The powder was amorphous throughout the storage as
324
confirmed by XRD. Quality of probiotic Sohiong juice powder could be maintained up to 36
325
days without packaging. Therefore, good quality non-dairy based probiotic Sohiong fruit
326
powder could be produced using spray drying technique with possible application in the food
327
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18
Table 1 Equilibrium moisture content versus specific water activity data at different temperature 25 oC 30 oC 35 oC 40 oC aw
Xeq
aw
Xeq
aw
Xeq
0.116 0.049±0.006 0.114 0.046±0.012 0.113 0.046±0.030 0.254 0.058±0.012 0.243 0.058±0.002 0.225 0.057±0.010 0.330 0.075±0.012 0.310 0.072±0.002 0.288 0.070±0.006 0.436 0.100±0.015 0.429 0.091±0.015 0.404 0.090±0.006 0.517 0.170±0.010 0.507 0.149±0.011 0.471 0.126±0.021 0.688 0.281±0.052 0.676 0.261±0.031 0.620 0.213±0.015 0.762 0.349±0.010 0.742 0.316±0.045 0.714 0.279±0.020 0.844 0.461±0.023 0.834 0.426±0.010 0.825 0.393±0.015 * aw is the water activity and Xeq is the equilibrium moisture content
45oC
aw
Xeq
aw
Xeq
0.113 0.201 0.255 0.380 0.453 0.598 0.691 0.817
0.043±0.012 0.055±0.015 0.058±0.006 0.073±0.029 0.095±0.003 0.189±0.015 0.250±0.001 0.357±0.025
0.112 0.198 0.236 0.356 0.422 0.583 0.681 0.811
0.040±0.026 0.049±0.030 0.051±0.021 0.068±0.015 0.084±0.010 0.163±0.015 0.221±0.024 0.282±0.005
Table 2 Model coefficients and fit parameters Model Temperat ure (oC) Constant 1 BET
Halsey
Henderson
Oswin
Iglesias and Chirife
Smith
Caurie
GAB
Peleg
25 30 35 40 45 25 30 35 40 45 25 30 35 40 45 25 30 35 40 45 25 30 35 40 45 25 30 35 40 45 25 30 35 40 45 25 30 35 40 45 25 30 35 40 45
7.023, C 6.252, C 7.446, C 6.259, C 10.14, C 5.986, h1 6.016, h1 6.400, h1 6.572, h1 9.227, h1 8.437, H1 8.738, H1 9.572, H1 9.998, H1 14.170, H1 0.150, M 0.144, M 0.142, M 0.131, M 0.121, M 0.052, A 0.047, A 0.048, A 0.041, A 0.044, A -0.014, A -0.012, A -0.0036, A -0.0037, A 0.008, A -3.614, A -3.658, A -3.581, A -3.688, A -3.579, A 0.755, C 0.837, C 1.542, C 1.087, C 0.461, C 0.029, k1 0.632, k1 0.045, k1 0.558, k1 0.007, k1
Model coefficients Constant 2
Constant 3
Constant 4
0.080, M 0.078, M 0.075, M 0.072, M 0.060, M 1.173, h2 1.148, h2 1.167, h2 1.134, h2 1.244, h2 0.885, H2 0.884, H2 0.931, H2 0.917, H2 1.055, H2 0.685, N 0.694, N 0.674, N 0.689, N 0.617, N 0.083, B 0.082, B 0.079, B 0.076, B 0.062, B -0.252, B -0.239, B -0.223, B -0.209, B -0.170, B 3.370, B 3.373, B 3.218, B 3.271, B 2.904, B 0.746, K 0.764, K 0.831, K 0.781, K 0.499, K 0.660, k2 0.039, k2 0.578, k2 0.024, k2 0.417, k2
0.304, M 0.261, M 0.161, M 0.198, M 0.718, M -0.215, n1 -0.071, n1 -0.0003, n1 -0.273, n1 -0.717, n1
2.583, n2 2.718, n2 2.641, n2 2.528, n2 1.912, n2
Goodness of fit parameters Adjusted RMSE R Square 0.962 0.030 0.966 0.026 0.970 0.022 0.961 0.023 0.928 0.024 0.969 0.027 0.971 0.024 0.976 0.020 0.965 0.021 0.943 0.022 0.988 0.016 0.989 0.015 0.990 0.013 0.979 0.017 0.976 0.014 0.982 0.024 0.983 0.018 0.987 0.014 0.977 0.018 0.964 0.017 0.952 0.034 0.957 0.029 0.959 0.025 0.952 0.025 0.913 0.027 0.089 0.985 0.018 0.983 0.014 0.987 0.018 0.975 0.977 0.014 0.995 0.011 0.995 0.009 0.997 0.007 0.011 0.990 0.012 0.982 0.989 0.017 0.015 0.988 0.013 0.989 0.977 0.018 0.015 0.974 0.995 0.011 0.996 0.009 0.998 0.005 0.993 0.010 0.989 0.009
Table 3 Colour values of the spray dried Sohiong powder Storage days. L* a* b* f b 0 43.880±0.197 34.487±0.356 6.390±0.369b 4 43.703±0.505ef 34.478±0.105b 6.231±0.265b 8 43.643±0.546ef 34.437±0.066b 6.185±0.314b 12 43.557±0.597ef 33.934±0.062a 6.297±0.044b 16 43.132±0.026de 36.374±0.154c 6.220±0.099b 20 42.890±0.192d 36.169±0.078c 6.111±0.058b 24 42.057±0.056c 36.951±0.175d 5.824±0.060a 28 40.817±0.343b 37.170±0.341d 5.810±0.187a 32 40.540±0.115ab 36.999±0.101d 5.446±0.167a 36 40.093±0.029a 38.104±0.058e 5.670±0.328a 40 40.090±0.036a 37.973±0.146e 5.744±0.092a a,b,c,d,e,f represents the significance along the column.
Table 4 Porosity and flow properties of spray dried Sohiong powder Storage days. Porosity Carr’s index Hausner ratio bc a 0 0.480±0.004 16.067±1.904 1.192±0.027abc 4 0.471±0.009ab 15.931±1.383a 1.190±0.020abc ab a 8 0.470±0.001 15.678±1.160 1.186±0.016a 12 0.461±0.016a 17.838±1.143ab 1.217±0.017bc bc ab 16 0.481±0.004 17.043±1.436 1.206±0.021abc ab a 20 0.470±0.004 15.721±1.089 1.187±0.015abc 24 0.470±0.005ab 15.836±0.113a 1.188±0.002abc c a 28 0.486±0.005 15.540±0.284 1.184±0.004ab 32 0.487±0.005c 18.886±1.498bc 1.233±0.023cd d c 36 0.509±0.006 21.132±0.545 1.268±0.009d 40 0.523±0.007e 23.670±3.187d 1.312±0.054e a,b,c,d,e represents the significance along the column.
Table 5 Densities of the spray dried Sohiong powder Storage days. Loose bulk density Tapped bulk density True density a a 0 0.521±0.001 0.621±0.014 1.003±0.008a 4 0.527±0.006a 0.627±0.014a 0.998±0.012a ab a 8 0.530±0.004 0.629±0.013 1.001±0.005a 12 0.540±0.004bc 0.658±0.011b 1.003±0.027a c b 16 0.548±0.006 0.661±0.009 1.056±0.016b d c 20 0.609±0.010 0.723±0.011 1.148±0.012c 24 0.652±0.005e 0.775±0.009d 1.230±0.005d f d 28 0.669±0.003 0.792±0.006 1.301±0.012e 32 0.678±0.004fg 0.836±0.014e 1.322±0.005e g f 36 0.686±0.004 0.869±0.011 1.396±0.016f 40 0.706±0.014h 0.926±0.034g 1.480±0.009g a,b,c,d,e,f,g,h represents the significance along the column.
Probiotic viability (log CFU/g)
7.300 7.100 6.900 6.700 6.500 6.300 6.100 5.900 5.700 5.500 0
5
10
15
20 25 30 Storage days
35
Fig 1. Effect of storage on Probiotic viability
40
45
Fig 2. Probiotic Sohiong powder (a) 0 days storage, (b) 36 days storage
Moisture content (%) w.b
9.000 8.000 7.000 6.000 5.000 4.000 3.000 0
5
10
15
20 25 30 Storage days
35
Fig 3. Effect of storage on moisture content
40
45
Hygroscopicity (%)
35.000 30.000 25.000 20.000 15.000 10.000 5.000 0.000 0
5
10
15
20 25 30 Storage days
35
Fig 4. Effect of storage on hygroscopicity
40
45
^ex o mW
SPB, 08.01.2019 11:51:03 SPB, 9.7000 mg
Glas s Transition Onset 41.00 °C Midpoint 57.04 °C
0
-2
-4
-6
-8 -20
0
20
40
60
80
100
120
140
160
180
200
220
°C
0
2
4
6
8
10
12
14
16
18
20
22
24
min
Lab : M ET T LE R
S T A R e S W 1 2. 10
Fig 5. Tg of probiotic Sohiong powder at 0 day of storage
b
a Encapsulated cells
10 kV
30µm
10 kV
5µm
d
c Encapsulated cells Encapsulated cells
10 kV
5µm
10 kV
5µm
Fig 6. FESEM images of (a) maltodextrin, (b) spray dried Sohiong powder at 0day, (c) Spray dried Sohiong powder at 18 day and (d) Spray dried Sohiong powder at 36 days
Highlights 1. A non-dairy based probiotic Sohiong fruit powder was developed. 2. Moisture sorption isotherms of probiotic Sohiong powder were modelled. 3. The storage stability of the powder in LDPE pouches was studied at 25oC and 50% RH. 4. Probiotic viability of 6.12 log CFU/g was obtained until 36 days of storage period. 5. The probiotic Sohiong powder was of good quality, structure and morphology.
Conflict of Interest and Authorship Conformation Form Please check the following as appropriate:
All authors have participated in (a) conception and design, or analysis and interpretation of the data; (b) drafting the article or revising it critically for important intellectual content; and (c) approval of the final version. This manuscript has not been submitted to, nor is under review at, another journal or other publishing venue. The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript The following authors have affiliations with organizations with direct or indirect financial interest in the subject matter discussed in the manuscript: Author’s name 1. Kambhampati Vivek 2. Sabyasachi Mishra 3. Rama Chandra Pradhan
Affiliation National Institute of Technology, Rourkela National Institute of Technology, Rourkela National Institute of Technology, Rourkela
S0023-6438(19)31041-2
DOI:
https://doi.org/10.1016/j.lwt.2019.108699
Reference:
YFSTL 108699
To appear in:
LWT - Food Science and Technology
Received Date: 29 May 2019 Revised Date:
28 September 2019
Accepted Date: 30 September 2019
Please cite this article as: Vivek, K., Mishra, S., Pradhan, R.C., Characterization of spray dried probiotic Sohiong fruit powder with Lactobacillus plantarum, LWT - Food Science and Technology (2019), doi: https://doi.org/10.1016/j.lwt.2019.108699. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
1
Characterization of spray dried probiotic Sohiong fruit powder with
2
Lactobacillus plantarum
3
Kambhampati Vivek, Sabyasachi Mishra*, Rama Chandra Pradhan
4
Department of Food Process Engineering, National Institute of Technology, Rourkela,
5
Odisha-769008, India.
6
*Corresponding author: Email: [email protected], Tel: +91-661-2462905.
7
ABSTRACT
8
Non-dairy based probiotic foods have gained consumer interest due to lactose intolerance,
9
casein allergy, and cholesterol associated risks with consumption of dairy-based products.
10
Probiotic fruit juice powder can be a suitable alternative for dairy-based probiotic powders.
11
Enzymatically extracted Sohiong juice was fermented with Lactobacillus plantarum and
12
spray dried to obtain probiotic Sohiong fruit powder. The storage stability of the powder was
13
determined at 25oC and 50% RH. Acceptable probiotic viability of 6.12 log CFU/g was
14
obtained at 36 days of storage period. Changes in color value, density, porosity, flow
15
properties, hygroscopicity, and moisture content were observed in acceptable ranges. FESEM
16
images confirmed proper encapsulation of probiotics. Glass transition temperature of the
17
powder decreased from 57.04 to 45.81oC during storage. Moisture sorption isotherms of the
18
powder were modeled using various sorption equations at different temperatures ranging
19
between 25 to 45oC. The Peleg model showed the best fitting with highest R2adj. (0.998) and
20
lowest RMSE (0.005) at 35oC.
21
maintained for 36 days without refrigeration and packaging suggesting potential application
22
in the food industry.
23
Keywords: Sohiong, spray drying, probiotic powder, moisture isotherm, storage stability.
Quality of probiotic Sohiong juice powder could be
1
24 25
1. Introduction
26
Consumer interest in functional foods has increased in recent years due to various
27
health benefits. Probiotics are such functional foods with live microbial supplements
28
(Arepally & Goswami, 2019). Dairy-based probiotic food products are commercially
29
available all over the world in different forms. However, casein allergy, lactose intolerance,
30
and cholesterol associated risks are the major setbacks to dairy products. Therefore, there is a
31
need to develop non-dairy (cereals, fruits and vegetables, and meat) based probiotic
32
functional food products. Among the various non-dairy products, fruits have a wide variety of
33
phytochemicals and have ability to decreases the risk of various diseases (Vivek, Mishra, &
34
Pradhan, 2017). Hence fruit juice could be an alternative option for probiotic food
35
formulation. Probiotification of fruit juice also enhances the digestibility, nutritional, shelf
36
life, and sensory attributes.
37
Sohiong (Prunus nepalensis) is an important, underutilized fruit in the northeastern
38
part of India. It belongs to the Rosaceae family and is also known as Khasi cherry. It is a
39
seasonal fruit available from August to October. The fruit is a good source of total phenols,
40
antioxidants, β-carotenes, anthocyanins, vitamins, and minerals (Vivek, Mishra, & Pradhan,
41
2017). This makes Sohiong juice an excellent substrate for probiotic fermentation (Vivek,
42
Mishra, Pradhan, & Jayabalan, 2019). The poor probiotic survivability in fermented juices is
43
a significant drawback because of free probiotic cells (Mansouripour, Esfandiari, & Nateghi,
44
2013). Therefore, encapsulation is required to protect the probiotic cells from adverse
45
environment, storage, and intestinal conditions.
46
Spray drying is an innovative and economical method for encapsulating the probiotic
47
micro-organisms in fruit matrix. This process produces dried probiotic fruit powders with 2
48
good viable probiotic cells (Anekella & Orsat, 2014). The fruit powders produced by spray
49
drying have low water activity with long shelf life and easy handling (Bhusari, Muzaffar, &
50
Kumar, 2014). The viability of probiotic micro-organisms and changes in physicochemical
51
properties of the powder mainly depends on the storage conditions, i.e. temperature and
52
relative humidity. The moisture sorption isotherms are essential in identifying the optimal
53
storage conditions of the probiotic powder. It describe the relationship between equilibrium
54
moisture content and water activity of powder. It is mainly used for shelf life and packaging
55
predictions and modeling the moisture changes that occur during storage (Koç, Yilmazer,
56
Balkır, & Ertekin, 2010). This work is aimed at characterization and physical stability
57
evaluation of spray dried probiotic Sohiong powder during storage period and measurement
58
of moisture isotherms at various tempratures and relative humidities.
59
2. Materials and methods
60
2.1. Sample preparation
61
Ten kilograms of uniform size, shape and maturity (ripe) of Sohiong fruits without
62
bruises were procured from Meghalaya, India. The fruits were washed under running tap
63
water and deseeded to obtain fresh fruit flesh. The flesh was grounded using a blender for
64
homogenous pulp. The pulp was then subjected to ultrasound pre-treatment (90% ultrasound
65
amplitude, 9.95 min ultrasound treatment time, and 0.05% w/w pectinase) to extract Sohiong
66
juice (Vivek, Mishra, & Pradhan, 2019).
67
Analytics, India) with the frequency of 20 kHz was used for the experiments. Ultra-sonicator
68
had the maximum output power of 250 watts with a maximum amplitude of 240 µm. The
69
juice was then pasteurized with the probe sonicator at 80% amplitude for 15 minutes and
70
fermented with L. plantarum for 72 hours at 37oC to get a bacterial load of 10 log CFU/ml
71
before spray drying (Vivek, Mishra, Pradhan, & Jayabalan, 2019).
Probe type ultra-sonicator (PKS-250F, PCI
3
72 73 74
2.2. Spray drying of fermented Sohiong juice with L. plantarum
75
A tabletop laboratory-scale mini spray dryer (LSD-48, JISL, India) with a concurrent
76
flow regime and a two flow nozzle (0.7 mm diameter) was employed for the
77
microencapsulation process. Based on the preliminary experiments the drying chamber
78
pressure, aspiration rate, and feed temperature were set at 2.5 kg/cm2, 50% and 26±1oC,
79
respectively. The fermented juice (100 ml) was mixed with an anti-caking agent (0.5% w/w
80
magnesium carbonate), and spray dried under optimum conditions of 120oC inlet temperature
81
12% w/w maltodextrin and 201 ml/hr inlet feed rate to obtain probiotic Sohiong fruit powder.
82
The powder was collected from the cyclone separator, according to Muzaffar and Kumar
83
(2015). The spray dried powder had 7.08 Log CFU/g.
84
2.3. Determination of moisture sorption isotherms
85
The gravimetric method was adopted for determining the moisture sorption isotherms
86
of probiotic Sohiong juice powder (Fabra, Talens, Moraga, & Martínez-Navarrete, 2009).
87
Ten grams of powder samples with known moisture content was kept in the desiccators.
88
Relative humidity (10 to 85 %) in the desiccators were maintained with the saturated salt
89
solutions (Polachini, Betiol, Lopes-Filho, & Telis-Romero, 2016). The desiccators were
90
placed inside the incubators to sustain over different temperatures, i.e., 25, 30, 35, 40, and
91
45oC. The sample weight was recorded (± 0.001 g) until constant weight. Then the moisture
92
content was determined as equilibrium moisture content (EMC). Potassium sorbate (5 mg)
93
was placed inside the desiccators to prevent mold growth during the study (Amreen, Khojare,
94
& Jadhao, 2017). All the experiments were done at atmospheric pressure and repeated thrice.
95
The moisture isotherm data at different temperatures and relative humidity were analyzed, 4
96
according to (Muzaffar & Kumar, 2015). A plot was made between equilibrium moisture
97
content (EMC) and water activity which is in conversion from relative humidity (Shah,
98
Tawakkul, & Khan, 2008) for producing the moisture sorption isotherms. The experimental
99
data were fitted to the nine different empirical mathematical models (Brunauer-Emmett-
100
Teller (BET), Harsey, Henderson, Oswin, Iglesias and Chirife, Smith, Caurie, Guggenheim,
101
Anderson and De Boer (GAB) and Peleg) for obtaining a good fit (Chang, Karim,
102
Abdulkarim, Kong, & Ghazali, 2019). Curve fitting and regression analysis were performed
103
by MATLAB, 2015a. The best fit of the model was determined using the adjusted coefficient
104
of determination (R2adj.), and root mean square error (RMSE).
105
2.5. Storage properties of Sohiong powder
106
The freshly prepared probiotic Sohiong powder samples were placed in the Petri
107
plates with open lid and stored at 25oC and 50% RH. The sample was periodically analysed
108
for different physicochemical properties, i.e. color, bulk density, true density, porosity, flow
109
properties, moisture content, hygroscopicity and degree of caking. These properties were
110
determined every four days until the viable cell reaches 6.0 log CFU/g in the powder sample.
111
2.5.1. Probiotic viability
112
The viability of L. plantarum was determined by following according to Vivek,
113
Mishra, Pradhan, and Jayabalan, (2019). One gram of the powder sample was serially diluted
114
in accordance with Koç et al. (2010). Then the aliquots were taken and spread on Petri dish
115
containing De Man, Rogosa and Sharpe (MRS) agar then the petri dish was incubated at 37oC
116
for 72 hours in anaerobic conditions. The number of colonies formed on the surface of the
117
agar was counted and used for calculating the colony-forming unit (CFU) per gram.
118
2.5.2. Colour measurement
5
119
The color of the spray dried powder samples were determined using the colorimeter
120
(Hunter lab, Colorflex EZ, USA). The values were expressed in L* (light to dark), a* (red to
121
green) and b* (yellow to blue).
122
2.5.3. Flow properties
123
The flow properties include Carr’s index (CI, and Hausner ratio (HR) are used to
124
estimate cohesiveness and flowability of the spray dried Sohiong powder (Arepally &
125
Goswami, 2019). The CI and HR are calculated using the following expression.
Carr s index % =
Tapped bulk density − Loose bulk density x 100 tapped bulk density
Hausner Ratio =
126
Tapped bulk density Loose bulk density
2.5.4. Moisture content
127
The moisture content of the spray dried powder samples were determined according
128
to Arepally and Goswami (2019) and expressed in % w/w, wet basis.
129
2.5.5. Bulk and true densities
130
The bulk density i.e., loose and tapped bulk density and true density of the spray-dried
131
probiotic powder were calculated using the following expressions (Arepally & Goswami,
132
2019).
Loose bulk density g/cm# =
Tapped bulk density g/cm# =
True density ml =
weight of powder bulk powdered volume
weight of powder Tapped powdered volume
1 Change in volume of toluene
6
133
2.5.7. Porosity
134
The porosity of the spray dried Sohiong powder is calculated using the following expression. Porosity = 1 −
)* )+
135
Where, ρb and ρt are loosed bulk density and true density, respectively.
136
2.5.8. Hygroscopicity and degree of caking
137
The hygroscopicity was determined by placing 1g of a powder sample in the
138
desiccator at ambient temperature (25oC). Saturated sodium chloride solution was kept in the
139
desiccator to maintain a relative humidity of 73.36% (Bhusari et al., 2014). The weight
140
change of sample was observed after a week. All the measurements were analysed in
141
triplicates, and the results were expressed in grams of adsorbed moisture per 100 g of dry
142
solids. The degree of caking was measured in accordance with Molina, Clemente, Scapim,
143
and Vagula (2014). The samples after the hygroscopicity test were kept in the oven at 70oC
144
until it reaches the constant weight and allowed to cool in a desiccator. After cooling the
145
sample was manually agitated in 500 µm sieve for five minutes. The powder left over on the
146
screen was measured for calculating the degree of caking using the following equation.
147
148
Degree of caking =
,-./01 .2 3.4567 76189065 .:67 1;6 <=7660 ,-./01 .2 3.4567 /<65 2.7 <96:90>
2.5.9. Glass transition temperature
149
Differential scanning calorimetry (DSC, Mettler Toledo, DSC 822, Switzerland) was
150
used for determining the glass transition temperature (Tg) of spray dried Sohiong powder.
151
Ten milligrams of the powder sample was taken in an aluminium pan covered with a lid.
152
While the empty container was used as a reference. The temperature was set in the range of -
153
20 to 250oC with a heating rate of 10oC/min (Koç et al., 2012). 7
154
2.5.10. Powder morphology and Degree of crystallinity
155
Morphology, surface and cross-sectional microstructures of spray dried Sohiong fruit
156
powder was examined using Field emission scanning electron microscopy (FESEM, Nova
157
NanoSEM, FEI, USA). The powder was fixed on the carbon adhesive tape and plasma coated
158
before analysis. The microscope was operated at a voltage of 10 kV. The degree of
159
crystallinity of spray-dried probiotic Sohiong powders was determined using X-ray
160
diffractometer (XRD, D8 Advance A25, Brucker, USA). The radiation was generated at 40
161
mA and 40 kV. The scattering angle (2θ) was measured from 10o to 99o at a step size of 0.013
162
(Papoutsis et al., 2018).
163
2.5.11. Statistical analysis
164
The analysis of variance (ANOVA) was carried out using SPSS version 20 (SPSS
165
Inc., Chicago, IL, USA) to determine significant differences (p<0.05) of properties among the
166
storage period. The Duncan multiple range test with a 95% confidence interval was used for
167
all the data analysis.
168
3. Results and discussion
169
3.1. Moisture sorption isotherm
170
Change in EMC at different water activity and temperatures are shown in Table 1.
171
The coefficients of all the sorption models were presented in Table 2. The adjusted
172
coefficient of determination (R2adj) and Relative Mean Square Error (RMSE) was used to
173
evaluate the best fit of the model. The data obtained from the isotherm study can be useful to
174
describe the energy requirements of a dehydration process. It can also be used to define
175
transport, storage, and process conditions, to predict the shelf life of the material and
176
adsorption or desorption behavior of the material. The Equilibrium moisture content
8
177
increased with the temperature at constant water activity. This indicates the characteristics of
178
a powder, which is amorphous and rich in hydrophilic components (Muzaffar and Kumar,
179
2015). The data of probiotic Sohiong powder followed a type III isotherm behavior,
180
indicating the formation of multilayer. This may be due to the presence of soluble
181
components i.e. sugars in the powder (Brunauer, Deming, Deming, & Teller, 1940).
182
Generally the isotherms are classified into three regions, monolayer moisture region,
183
multilayer moisture region and free water region. The isotherm data of probiotic Sohiong
184
powder showed no flattish portion in the curve indicating no monolayer formation (Polachini
185
et al., 2016). The equilibrium moisture of the powder was increased with water activity at all
186
the temperatures. This might be due to the increase in soluble compounds and porosity of the
187
probiotic Sohiong powder (Molina et al., 2014). The adsorption behavior decreased when
188
water activity kept constant at high temperatures. At greater temperatures, the water-binding
189
potential is less compared to that at a lower temperature. Similar results were reported on
190
pineapple, yogurt and kiwi fruit powders (Gabas, Telis, Sobral, & Telis-Romero, 2007;
191
Moraga, Martínez-Navarrete, & Chiralt, 2006; Seth, Dash, Mishra, & Deka, 2018). Among
192
all the models Peleg model showed the highest R2adj with less RMSE at all the temperatures
193
compared to other models (Table 2). This indicates the data fits exceptionally well to the
194
model. The goodness of fit parameters among pelage model was best at 35oC. The pelage
195
model is suitable to describe type II and III types of isotherm data. Therefore the Peleg model
196
is the best form to represent the moisture isotherms for probiotic Sohiong powder.
197
3.2. Effect of storage on probiotic viability
198
The viability of L. plantarum in spray-dried Sohiong powder showed a decreasing
199
trend during the storage period (Fig. 1). This may be due to the stress involved during spray
200
drying, thermal shock (storage at ambient temperatures), and exposure of bacteria to an
201
oxygen-containing environment and water adsorption of powder (Anekella & Orsat, 2014). A 9
202
significant decrease in probiotic viability was observed from 16 days of storage period. The
203
probiotic viability reduced below 6 log CFU/g after 36 days of storage period. It is important
204
for any probiotic formulation to maintain the minimum required standard counts of >106
205
CFU/g throughout the shelf life of the product (Pinto et al., 2015). The survival of the
206
probiotic microorganism is a strain-specific and depends on storage temperature and relative
207
humidity. The results of this study agree with the findings of other similar study reported by
208
Koç et al. (2010); Anekella and Orsat (2014) on yogurt and probiotic raspberry powder,
209
respectively.
210
3.3. Effect of storage on color values of probiotic Sohiong powder
211
The color values (L*, a* and b*) of spray dried probiotic Sohiong powder was
212
presented in Table 3. The L* and b* values decreased, and a* value increased during the
213
storage period. A significant difference in L* values was observed between 20-24 days and
214
24-28 days of storage period. A similar result was observed for aloe vera powder, where
215
lightness decreased during the storage period (Ramachandra & Rao, 2011). A significant
216
difference in b* values was observed between 20-24 days of storage period. This might be
217
due to the increase in the a* values (Fig. 2). The a* values increased significantly from 16
218
days of storage period. This might be due to the increase in the moisture content of the
219
powder during storage. This phenomenon might be due to the release in water-soluble
220
pigments, i.e. anthocyanins, which increases a* values (Table 3). Therefore, encapsulated
221
anthocyanins in the powder can be protected until 16 days of storage period. The change in
222
color during storage may be due to the physical and chemical reactions during the storage
223
period (Chang, Karim, Abdulkarim, & Ghazali, 2018).
224
3.4. Effect of storage on flow properties of probiotic Sohiong powder
10
225
Carr’s index (CI) and Hausner ratio (HR) are important flow properties in the
226
handling and transportation of powders. The CI estimates powder stability and bridge
227
strength, while HR estimates the inter particulate friction (Shah et al., 2008). The CI and HR
228
values of spray dried Sohiong powder ranged from 16.07 to 23.67 and 1.19 to 1.31,
229
respectively, during the storage period. The CI and HR values increased with the storage
230
period. The significant difference for CI was observed from 32 days, while a significant
231
difference for HR was observed at 8 and 32 to 40 days (Table 4). The increase in values of CI
232
and HR during storage may be due to the increase in the amount of water adsorbed by the
233
powder. This increased the cohesiveness of the powder particles, which results in decreased
234
flowability during the storage period. The flow properties of Sohiong powder falls in the fair
235
and possible category.
236
properties of a powder (Carr, 1965; Hausner, 1967). The flow energy of powder increases
237
when the powder tends to compress readily by tapping (Shah et al., 2008). Similar results
238
were reported by Bhusari et al. (2014) and Arepally and Goswami (2019).
239
3.5. Effect of storage on the moisture content of probiotic Sohiong powder
A lower CI and HR values are recommended for better flow
240
The moisture content is a fundamental property for spray-dried powders for
241
determining its stability and storage. It also influences the probiotic viability during storage
242
(Arepally & Goswami, 2019). The moisture content of spray dried Sohiong powder increased
243
from 3.98 to 7.67 % during the storage period. It showed a non-linear increasing trend (Fig.
244
3). A Significant increase in moisture was observed from 12 days of storage period. This may
245
be due to the sugars present in fruit and carrier agent, i.e. maltodextrin, which increased the
246
water adsorption of powder (Molina et al., 2014). The degree of increase or decrease in the
247
moisture content of powder may also depend on the concentration of maltodextrin and
248
anticaking agent used in the feed. Similar results were reported for spray dried yogurt and
249
tamarind powders (Bhusari et al., 2014; Koç et al., 2010). 11
250
3.6. Effect of storage on density and porosity of probiotic Sohiong powder
251
Loose bulk density, tapped bulk density, and true density of spray dried probiotic
252
Sohiong powder was measured, and the results were shown in Table 5. These densities
253
influence storage space, packaging, and transportation cost of the product. It was observed
254
that all the three densities increased with storage period. A significant increase in loose
255
density and true density was observed from 16 of storage period. While a significant increase
256
in tapped density was observed from 12 days of storage period. This may be due to the
257
increase in powder moisture content during storage. Porosity is the measure of void space in
258
the powder sample. It plays an important role during the reconstitution of powder samples.
259
The porosity of probiotic Sohiong powder increased during the storage period. A significant
260
increase in porosity was observed from 28 days of storage period. This may be due to the
261
increase in the amount of water adsorbed by the powder during storage from the surrounding
262
environment through the packaging material. This results in the agglomeration (lumps) of
263
powder. The formation of lumps responsible for increases in density and porosity of the
264
powder. Similar results were reported by Bhusari et al. (2014) and Arepally and Goswami
265
(2019). The reconstitution of Low-density powders (lighter powders) is rapid compared to
266
high-density powders.
267
3.7. Effect of storage on hygroscopicity and degree of caking of probiotic Sohiong powder
268
The hygroscopicity of Sohiong powder describes the products ability to uptake water
269
from the ambient atmosphere. It ranges from 11.3 to 31.16 % during the storage period (Fig.
270
4). A significant increase in hygroscopicity was observed from 16 days of storage period. The
271
increase in hygroscopicity may be due to the wider contact surface of the powder particles
272
and the use of higher dextrose equivalent (DE-17 to 20) maltodextrin (Molina et al., 2014).
273
The fine particles with a wider contact surface have a large number of active sites for water
12
274
adsorption. A significant increase in the degree of caking was observed from 12 days of
275
storage period. The degree of caking mainly depends on the anticaking agent added in the
276
feed and water adsorption during storage. Lower hygroscopicity and degree of caking can be
277
achieved by using the high concentration of maltodextrin (low DE) and anticaking agents.
278
This is recommended to get non-sticky free-flowing powders. Similar results were observed
279
for spray dried probiotic lychee juice (Kingwatee et al., 2015).
280
3.8. Effect of storage on the glass transition temperature of probiotic Sohiong powder
281
The glass transition temperature (Tg) is the temperature where the material undergoes
282
a physical transition from glassy to a rubbery state. The Tg of spray dried Sohiong powder
283
was measured on 0, 20 and 40 days of storage period. The glass transition temperature was
284
decreased during the storage period. The 0, 20 and 40 days stored powder showed 57.04oC,
285
49.12oC and 45.81oC, respectively. The adsorption of moisture during storage period leads to
286
the mobilization of molecules to exhibit a higher tendency to undergo physical transition.
287
This phenomenon decreases the Tg of spray dried probiotic Sohiong powder. The plasticizing
288
effect of water leads to depressing the Tg of amorphous materials (Fabra et al., 2009). The
289
glass transition temperatures depend on the fruit solids, maltodextrin, and anticaking agents.
290
Anticaking agents increase the stability of spray dried fruit powders (Chang et al., 2018). The
291
results showed that the obtained Tg values are higher than the room temperature (Tg>25oC);
292
therefore, the powder is in the glassy state (Fig. 5). The addition of maltodextrin in feed
293
solution increased the Tg values of powder, which has a higher molecular weight (Jaya &
294
Das, 2004). Similar results were observed for spray dried sucrose and mango powder
295
(Adhikari, Howes, Bhandari, & Langrish, 2009).
296
3.9. Effect of storage on powder morphology and degree of crystallinity of probiotic Sohiong
297
powder
13
298
The morphology of spray dried Sohiong powder was determined at 0, 18 and 36 days
299
of storage period. The maltodextrin added in feed was used as a control (Fig.6). The
300
magnification level used for determining the microstructure for maltodextrin and spray dried
301
probiotic Sohiong powder were 2500x and 10000x, respectively. The spray dried powder
302
resulted in almost spherical with smaller size. While the maltodextrin particles were irregular,
303
hollow and shriveled with larger size (Fig.6). No free cells were visible in FESEM images,
304
confirming the formation of good microcapsules. Similar results were observed for spray
305
drying of mango (Caparino et al., 2012) and lychee (Kingwatee et al., 2015). The smoothness
306
and spherical shape decreased with increase in storage period. This may be due to the
307
formation of liquid bridges resulting in agglomeration of particles. The degree of crystallinity
308
of spray-dried probiotic Sohiong powder at 0, 20, and 40 days of storage was observed for the
309
crystalline-amorphous state. The x-ray diffraction patterns did not show a crystalline peak
310
formation. It showed a broad background pattern which indicates the amorphous
311
characteristics (Caparino et al., 2012). This may be due to the low molecular weight
312
compounds, i.e. organic acids and fruit sugars present in the Sohiong fruit. These compounds
313
have insufficient time to crystallize, thus produces an amorphous metastable state of dried
314
powders. Similar results were observed for spray dried mango powder (Caparino et al.,
315
2012).
316
4. Conclusion
317
The enzymatically extracted Sohiong juice was fermented with L. plantarum and
318
subjected to spray drying to get the probiotic Sohiong powder. The moisture sorption
319
isotherms of the powder were determined using various sorption models in the range of 25 to
320
45oC. The equilibrium moisture content of spray dried Sohiong powder increased with water
321
activity. Peleg model was recommended as the best model due to highest goodness of fit
322
parameters. The product stability at 25oC and 50% RH was determined until the probiotic 14
323
viability reduces beyond 6 Log CFU/g. The powder was amorphous throughout the storage as
324
confirmed by XRD. Quality of probiotic Sohiong juice powder could be maintained up to 36
325
days without packaging. Therefore, good quality non-dairy based probiotic Sohiong fruit
326
powder could be produced using spray drying technique with possible application in the food
327
industry.
328 329 330
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18
Table 1 Equilibrium moisture content versus specific water activity data at different temperature 25 oC 30 oC 35 oC 40 oC aw
Xeq
aw
Xeq
aw
Xeq
0.116 0.049±0.006 0.114 0.046±0.012 0.113 0.046±0.030 0.254 0.058±0.012 0.243 0.058±0.002 0.225 0.057±0.010 0.330 0.075±0.012 0.310 0.072±0.002 0.288 0.070±0.006 0.436 0.100±0.015 0.429 0.091±0.015 0.404 0.090±0.006 0.517 0.170±0.010 0.507 0.149±0.011 0.471 0.126±0.021 0.688 0.281±0.052 0.676 0.261±0.031 0.620 0.213±0.015 0.762 0.349±0.010 0.742 0.316±0.045 0.714 0.279±0.020 0.844 0.461±0.023 0.834 0.426±0.010 0.825 0.393±0.015 * aw is the water activity and Xeq is the equilibrium moisture content
45oC
aw
Xeq
aw
Xeq
0.113 0.201 0.255 0.380 0.453 0.598 0.691 0.817
0.043±0.012 0.055±0.015 0.058±0.006 0.073±0.029 0.095±0.003 0.189±0.015 0.250±0.001 0.357±0.025
0.112 0.198 0.236 0.356 0.422 0.583 0.681 0.811
0.040±0.026 0.049±0.030 0.051±0.021 0.068±0.015 0.084±0.010 0.163±0.015 0.221±0.024 0.282±0.005
Table 2 Model coefficients and fit parameters Model Temperat ure (oC) Constant 1 BET
Halsey
Henderson
Oswin
Iglesias and Chirife
Smith
Caurie
GAB
Peleg
25 30 35 40 45 25 30 35 40 45 25 30 35 40 45 25 30 35 40 45 25 30 35 40 45 25 30 35 40 45 25 30 35 40 45 25 30 35 40 45 25 30 35 40 45
7.023, C 6.252, C 7.446, C 6.259, C 10.14, C 5.986, h1 6.016, h1 6.400, h1 6.572, h1 9.227, h1 8.437, H1 8.738, H1 9.572, H1 9.998, H1 14.170, H1 0.150, M 0.144, M 0.142, M 0.131, M 0.121, M 0.052, A 0.047, A 0.048, A 0.041, A 0.044, A -0.014, A -0.012, A -0.0036, A -0.0037, A 0.008, A -3.614, A -3.658, A -3.581, A -3.688, A -3.579, A 0.755, C 0.837, C 1.542, C 1.087, C 0.461, C 0.029, k1 0.632, k1 0.045, k1 0.558, k1 0.007, k1
Model coefficients Constant 2
Constant 3
Constant 4
0.080, M 0.078, M 0.075, M 0.072, M 0.060, M 1.173, h2 1.148, h2 1.167, h2 1.134, h2 1.244, h2 0.885, H2 0.884, H2 0.931, H2 0.917, H2 1.055, H2 0.685, N 0.694, N 0.674, N 0.689, N 0.617, N 0.083, B 0.082, B 0.079, B 0.076, B 0.062, B -0.252, B -0.239, B -0.223, B -0.209, B -0.170, B 3.370, B 3.373, B 3.218, B 3.271, B 2.904, B 0.746, K 0.764, K 0.831, K 0.781, K 0.499, K 0.660, k2 0.039, k2 0.578, k2 0.024, k2 0.417, k2
0.304, M 0.261, M 0.161, M 0.198, M 0.718, M -0.215, n1 -0.071, n1 -0.0003, n1 -0.273, n1 -0.717, n1
2.583, n2 2.718, n2 2.641, n2 2.528, n2 1.912, n2
Goodness of fit parameters Adjusted RMSE R Square 0.962 0.030 0.966 0.026 0.970 0.022 0.961 0.023 0.928 0.024 0.969 0.027 0.971 0.024 0.976 0.020 0.965 0.021 0.943 0.022 0.988 0.016 0.989 0.015 0.990 0.013 0.979 0.017 0.976 0.014 0.982 0.024 0.983 0.018 0.987 0.014 0.977 0.018 0.964 0.017 0.952 0.034 0.957 0.029 0.959 0.025 0.952 0.025 0.913 0.027 0.089 0.985 0.018 0.983 0.014 0.987 0.018 0.975 0.977 0.014 0.995 0.011 0.995 0.009 0.997 0.007 0.011 0.990 0.012 0.982 0.989 0.017 0.015 0.988 0.013 0.989 0.977 0.018 0.015 0.974 0.995 0.011 0.996 0.009 0.998 0.005 0.993 0.010 0.989 0.009
Table 3 Colour values of the spray dried Sohiong powder Storage days. L* a* b* f b 0 43.880±0.197 34.487±0.356 6.390±0.369b 4 43.703±0.505ef 34.478±0.105b 6.231±0.265b 8 43.643±0.546ef 34.437±0.066b 6.185±0.314b 12 43.557±0.597ef 33.934±0.062a 6.297±0.044b 16 43.132±0.026de 36.374±0.154c 6.220±0.099b 20 42.890±0.192d 36.169±0.078c 6.111±0.058b 24 42.057±0.056c 36.951±0.175d 5.824±0.060a 28 40.817±0.343b 37.170±0.341d 5.810±0.187a 32 40.540±0.115ab 36.999±0.101d 5.446±0.167a 36 40.093±0.029a 38.104±0.058e 5.670±0.328a 40 40.090±0.036a 37.973±0.146e 5.744±0.092a a,b,c,d,e,f represents the significance along the column.
Table 4 Porosity and flow properties of spray dried Sohiong powder Storage days. Porosity Carr’s index Hausner ratio bc a 0 0.480±0.004 16.067±1.904 1.192±0.027abc 4 0.471±0.009ab 15.931±1.383a 1.190±0.020abc ab a 8 0.470±0.001 15.678±1.160 1.186±0.016a 12 0.461±0.016a 17.838±1.143ab 1.217±0.017bc bc ab 16 0.481±0.004 17.043±1.436 1.206±0.021abc ab a 20 0.470±0.004 15.721±1.089 1.187±0.015abc 24 0.470±0.005ab 15.836±0.113a 1.188±0.002abc c a 28 0.486±0.005 15.540±0.284 1.184±0.004ab 32 0.487±0.005c 18.886±1.498bc 1.233±0.023cd d c 36 0.509±0.006 21.132±0.545 1.268±0.009d 40 0.523±0.007e 23.670±3.187d 1.312±0.054e a,b,c,d,e represents the significance along the column.
Table 5 Densities of the spray dried Sohiong powder Storage days. Loose bulk density Tapped bulk density True density a a 0 0.521±0.001 0.621±0.014 1.003±0.008a 4 0.527±0.006a 0.627±0.014a 0.998±0.012a ab a 8 0.530±0.004 0.629±0.013 1.001±0.005a 12 0.540±0.004bc 0.658±0.011b 1.003±0.027a c b 16 0.548±0.006 0.661±0.009 1.056±0.016b d c 20 0.609±0.010 0.723±0.011 1.148±0.012c 24 0.652±0.005e 0.775±0.009d 1.230±0.005d f d 28 0.669±0.003 0.792±0.006 1.301±0.012e 32 0.678±0.004fg 0.836±0.014e 1.322±0.005e g f 36 0.686±0.004 0.869±0.011 1.396±0.016f 40 0.706±0.014h 0.926±0.034g 1.480±0.009g a,b,c,d,e,f,g,h represents the significance along the column.
Probiotic viability (log CFU/g)
7.300 7.100 6.900 6.700 6.500 6.300 6.100 5.900 5.700 5.500 0
5
10
15
20 25 30 Storage days
35
Fig 1. Effect of storage on Probiotic viability
40
45
Fig 2. Probiotic Sohiong powder (a) 0 days storage, (b) 36 days storage
Moisture content (%) w.b
9.000 8.000 7.000 6.000 5.000 4.000 3.000 0
5
10
15
20 25 30 Storage days
35
Fig 3. Effect of storage on moisture content
40
45
Hygroscopicity (%)
35.000 30.000 25.000 20.000 15.000 10.000 5.000 0.000 0
5
10
15
20 25 30 Storage days
35
Fig 4. Effect of storage on hygroscopicity
40
45
^ex o mW
SPB, 08.01.2019 11:51:03 SPB, 9.7000 mg
Glas s Transition Onset 41.00 °C Midpoint 57.04 °C
0
-2
-4
-6
-8 -20
0
20
40
60
80
100
120
140
160
180
200
220
°C
0
2
4
6
8
10
12
14
16
18
20
22
24
min
Lab : M ET T LE R
S T A R e S W 1 2. 10
Fig 5. Tg of probiotic Sohiong powder at 0 day of storage
b
a Encapsulated cells
10 kV
30µm
10 kV
5µm
d
c Encapsulated cells Encapsulated cells
10 kV
5µm
10 kV
5µm
Fig 6. FESEM images of (a) maltodextrin, (b) spray dried Sohiong powder at 0day, (c) Spray dried Sohiong powder at 18 day and (d) Spray dried Sohiong powder at 36 days
Highlights 1. A non-dairy based probiotic Sohiong fruit powder was developed. 2. Moisture sorption isotherms of probiotic Sohiong powder were modelled. 3. The storage stability of the powder in LDPE pouches was studied at 25oC and 50% RH. 4. Probiotic viability of 6.12 log CFU/g was obtained until 36 days of storage period. 5. The probiotic Sohiong powder was of good quality, structure and morphology.
Conflict of Interest and Authorship Conformation Form Please check the following as appropriate:
All authors have participated in (a) conception and design, or analysis and interpretation of the data; (b) drafting the article or revising it critically for important intellectual content; and (c) approval of the final version. This manuscript has not been submitted to, nor is under review at, another journal or other publishing venue. The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript The following authors have affiliations with organizations with direct or indirect financial interest in the subject matter discussed in the manuscript: Author’s name 1. Kambhampati Vivek 2. Sabyasachi Mishra 3. Rama Chandra Pradhan
Affiliation National Institute of Technology, Rourkela National Institute of Technology, Rourkela National Institute of Technology, Rourkela