Results of Heat Challenge Experiment and Related Work

Direct vat set (DVS) probiotic dairy starters can be produced to address the issue of starter
contamination and the associated poor performance of the starter. The current study was
aimed suitable protective agents and to optimize the spray dried parameters for the
production of probiotic DVS with better survivability. The DVS starter observations w.r.t.
purity, selection of protective agents and their levels, optimization spray drying conditions,
techno-functional evaluation of probiotic DVS prepared, selection of packaging material and
storage stability have been presented in this chapter in the form of tables and figures.
Maintenance, preservation and propagation of Lactiplantibacillus plantarum strains
Lactiplantibacillus plantarum CRD7, Lactiplantibacillus plantarum HD48 and
Lactiplantibacillus plantarum HD 51 was maintained in newly developed cost-effective
whey-based medium (WBM). Loopful of overnight grown culture was streaked on WBM
agar plates to obtain well isolated pure colonies. Long term preservation of cultures was
carried out in 40% glycerol in cryovials at -20°C. Culture was activated by sub culturing
thrice before use. The active culture was inoculated in sterile skim milk @1% inoculum and
incubated at 37°C/16-20 h twice to get active culture before study. The purity of culture was
determined by microscopic examination (Gram’s and negative staining) as well as catalase
test.
RESULTS
Three probiotic Lactiplantibacillus cultures listed in Table 1 were selected based on their
health benefits features. Evaluated earlier at Synbiotic Functional Food and Bioremediation
Research Laboratory, Dairy Microbiology Division, ICAR-National Dairy Research Institute
(Deemed University), Karnal-132001, Haryana, India. Selected probiotic strains were sub-
cultured in whey-based medium, skim milk for their microscopic and catalase test for purity
evaluation. Morphological and biochemical parameters details of selected strains have been
presented in Table 1 and figure 1.
Table 1: Morphological and biochemical parameters
Probiotic strains Negative staining Grams staining Catalase

Lp. plantarum CRD7 Short rod in chains +ve -ve
Lp. plantarum HD48 Short rod in chains +ve -ve
Lp. plantarum HD51 Short rod in chains +ve -ve
The selected probiotic Lactiplantibacilli plantarum strains were found Gram’s positive, long
or short rods arranged in single chains. Selected probiotic Lactobacilli were found catalase
negative. These morphological and biochemical observations reveled their purity for further
investigations.
1
Fig 1: Microscopic and catalase test observations of probiotic Lactiplantibacillus strains
TECHNO-FUNCTIONAL ATTRIBUTES OF PROBIOTIC DAHI PREPARED BY

SINGLE LACTOBACILLUS CULTURE
Data on techno-functional attributes of dahi prepared with single strains have been presented
in Table 2 and Figure 2. Probiotic dahi was prepared with standardized milk, heated to
90°C/10min. Milk was cooled to 37°C and inoculated with 1% inoculum. This was incubated
at 37°C and observed for curd setting, pH, titratable acidity and probiotic counts. Probiotic
dahi was also analysed for sensory and textural attributes. The curd setting time taken by
single probiotic Lactiplantibacillus plantarum strains for the preparation of dahi ranged
14.00±0.02 -15.00±0.03 h, pH and titratable acidity ranged between 4.12±0.01-4.37±0.01 and
0.72±0.03-0.75±0.02 (% LA) and total probiotic counts ranged from 8.71±0.01 to
9.21±0.01(Log CFU/mL). These observations revealed their survivability to be used as
probiotic dairy starters. On basis of minimum curd setting time, faster reduction in pH and
acidity development, and highest probiotic counts, selected probiotic strains were explored to
optimize spray drying conditions and selection of suitable protective agents for the
preparation of DVS starters.
Techno-functional attributes of probiotic dahi prepared with lactobacillus strains
Total probiotic
Curd setting Titratable acidity
Probiotic strains pH counts
time (h) (% LA)
(Log CFU/mL)
Lp. plantarum 4.26±0.01
CRD7 14.30±0.02a a 0.72±0.00a,b 8.71±0.01a
Lp. plantarum HD48 15.00±0.03b 4.12±0.01 0.74±0.00a 8.66±0.01a
2
a
4.37±0.01
Lp. plantarum HD51 14.00±0.02a a 0.75±0.01b 9.21±0.01a
Data are represented as Mean ± SE; (p<0.05); n=3.
a-b (along the column) values with different superscripts are significantly different from each other’s .
Techno-functional attributes of probiotic dahi prepared with lactobacillus strains
Sensory scores
Observations related to sensory parameters such as colour and appearance (10), flavour (10),
body and texture (10), overall acceptability scores of probiotic dahi prepared using individual
probiotic Lactiplantibacillus plantarum cultures are mentioned in Table 3 and Figure 3.
The highest score for overall acceptability i.e., 9.3±2.03 and 9.0±1.02 was obtained
for probiotic dahi prepared from Lp. plantarum CRD7 and Lp. plantarum HD48,
respectively. Significant difference in the scores was obtained for probiotic dahi prepared
with different Lp. Plantarum cultures.
Sensory scores of probiotic dahi prepared with lactobacillus strains
Colour and Body and Overall
Probiotic strains Flavour
appearance texture acceptability
Lp. plantarum CRD7 9.3±0.70 a 8.6±0.36a 9.3±1.02a 9.3±2.03a
Lp. plantarum HD48 8.6±0.28b 7.3±0.75b 9.0±0.65b 9.0±1.02b
Lp. plantarum HD51 9.3±0.70 a 8.8±0.32a 9.4±1.04a 9.4±0.03a
a-b (along the column) values with different superscripts are significantly different from each other’s .
Textural attributes of probiotic dahi prepared with lactobacillus strains
Hardness or firmness, is the most important characteristics in determining texture of dairy

products vis-à-vis fermented dairy foods. It is regarded as the force required to attain a certain
deformation and is considered a measure of hardness of fermented milk products such as
3
yoghurt. Results obtained on texture profile have been presented in Table 4 and Figure 4.
Texture parameters such as firmness (N), consistency (N.s), cohesiveness (N) and work of
cohesion (N.s) of probiotic dahi prepared by application of individual probiotic
Lactiplantibacillus plantarum cultures ranged from 2.15±0.03 to 2.74±0.03. The highest
score for firmness was obtained for dahi prepared with Lp. Plantarum HD51 (2.74±0.03)
Textural attributes of probiotic dahi prepared with lactobacillus strains
Consistency Cohesiveness Work of

Probiotic strains Firmness (N)
(N.s) (N) cohesion (N.s)
CRD7 2.74±0.03a 24.55±0.25a -0.55±0.01b -4.03±0.28b
HD48 2.15±0.02b 28.23±0.75b -0.72±0.03a -3.54±0.32a
HD51 2.42±0.02b 39.23±0.75b -0.34±0.03a -2.54±0.32a
a-b (along the column) values with different superscripts are significantly different from each others.
Sensory scores of probiotic dahi prepared with lactobacillus strains.
Texture profile of probiotic dahi prepared with lactobacillus strains
4
HEAT TOLERANCE DETERMINATION OF PROBIOTIC LACTIPLANTIBACILLUS
PLANTARUM HD 51 TO SUB-LETHAL HEAT STRESS
Heat tolerance of probiotic strains was determined by exposing it to sublethal stress for varied
time-temperature combinations, different pH levels and varied salt concentrations as
mentioned in Table 5.1 and analyzing for survivability by pour plating technique.
Experimental design to delineate effects of stress conditions on heat tolerance of Lp. plantarum
strains
Cell count
Stress type Control Stress levels Exposure time (min)
(CFU/g)
Temp (°C) 37 55,60 & 65 0, 2.5, 5.0, 7.5 and 10 10 , 1011 & 1012
10
5.5,6.0,6.5,7.0
pH 6.5 0, 2.5, 5.0, 7.5 and 10 1010, 1011 & 1012
& 7.5
Salt (w/v %) 0 1, 2, 3,4 & 5 0, 2.5, 5.0, 7.5 and 10 1010, 1011 & 1012
Heat challenge experiment was conducted in 35 and 45% TS reconstituted skim milk (RSM)
to delineate effect of storage conditions on heat tolerance of probiotic strains according to
Bommasamudram et al., (2021) in 100 mL sample tubes as proposed in experimental design
using a water bath adjusted at 55°C, 60°C and 65°C for 0, 2.5, 5.0, 7.5 and 10 min. Treated
samples was immediately cooled to room temperature by keeping in chilled water. After
completion, viability of lactobacilli was checked by pour plate technique in whey-based
medium. The cell biomass of selected probiotic Lactiplantibacillus strains i.e. Lp. plantarum
CRD7, Lp. plantarum HD48 and Lp. plantarum HD51 was obtained individually by
centrifugation at 8000 rpm/10 min at 4oC from cultures grown under optimized cultural
growth conditions (whey-based medium, pH 6.5±0.02, incubation at 37 oC/16 h) by batch
fermentation. The harvested concentrated cell biomass of selected probiotic strains was re-
suspended in 35 and 45% sterilized reconstituted skim milk (RSM) supplemented with
varied concentrations (0, 0.5, 2.5, 5.0, 7.5 and 10% w/v) of respective protective agents i.e.
maltodextrin, cornstarch and gum acacia. The re-suspended biomass in RSM (50 mL) was
dispersed in 100 mL sample bottles and agitated by magnetic stirrer bars. This was placed in
water bath at appropriate test temperatures of 37 (control), 55, 60, 65 and 70oC. One bottle
was used to monitor the temperature. After temperature equilibration, inoculum of selected
culture was added to the sample bottles to attained 10 10, 1011 & 1012 CFU/mL. One mL of
aliquots were withdrawn at intervals 0. 2.5, 5.0, 7.5 and 10 min and analysed for total viable
probiotic counts.
5
Effect of heat tolerance on survivability of Lactiplantibacillus plantarum CRD7 (55 ᴼC) against different pH and salt concentrations
L. plantarum CRD7 (1010) L. plantarum CRD7 (1011) L. plantarum CRD7 (1012)

Final
Initial Final
Time Initial count Final count count
pH salt Survivabilit (Log (Log Survivabilit Initial count
(Min) (Log (Log (Log Survivability (%)
y (%) CFU/m CFU/m y (%) (Log CFU/mL)
CFU/mL) CFU/mL) CFU/m
L) L)
L)
0 11.18±0.0 11.18±0.0 12.12±0.0
0 10.07±0.05 10.07±0.05 100.00±0.05
4 4
100.00±0.00 12.12±0.07
7
100.00±0.11
1 11.37±0.0 10.48±0.0 11.38±0.0

2.5 10.19±0.04 9.44±0.12 93.69±0.12
5 3
93.82±0.04 12.11±0.09
2
96.77±0.12
2 11.23±0.0 10.51±0.0 10.82±0.0

5.5 5.0 10.21±0.15 9.17±0.05 91.01±0.05
0 4
91.97±0.00 12.12±0.02
7
93.17±0.11
3 11.16±0.0 10.17±0.0
7.5 10.16±0.12 8.69±0.14 86.25±0.14
3
9.60±0.06 87.48±0.11 12.25±0.21
5
90.25±0.07
4 11.13±0.1
10.0 10.18±0.03 8.35±0.01 82.92±0.01
1
9.19±0.04 84.34±0.10 12.20±0.16 9.82±0.04 87.11±0.04
0 11.18±0.0 11.37±0.0 12.11±0.0

0 10.07±0.05 9.80±0.27 100.00±0.04
4 5
100.00±0.04 12.12±0.07
9
100.00±0.04
1 11.37±0.0 10.76±0.0 11.73±0.0

2.5 10.19±0.04 9.42±0.12 93.85±0.06
5 2
92.08±0.10 12.11±0.09
1
91.50±0.04
2 11.23±0.0 10.46±0.0 11.09±0.0

6.0 5.0 10.21±0.15 8.87±0.05 92.10±0.04
0 5
90.60±0.04 12.12±0.02
9
91.85±0.04
3 11.16±0.0 10.42±0.0
7.5 10.16±0.12 8.37±0.05 87.00±0.05
3
9.90±0.03 85.91±0.06 12.25±0.21
9
88.74±0.07
4 11.13±0.1 10.01±0.0
10.0 10.18±0.03 9.80±0.27 82.14±0.05
1
9.39±0.68 82.19±0.04 12.20±0.16
2
85.81±0.04
0 11.18±0.0 11.37±0.0 12.12±0.0

0 10.07±0.05 9.88±0.10 100.00±0.15
4 5
100.00±0.05 12.12±0.07
2
100.00±0.07
1 11.37±0.0 10.80±0.2 11.73±0.0

2.5 10.19±0.04 9.42±0.04 96.17±0.27
5 1
96.74±0.03 12.11±0.09
4
95.59±0.05
2 11.23±0.0 10.46±0.0 11.18±0.0

6.5 5.0 10.21±0.15 9.12±0.00 92.46±0.15
0 5
91.95±0.05 12.12±0.02
2
89.27±0.07
3 11.16±0.0 10.47±0.1
7.0 10.16±0.12 8.48±0.03 89.28±0.00
3
9.90±0.03 87.03±0.03 12.25±0.21
3
83.87±0.05
4 11.13±0.1 10.10±0.0
10.0 10.18±0.03 9.88±0.10 83.06±0.03
1
9.39±0.68 82.54±0.68 12.20±0.16
1
80.98±0.04
6
0 11.18±0.0 11.16±0.0 12.25±0.2
0 10.07±0.05 9.84±0.06 100.00±0.12
4 3
100.00±0.00 12.12±0.07
1
100.00±0.09
1 11.37±0.0 10.76±0.0 11.73±0.0

2.5 10.19±0.04 9.42±0.12 93.85±0.06
5 2
92.08±0.10 12.11±0.09
1
91.50±0.04
2 11.23±0.0 10.37±0.0 11.34±0.2

7.0 5.0 10.21±0.15 9.13±0.02 92.72±0.12
0 3
92.43±0.00 12.12±0.02
1
91.54±0.09
3 11.16±0.0 10.70±0.0
7.5 10.16±0.12 8.56±0.11 89.86±0.02
3
9.93±0.06 88.64±0.10 12.25±0.21
4
86.00±0.09
4 11.13±0.1 10.35±0.1
10.0 10.18±0.03 9.84±0.06 84.20±0.11
1
9.38±0.07 83.07±0.11 12.20±0.16
1
82.62±0.02
0 11.18±0.0 11.13±0.1 12.20±0.1

0 10.07±0.05 9.87±0.05 100.00±0.03
4 1
100.00±0.03 12.12±0.07
6
100.00±0.02
1 11.37±0.0 10.77±0.1 11.72±0.1

2.5 10.19±0.04 9.49±0.03 82.95±0.05
5 2
96.42±0.02 12.11±0.09
1
95.67±0.09
2 11.23±0.0 10.37±0.1 11.37±0.1

7.5 5.0 10.21±0.15 9.18±0.04 81.22±0.03
0 1
92.92±0.03 12.12±0.02
6
92.24±0.02
3 11.16±0.0 10.05±0.0 10.94±0.1

7.5 10.16±0.12 8.80±0.15 80.13±0.04
3 7
88.93±0.06 12.25±0.21
2
86.34±0.13
4 11.13±0.1 10.60±0.0
10.0 10.18±0.03 9.87±0.05 79.39±0.15
1
9.70±0.04 84.05±0.07 12.20±0.16
5
83.29±0.01

Final
Time
pH salt Initial count Final count Survivabil Initial (Log Final (Log Survivabil Initial count count Survivability
(Min)
(Log CFU/mL) (Log CFU/mL) ity (%) CFU/mL) CFU/mL) ity (%) (Log CFU/mL) (Log (%)
CFU/mL)
0 0 10.01±0.05 10.01±0.05 100.00±0.05 11.18±0.04 11.18±0.04 100.00±0.00 12.12±0.07 12.12±0.07 100.00±0.11
1 2.5 10.20±0.04 10.05±0.12 91.69±0.12 11.37±0.05 10.48±0.03 93.82±0.04 12.11±0.09 11.38±0.02 90.77±0.12
5.5 2 5.0 10.04±0.15 9.27±0.05 91.01±0.05 11.23±0.00 10.13±0.04 91.97±0.00 12.12±0.02 10.82±0.07 89.17±0.11
3 7.5 10.06±0.12 8.59±0.14 86.25±0.14 11.16±0.03 9.60±0.06 87.48±0.11 12.25±0.21 10.17±0.05 85.25±0.07
4 10.0 10.05±0.03 8.45±0.01 82.92±0.01 11.13±0.11 9.19±0.04 84.34±0.10 12.20±0.16 9.82±0.04 81.11±0.04
6.0 0 0 10.01±0.05 10.00±0.27 99.00±0.04 11.18±0.04 11.37±0.05 100.00±0.04 12.12±0.07 12.11±0.09 100.00±0.04
1 2.5 10.20±0.04 9.24±0.04 96.17±0.27 11.37±0.05 10.80±0.21 93.74±0.03 12.11±0.09 11.57±0.04 90.59±0.05
2 5.0 10.04±0.15 8.64±0.02 92.10±0.04 11.23±0.00 10.46±0.05 90.60±0.04 12.12±0.02 11.09±0.09 89.85±0.04
3 7.5 10.06±0.12 8.27±0.05 87.00±0.05 11.16±0.03 9.90±0.03 85.91±0.06 12.25±0.21 10.42±0.09 88.74±0.07
7
4 10.0 10.05±0.03 8.11±0.27 82.14±0.05 11.13±0.11 9.39±0.68 82.19±0.04 12.20±0.16 10.01±0.02 85.81±0.04
0 0 10.01±0.05 10.01±0.10 100.00±0.15 11.18±0.04 11.37±0.05 100.00±0.05 12.12±0.07 12.12±0.02 100.00±0.07
1 2.5 10.20±0.04 9.95±0.15 92.77±0.10 11.37±0.05 10.80±0.21 94.99±0.21 12.11±0.09 11.60±0.09 87.89±0.02
6.5 2 5.0 10.04±0.15 9.87±0.00 93.46±0.15 11.23±0.00 10.46±0.05 93.95±0.05 12.12±0.02 11.18±0.02 94.27±0.07
3 7.0 10.06±0.12 8.38±0.03 89.28±0.00 11.16±0.03 9.90±0.03 87.03±0.03 12.25±0.21 10.47±0.13 83.87±0.05
4 10.0 10.05±0.03 9.78±0.10 83.06±0.03 11.13±0.11 9.39±0.68 82.54±0.68 12.20±0.16 10.10±0.01 80.98±0.04
0 0 10.01±0.05 9.99±0.06 99.02±0.12 11.18±0.04 11.16±0.03 100.00±0.00 12.12±0.07 12.25±0.21 100.00±0.09
1 2.5 10.20±0.04 9.65±0.12 91.85±0.06 11.37±0.05 10.76±0.02 92.08±0.10 12.11±0.09 11.73±0.01 92.50±0.04
7.0 2 5.0 10.04±0.15 9.23±0.02 90.72±0.12 11.23±0.00 10.37±0.03 91.43±0.00 12.12±0.02 11.34±0.21 91.54±0.09
3 7.5 10.06±0.12 8.92±0.11 89.86±0.02 11.16±0.03 9.93±0.06 88.64±0.10 12.25±0.21 10.70±0.04 86.00±0.09
4 10.0 10.05±0.03 8.75±0.06 84.20±0.11 11.13±0.11 9.38±0.07 83.07±0.11 12.20±0.16 10.35±0.11 82.62±0.02
0 0 10.01±0.05 10.01±0.05 100.00±0.03 11.18±0.04 11.13±0.11 100.00±0.03 12.12±0.07 12.20±0.16 100.00±0.02
1 2.5 10.20±0.04 9.49±0.03 91.95±0.05 11.37±0.05 10.77±0.12 91.42±0.02 12.11±0.09 11.72±0.11 91.67±0.09
7.5 2 5.0 10.04±0.15 9.18±0.04 89.22±0.03 11.23±0.00 10.37±0.11 89.92±0.03 12.12±0.02 11.37±0.16 88.24±0.02
3 7.5 10.06±0.12 8.80±0.15 89.13±0.04 11.16±0.03 10.05±0.07 88.93±0.06 12.25±0.21 10.94±0.12 86.34±0.13
4 10.0 10.05±0.03 9.87±0.05 86.39±0.15 11.13±0.11 9.70±0.04 84.05±0.07 12.20±0.16 10.60±0.05 83.29±0.01

Time
pH Salt Initial count Final count Survivabil Initial (Log Final (Log Survivabil Initial count Final count Survivabil
(Min)
(Log CFU/mL) (Log CFU/mL) ity (%) CFU/mL) CFU/mL) ity (%) (Log CFU/mL) (Log CFU/mL) ity (%)
0 0 10.08±0.05 10.01±0.05 100.00±0.05 11.11±0.04 11.18±0.04 100.00±0.00 12.12±0.07 12.12±0.07 100.00±0.11
1 2.5 10.90±0.04 9.97±0.12 91.69±0.12 11.32±0.05 10.48±0.03 83.82±0.04 12.11±0.09 11.38±0.02 91.77±0.12
5.5 2 5.0 10.02±0.15 9.21±0.05 87.01±0.05 11.21±0.00 10.13±0.04 86.97±0.00 12.12±0.02 10.82±0.07 74.17±0.11
3 7.5 10.05±0.12 8.87±0.14 85.25±0.14 11.12±0.03 9.60±0.06 85.48±0.11 12.25±0.21 10.17±0.05 81.25±0.07
4 10.0 10.14±0.03 8.41±0.01 82.92±0.01 11.11±0.11 9.19±0.04 86.34±0.10 12.20±0.16 9.82±0.04 82.11±0.04
0 0 10.01±0.05 10.00±0.27 99.00±0.04 11.11±0.04 11.37±0.05 100.00±0.04 12.12±0.07 12.11±0.09 100.00±0.04
1 2.5 10.18±0.04 9.21±0.04 88.20±0.27 11.31±0.05 10.80±0.21 88.74±0.03 12.11±0.09 11.57±0.04 94.59±0.05
6.0 2 5.0 10.08±0.15 8.68±0.02 88.01±0.04 11.22±0.00 10.46±0.05 83.60±0.04 12.12±0.02 11.09±0.09 87.85±0.04
3 7.5 10.07±0.12 8.21±0.05 86.00±0.05 11.17±0.03 9.90±0.03 82.91±0.06 12.25±0.21 10.42±0.09 82.74±0.07
4 10.0 10.04±0.03 8.13±0.27 81.14±0.05 11.18±0.11 9.39±0.68 81.19±0.04 12.20±0.16 10.01±0.02 81.81±0.04
8
0 0 10.04±0.05 10.00±0.10 100.00±0.15 11.18±0.04 11.37±0.05 100.00±0.05 12.12±0.07 12.12±0.02 100.00±0.07
1 2.5 10.22±0.04 9.91±0.15 91.77±0.10 11.34±0.05 10.80±0.21 93.99±0.21 12.11±0.09 11.60±0.09 86.89±0.02
6.5 2 5.0 10.02±0.15 9.82±0.00 89.46±0.15 11.25±0.00 10.46±0.05 90.295±0.05 12.12±0.02 11.18±0.02 87.27±0.07
3 7.0 10.05±0.12 8.31±0.03 88.28±0.00 11.11±0.03 9.90±0.03 86.03±0.03 12.25±0.21 10.47±0.13 83.87±0.05
4 10.0 10.05±0.03 9.78±0.10 81.06±0.03 11.15±0.11 9.39±0.68 81.54±0.68 12.20±0.16 10.10±0.01 80.98±0.04
0 0 10.01±0.05 9.78±0.06 99.92±0.12 11.14±0.04 11.16±0.03 100.00±0.00 12.12±0.07 12.25±0.21 100.00±0.09
1 2.5 10.18±0.04 9.56±0.12 82.85±0.06 11.35±0.05 10.76±0.02 91.08±0.10 12.11±0.09 11.73±0.01 88.50±0.04
7.0 2 5.0 10.03±0.15 9.21±0.02 86.72±0.12 11.24±0.00 10.37±0.03 89.43±0.00 12.12±0.02 11.34±0.21 86.54±0.09
3 7.5 10.05±0.12 8.29±0.11 86.86±0.02 11.12±0.03 9.93±0.06 84.64±0.10 12.25±0.21 10.70±0.04 82.00±0.09
4 10.0 10.04±0.03 8.57±0.06 83.20±0.11 11.11±0.11 9.38±0.07 83.07±0.11 12.20±0.16 10.35±0.11 81.62±0.02
0 0 10.018±0.05 10.01±0.05 100.00±0.03 11.13±0.04 11.13±0.11 100.00±0.03 12.12±0.07 12.20±0.16 100.00±0.02
1 2.5 10.29±0.04 9.94±0.03 92.95±0.05 11.31±0.05 10.77±0.12 91.42±0.02 12.11±0.09 11.72±0.11 90.67±0.09
7.5 2 5.0 10.14±0.15 9.14±0.04 88.22±0.03 11.22±0.00 10.37±0.11 88.92±0.03 12.12±0.02 11.37±0.16 87.24±0.02
3 7.5 10.12±0.12 8.08±0.15 88.13±0.04 11.11±0.03 10.05±0.07 84.93±0.06 12.25±0.21 10.94±0.12 85.34±0.13
4 10.0 10.25±0.03 9.27±0.05 87.39±0.15 11.12±0.11 9.70±0.04 83.05±0.07 12.20±0.16 10.60±0.05 82.29±0.01
Effect of heat tolerance on survivability of Lactiplantibacillus plantarum HD48 (55 ᴼC) against different salt and pH concentrations
Probiotic viable count (Log CFU/g or mL)

Probiotic Salt
pH
Lactobacillus strain (%w/v) Before heat
0 min 2.5 min 5.0 min 7.5 min 10 min
treatment
0.0 (control) 6.0 (control) 9.21±0.01a 9.19±0.01a 9.10±0.011b 8.85±0.02e 8.35±0.02e 8.01±0.02e
HD48 (9 Log 1.0 4.5 9.10±0.02b 9.10±0.02b 9.01±0.00b 8.78±0.02cd 8.24±0.02cd 7.99±0.02cd
CFU/mL)
2.5 6.5 9.23±0.02b 9.23±0.02b 9.13±0.03a 8.61±0.03b 8.32±0.03b 8.09±0.03b
5.0 7.0 9.11±0.03c 9.11±0.03c 9.02±0.011b 8.89±0.02a 8.25±0.02a 8.00±0.02a
0.0 (control) 6.0 (control) 10.30±0.03c 10.30±0.03c 9.99±0.02e 9.67±0.04c 8.81±0.04c 8.34±0.04c
HD48 (10 Log
CFU/mL)
1.0 4.5 10.21±0.01a 10.21±0.01a 10.01±0.02cd 9.53±0.01d 8.22±0.01d 8.00±0.01d
9
2.5 6.5 10.10±0.02b 10.10±0.02b 9.89±0.03b 9.54±0.02d 8.22±0.02d 8.02±0.02d
5.0 7.0 10.01±0.03c 10.01±0.03c 9.88±0.02a 9.66±0.02ab 8.21±0.02ab 8.11±0.02ab
0.0 (control) 6.0 (control) 11.07±0.04d 11.05±0.04d 10.98±0.04c 10.64±0.03ab 10.02±0.03ab 9.23±0.03ab
HD48 (11 Log 1.0 4.5 11.21±0.05e 11.19±0.05e 10.78±0.01d 10.23±0.01b 10.09±0.01b 9.65±0.01b
CFU/mL)
2.5 6.5 11.13±0.02b 11.12±0.02b 10.89±0.02d 10.13±0.02a 9.89±0.02a 9.29±0.02a
5.0 7.0 11.33±0.01a 11.21±0.01a 10.85±0.02ab 10.43±0.01c 9.92±0.01c 9.33±0.01c
0.0 (control) 6.0 (control) 12.10±0.04d 12.09±0.04d 11.84±0.03ab 10.50±0.01c 9.50±0.01c 8.99±0.01c
1.0 4.5 12.15±0.02b 12.12±0.02b 11.62±0.01b 10.62±0.02b 10.02±0.02b 9.08±0.02b

HD48 (12 Log
CFU/mL)
2.5 6.5 12.20±0.02b 12.21±0.02b 11.73±0.02a 10.78±0.02a 9.78±0.02a 9.18±0.02a
5.0 7.0 12.08±0.01a 12.09±0.01a 11.23±0.01c 10.61±0.01b 9.78±0.01b 9.10±0.01b

Salt Survivability (%)

Probiotic Lactobacillus strain pH
(%w/v) 2.5 min 5.0 min 7.5 min 10 min
c c c
0.0 (control) 6.0 (control) 82.75±0.02 82.75±0.02 80.75±0.02 82.75±0.02c
HD48 (9 Log CFU/mL) 1.0 4.5 89.13±0.05b 82.13±0.05b 80.13±0.05b 82.13±0.05b

2.5 6.5 91.62±0.06c 81.62±0.06c 79.62±0.06c 78.62±0.06c
5.0 7.0 88.69±0.03a 84.69±0.03a 79.69±0.03a 94.69±0.03a
0.0 (control) 6.0 (control) 89.83±0.09b 81.83±0.09b 76.83±0.09b 92.83±0.09b
1.0 4.5 88.04±0.02b 80.04±0.02b 79.04±0.02b 92.04±0.02b

HD48 (10 Log CFU/mL)
2.5 6.5 93.45±0.00c 82.45±0.00c 81.45±0.00c 78.45±0.00c
5.0 7.0 88.56±0.04a 79.56±0.04a 77.56±0.04a 70.56±0.04a
HD48 (11 Log CFU/mL) 0.0 (control) 6.0 (control) 91.65±0.01a 89.65±0.01a 81.65±0.01a 78.65±0.01a
10
1.0 4.5 91.75±0.12a 86.75±0.12a 72.75±0.12a 69.75±0.12a
2.5 6.5 93.75±0.03b 83.75±0.03b 82.75±0.03b 79.75±0.03b

5.0 7.0 86.01±0.02b 78.01±0.02b 91.01±0.02b 88.01±0.02b
0.0 (control) 6.0 (control) 85.97±0.00c 85.97±0.00c 80.67±0.00c 78.97±0.00c
1.0 4.5 86.18±0.02a 83.18±0.02a 79.18±0.02a 74.18±0.02a

2.5 6.5 90.61±0.01a 88.61±0.01a 84.61±0.01a 79.61±0.01a
5.0 7.0 78.82±0.17a 76.82±0.17a 74.82±0.17d 70.82±0.17a


Probiotic Salt
pH Before heat
Lactobacillus strain (%w/v) 0 min 2.5 min 5.0 min 7.5 min 10 min
treatment
HD48 (9 Log 1.0 4.5 9.10±0.02b 9.10±0.02b 8.78±0.00b 8.45±0.02cd 8.24±0.02cd 7.89±0.02cd
CFU/mL)
2.5 6.5 9.23±0.02b 9.13±0.02b 8.95±0.03a 8.21±0.03b 8.22±0.03b 7.17±0.03b
5.0 7.0 9.11±0.03c 9.01±0.03c 8.23±0.011b 8.10±0.02a 8.12±0.02a 8.05±0.02a
HD48 (10 Log
CFU/mL)
1.0 4.5 10.21±0.01a 10.21±0.01a 10.04±0.02cd 9.23±0.01d 8.32±0.01d 8.07±0.01d
2.5 6.5 10.10±0.02b 10.10±0.02b 9.89±0.03b 9.45±0.02d 8.09±0.02d 7.89±0.02d
11
5.0 7.0 10.01±0.03c 10.01±0.03c 9.78±0.02a 9.21±0.02ab 8.11±0.02ab 7.92±0.02ab
HD48 (11 Log 1.0 4.5 11.21±0.05e 11.19±0.05e 10.67±0.01d 10.23±0.01b 10.03±0.01b 8.65±0.01b
CFU/mL)
2.5 6.5 11.13±0.02b 11.12±0.02b 10.78±0.02d 10.13±0.02a 9.78±0.02a 9.29±0.02a
5.0 7.0 11.33±0.01a 11.21±0.01a 10.80±0.02ab 10.33±0.01c 9.89±0.01c 8.33±0.01c
HD48 (12 Log

1.0 4.5 12.15±0.02b 12.12±0.02b 11.6±0.01b 10.34±0.02b 10.12±0.02b 9.12±0.02b
CFU/mL)
2.5 6.5 12.20±0.02b 12.21±0.02b 11.32±0.02a 10.45±0.02a 9.67±0.02a 9.56±0.02a
5.0 7.0 12.08±0.01a 12.09±0.01a 11.45±0.01c 10.55±0.01b 9.89±0.01b 9.09±0.01b

(%w/v) 2.5 min 5.0 min 7.5 min 10 min
HD48 (9 Log CFU/mL)

1.0 4.5 85.13±0.05b 82.13±0.05b 80.13±0.05b 82.13±0.05b
2.5 6.5 90.62±0.06c 88.62±0.06c 78.62±0.06c 78.62±0.06c
5.0 7.0 86.69±0.03a 83.69±0.03a 77.69±0.03a 72.69±0.03a
1.0 4.5 88.04±0.02b 80.04±0.02b 79.04±0.02b 78.04±0.02b
2.5 6.5 92.67±0.00c 84.45±0.00c 81.45±0.00c 77.45±0.00c
5.0 7.0 85.56±0.04a 79.56±0.04a 77.56±0.04a 70.56±0.04a
HD48 (11 Log CFU/mL) 0.0 (control) 6.0 (control) 89.65±0.01a 86.65±0.01a 81.65±0.01a 78.65±0.01a
12
1.0 4.5 88.75±0.12a 80.75±0.12a 72.75±0.12a 69.75±0.12a
2.5 6.5 91.75±0.03b 82.75±0.03b 80.75±0.03b 76.75±0.03b
5.0 7.0 86.01±0.02b 75.01±0.02b 71.01±0.02b 69.01±0.02b
1.0 4.5 86.18±0.02a 83.18±0.02a 79.18±0.02a 74.18±0.02a

2.5 6.5 90.61±0.01a 88.61±0.01a 84.61±0.01a 79.61±0.01a
5.0 7.0 79.82±0.17a 75.82±0.17a 73.82±0.17d 71.77±0.15a

Probiotic Salt
pH Before heat
Lactobacillus strain (%w/v) 0 min 2.5 min 5.0 min 7.5 min 10 min
treatment
HD48 (9 Log 1.0 4.5 9.10±0.02b 9.10±0.02b 8.98±0.00b 8.12±0.02cd 8.00±0.02cd 7.23±0.02cd
CFU/mL)
2.5 6.5 9.23±0.02b 9.23±0.02b 9.00±0.03a 8.34±0.03b 8.06±0.03b 7.00±0.03b
5.0 7.0 9.11±0.03c 9.11±0.03c 8.78±0.011b 8.09±0.02a 7.98±0.02a 7.11±0.02a
HD48 (10 Log

1.0 4.5 10.21±0.01a 10.21±0.01a 10.01±0.02cd 9.53±0.01d 8.22±0.01d 8.00±0.01d
CFU/mL)
2.5 6.5 10.10±0.02b 10.10±0.02b 9.89±0.03b 9.54±0.02d 8.22±0.02d 8.02±0.02d
5.0 7.0 10.01±0.03c 10.01±0.03c 9.88±0.02a 9.66±0.02ab 8.21±0.02ab 8.11±0.02ab
13
HD48 (11 Log 1.0 4.5 11.21±0.05e 11.19±0.05e 10.78±0.01d 10.23±0.01b 10.09±0.01b 9.65±0.01b
CFU/mL)
2.5 6.5 11.13±0.02b 11.12±0.02b 10.89±0.02d 10.13±0.02a 9.89±0.02a 9.29±0.02a
5.0 7.0 11.33±0.01a 11.21±0.01a 10.85±0.02ab 10.43±0.01c 9.92±0.01c 9.33±0.01c
HD48 (12 Log
CFU/mL)
1.0 4.5 12.15±0.02b 12.12±0.02b 11.62±0.01b 10.62±0.02b 10.02±0.02b 9.08±0.02b
2.5 6.5 12.20±0.02b 12.21±0.02b 11.73±0.02a 10.78±0.02a 9.78±0.02a 9.18±0.02a
5.0 7.0 12.08±0.01a 12.09±0.01a 11.53±0.01c 10.61±0.01b 9.78±0.01b 9.10±0.01b

(%w/v) 2.5 min 5.0 min 7.5 min 10 min
HD48 (9 Log CFU/mL)

1.0 4.5 84.13±0.05b 81.13±0.05b 79.13±0.05b 77.13±0.05b
2.5 6.5 90.52±0.06c 87.62±0.06c 75.62±0.06c 74.62±0.06c
5.0 7.0 85.69±0.03a 82.69±0.03a 71.69±0.03a 70.69±0.03a
1.0 4.5 84.04±0.02b 79.04±0.02b 76.04±0.02b 75.04±0.02b
2.5 6.5 89.67±0.00c 83.45±0.00c 80.45±0.00c 76.45±0.00c
5.0 7.0 85.56±0.04a 78.56±0.04a 78.06±0.04a 70.06±0.04a
0.0 (control) 6.0 (control) 88.65±0.01a 83.65±0.01a 80.65±0.01a 77.65±0.01a
1.0 4.5 86.75±0.12a 78.75±0.12a 70.75±0.12a 68.75±0.12a
14
2.5 6.5 90.75±0.03b 81.75±0.03b 79.75±0.03b 74.75±0.03b
5.0 7.0 85.01±0.02b 72.01±0.02b 70.01±0.02b 68.01±0.02b
1.0 4.5 83.18±0.02a 81.18±0.02a 77.18±0.02a 75.18±0.02a

2.5 6.5 89.61±0.01a 86.61±0.01a 83.61±0.01a 78.61±0.01a
5.0 7.0 78.82±0.17a 72.82±0.17a 71.82±0.17d 69.77±0.15a
Effect of heat tolerance on survivability of Lactiplantibacillus plantarum HD51 (55 ᴼC) against different pH concentrations
Survivability (%)
Probiotic Lactobacillus strain pH 0 min 2.5 min 5.0 min 7.5 min 10 min
5.5 100.00 81.75±0.02c 81.75±0.02c 81.75±0.02c 81.75±0.02c
Lp. plantarum HD 51 6.0 100.00 88.13±0.05b 80.13±0.05b 81.13±0.05b 80.13±0.05b
(109 Log CFU/mL) 6.5 100.00 90.62±0.06c 80.62±0.06c 72.62±0.06c 74.62±0.06c
7.0 100.00 87.69±0.03a 83.69±0.03a 71.69±0.03a 91.69±0.03a
5.5 100.00 88.83±0.09b 80.83±0.09b 78.83±0.09b 92.83±0.09b

(1010 Log CFU/mL) 6.5 100.00 91.45±0.00c 81.45±0.00c 82.45±0.00c 72.45±0.00c
7.0 100.00 84.56±0.04a 78.56±0.04a 71.56±0.04a 70.56±0.04a
Lp. plantarum HD 51 5.5 100.00 90.65±0.01a 82.65±0.01a 84.65±0.01a 74.65±0.01a
15
6.0 100.00 9.75±0.12a 84.75±0.12a 77.75±0.12a 62.75±0.12a
6.5 100.00 92.75±0.03b 82.75±0.03b 81.75±0.03b 71.75±0.03b
11
(10 Log CFU/mL)
7.0 100.00 85.01±0.02b 71.01±0.02b 91.01±0.02b 88.01±0.02b
5.5 100.00 89.97±0.00c 82.97±0.00c 88.67±0.00c 72.97±0.00c
(1012 Log CFU/mL) 6.5 100.00 90.61±0.01a 87.61±0.01a 84.61±0.01a 70.61±0.01a
7.0 100.00 78.82±0.17a 72.82±0.17a 73.82±0.17d 71.82±0.17a
Effect of survivability of Lactiplantibacillus plantarum HD51 (60 ᴼC) against different pH concentrations under different time intervals
Survivability (%)
5.5 100.00 81.75±0.02c 80.75±0.02c 8.75±0.02c 81.75±0.02c
(109 Log CFU/mL) 6.5 100.00 91.62±0.06c 88.62±0.06c 74.62±0.06c 74.62±0.06c
7.0 100.00 81.69±0.03a 83.69±0.03a 72.69±0.03a 71.69±0.03a
5.5 100.00 84.83±0.09b 85.83±0.09b 71.83±0.09b 70.83±0.09b

(1010 Log CFU/mL) 6.5 100.00 93.67±0.00c 82.45±0.00c 71.45±0.00c 73.45±0.00c
7.0 100.00 84.56±0.04a 72.56±0.04a 72.56±0.04a 71.56±0.04a
(1011 Log CFU/mL) 6.0 100.00 85.75±0.12a 82.75±0.12a 71.75±0.12a 61.75±0.12a
16
6.5 100.00 91.75±0.03b 83.75±0.03b 81.75±0.03b 75.75±0.03b
7.0 100.00 82.01±0.02b 71.01±0.02b 72.01±0.02b 62.01±0.02b
5.5 100.00 81.97±0.00c 82.97±0.00c 81.67±0.00c 76.97±0.00c
(1012 Log CFU/mL) 6.5 100.00 90.61±0.01a 82.61±0.01a 76.61±0.01a 71.61±0.01a
7.0 100.00 71.82±0.17a 73.82±0.17a 72.82±0.17d 69.77±0.15a
Effect of survivability of Lactiplantibacillus plantarum HD51 (65 ᴼC) against different pH concentrations under different time intervals
Survivability (%)
5.5 100.00 82.35±0.02c 79.75±0.02c 75.75±0.02c 73.75±0.02c
(109 Log CFU/mL) 6.5 100.00 90.52±0.06c 85.62±0.06c 80.62±0.06c 74.62±0.06c
7.0 100.00 85.69±0.03a 82.69±0.03a 71.69±0.03a 70.69±0.03a
5.5 100.00 86.83±0.09b 84.83±0.09b 75.83±0.09b 74.83±0.09b

(1010 Log CFU/mL) 6.5 100.00 89.67±0.00c 83.45±0.00c 80.45±0.00c 76.45±0.00c
7.0 100.00 85.56±0.04a 78.56±0.04a 78.06±0.04a 70.06±0.04a
5.5 100.00 88.65±0.01a 83.65±0.01a 80.65±0.01a 77.65±0.01a
Lp. plantarum HD 51
6.0 100.00 86.75±0.12a 78.75±0.12a 70.75±0.12a 68.75±0.12a
(1011 Log CFU/mL)
6.5 100.00 90.75±0.03b 84.75±0.03b 79.75±0.03b 74.75±0.03b
17
7.0 100.00 85.01±0.02b 72.01±0.02b 70.01±0.02b 68.01±0.02b
5.5 100.00 84.97±0.00c 83.97±0.00c 72.67±0.00c 68.97±0.00c
(1012 Log CFU/mL) 6.5 100.00 89.61±0.01a 86.61±0.01a 81.61±0.01a 75.61±0.01a
7.0 100.00 78.82±0.17a 72.82±0.17a 71.82±0.17d 69.77±0.15a
Effect of survivability of Lactiplantibacillus plantarum HD51 (55 ᴼC) against different salt concentrations under different time intervals
Survivability (%)
Probiotic Lactobacillus strain Salt conc (%w/v) 0 min 2.5 min 5.0 min 7.5 min 10 min
0.0 (control) 100.00 95.49±0.2 92.41±0.21 90.21±0.14 83.61±0.24
Lp. plantarum HD 51 1.0 100.00 96.13±0.5 90.61±0.21 89.94±0.25 83.87±0.36
(109 Log CFU/mL) 2.0 100.00 98.78±0.6 94.90±0.36 91.12±0.26 89.90±0.24
3.0 100.00 96.70±0.1 90.11±0.24 89.56±0.36 76.92±.25
0.0 (control) 100.00 95.90±0.5 91.91±0.24 88.91±0.24 81.32±0.21

Lp. plantarum HD 51 1.0 100.00 95.13±0.2 94.73±.25 81.71±0.25 79.52±0.36
(1010 Log CFU/mL) 2.0 100.00 97.13±0.14 93.26±0.36 90.19±0.14 89.02±.25
3.0 100.00 95.81±0.15 91.82±0.14 87.82±0.22 84.23±0.24
0.0 (control) 100.00 96.01±0.15 90.58±0.25 85.14±0.21 80.62±0.21

Lp. plantarum HD 51 1.0 100.00 92.71±1.02 91.81±0.24 90.10±0.15 82.81±.24
(1011 Log CFU/mL) 2.0 100.00 97.93±.002 93.78±0.26 87.93±0.2 84.77±.25
3.0 100.00 93.44±0.14 90.75±0.24 91.73±1.02 81.76±.24
18
0.0 (control) 100.00 95.44±0.02 92.95±.24 91.29±0.02 87.14±0.21
Lp. plantarum HD 51 1.0 100.00 96.75±0.85 95.50±0.26 92.34±0.05 84.93±0.25
(1012 Log CFU/mL) 2.0 100.00 97.02±0.47 93.16±0.25 91.37±.026 89.06±1.02
3.0 100.00 95.20±0.69 91.89±0.23 89.24±.0.56 83.61±0.1.01

Effect of survivability of Lactiplantibacillus plantarum HD51 (60 ᴼC) against different salt concentrations
Survivability (%)
0.0 (control) 100.00 95.65±0.02 90.22±0.11 89.13±0.21 88.59±0.14
Lp. plantarum HD 51 1.0 100.00 95.08±0.21 90.71±0.21 89.18±0.24 86.23±0.08
(109 Log CFU/mL) 2.0 100.00 97.48±0.21 93.64±0.21 90.02±0.14 86.82±0.12
3.0 100.00 95.29±0.26 93.10±0.25 89.27±0.26 84.34±0.15
0.0 (control) 100.00 94.63±0.24 92.54±0.05 86.57±0.11 81.00±.14

Lp. plantarum HD 51 1.0 100.00 94.15±0.21 92.17±0.01 81.47±0.31 79.29±0.25
(1010 Log CFU/mL) 2.0 100.00 97.23±0.24 92.98±0.24 89.92±0.11 86.76±14
3.0 100.00 94.77±26 92.80±0.14 86.79±0.22 83.23±.26
0.0 (control) 100.00 95.04±.21 93.06±0.12 91.07±0.14 82.06±.21

Lp. plantarum HD 51 1.0 100.00 93.72±0.02 91.56±0.15 91.02±0.21 88.15±07
(1011 Log CFU/mL) 2.0 100.00 97.66±0.24 93.53±0.15 89.69±0.11 85.55±0.24
3.0 100.00 94.77±0.11 93.32±0.26 92.15±0.02 90.25±0.24
Lp. plantarum HD 51 0.0 (control) 100.00 94.30±0.05 93.39±0.25 91.82±0.14 74.30±21
19
1.0 100.00 95.55±0.24 94.56±0.14 91.43±0.11 91.34±24
2.0 100.00 97.18±0.11 93.31±0.02 90.16±0.12 86.34±11

12
(10 Log CFU/mL)
3.0 100.00 96.52±0.16 96.27±0.23 89.39±0.01 83.75±17
Effect of survivability of Lactiplantibacillus plantarum HD51 (65 ᴼC) against different salt concentrations under different time intervals
Survivability (%)
0.0 (control) 100.00 94.67±0.2 88.14±0.5 83.79±0.5 76.71±0.4

Lp. plantarum HD 51 1.0 100.00 95.60±0.4 87.91±0.5 84.62±0.4 79.45±0.6
(109 Log CFU/mL) 2.0 100.00 95.56±0.2 93.36±0.6 88.32±0.6 75.84±0.1
3.0 100.00 91.11±0.8 88.80±0.1 82.99±0.8 78.05±0.4
0.0 (control) 100.00 92.82±1.2 89.42±0.4 85.53±0.7 77.86±0.3
Lp. plantarum HD 51 1.0 100.00 94.42±0.9 93.34±0.8 80.51±0.6 78.35±0.8
(1010 Log CFU/mL) 2.0 100.00 96.83±0.4 94.46±1.2 89.39±0.4 77.13±0.7
3.0 100.00 93.51±0.5 91.01±0.9 82.02±0.6 81.02±0.1
0.0 (control) 100.00 94.21±0.8 96.29±1.0 90.68±0.7 83.53±0.3
Lp. plantarum HD 51 1.0 100.00 92.23±0.6 91.42±0.5 90.17±0.6 86.24±0.5
(1011 Log CFU/mL) 2.0 100.00 95.86±0.7 91.10±0.7 88.94±0.6 83.54±0.4
3.0 100.00 93.22±0.8 93.04±0.6 88.49±0.7 83.23±0.2
0.0 (control) 100.00 97.93±0.6 86.85±0.5 78.58±0.5 74.36±.5
Lp. plantarum HD 51
1.0 100.00 95.87±0.4 87.62±0.8 82.67±0.6 76.57±0.7
(1012 Log CFU/mL)
2.0 100.00 96.07±0.6 93.29±0.7 89.10±0.7 75.18±0.5
20
3.0 100.00 95.37±0.8 87.76±0.6 80.89±0.8 75.27±0.5
Effect of maltodextrin on survivability of probiotic Lactiplantibacillus plantarum HD48 (55℃)
Survivability (%)
Probiotic Lactobacillus Maltodextrin 0 min (Before Heat

2.5 min 5.0 min 7.5 min 10 min
strain concentration (%w/v) treatment)
0 100.00±0.05 93.69±0.12 91.01±0.05 86.25±0.14 82.92±0.01
0.5 100.00±0.04 96.17±0.27 92.10±0.04 87.00±0.05 82.14±0.05

Lp. plantarum HD48 2.5 100.00±0.15 96.77±0.10 92.46±0.15 89.28±0.00 83.06±0.03
10
(10 Log CFU/mL) 5.0 100.00±0.12 96.85±0.06 92.72±0.12 89.86±0.02 84.20±0.11
7.5 100.00±0.03 96.95±0.05 93.22±0.03 90.13±0.04 86.39±0.15
10 100.00±0.00 93.82±0.04 91.97±0.00 87.48±0.11 84.34±0.10
0 100.00±0.04 93.74±0.03 90.60±0.04 85.91±0.06 82.19±0.04
0.5 100.00±0.05 94.99±0.21 91.95±0.05 87.03±0.03 82.54±0.68
Lp. plantarum HD48 2.5 100.00±0.00 96.08±0.10 92.43±0.00 88.64±0.10 83.07±0.11
(1011 Log CFU/mL) 5.0 100.00±0.03 96.42±0.02 92.92±0.03 88.93±0.06 84.05±0.07
7.5 100.00±0.11 96.77±0.12 93.17±0.11 90.25±0.07 87.11±0.04
10 100.00±0.04 94.59±0.05 91.85±0.04 88.74±0.07 85.81±0.04
Lp. plantarum HD48 0 100.00±0.07 93.89±0.02 89.27±0.07 83.87±0.05 80.98±0.04
(1012 Log CFU/mL) 0.5 100.00±0.09 95.50±0.04 91.54±0.09 86.00±0.09 82.62±0.02
2.5 100.00±0.02 95.67±0.09 92.24±0.02 86.34±0.13 83.29±0.01
5.0 100.00±0.21 95.79±0.01 92.57±0.21 87.34±0.04 84.48±0.11
21
7.5 100.00±0.16 96.02±0.11 93.20±0.16 89.63±0.12 86.89±0.05
10 100.00±0.03 93.19±0.13 91.46±0.03 88.69±0.09 75.69±0.07
Effect of maltodextrin on heat tolerance of probiotic Lactiplantibacillus plantarum HD48 (60℃)
Survivability (%)

0 100.00±0.08 92.97±0.06 90.74±0.05 85.54±0.19 82.12±0.07
0.5 100.00±0.01 96.53±0.36 91.06±0.09 86.67±0.06 80.86±0.04

Lp. plantarum HD48 2.5 100.00±0.14 95.99±0.17 92.57±0.15 88.80±0.03 82.44±0.08
(1010 Log CFU/mL) 5.0 100.00±0.08 92.65±0.33 91.32±0.08 90.34±0.11 83.82±0.11
7.5 100.00±0.04 96.61±0.09 92.83±0.00 90.71±0.02 85.95±0.09
10 100.00±0.01 94.72±0.14 96.36±0.57 85.06±0.12 83.67±0.04
0 100.00±0.00 89.28±0.50 91.39±0.04 85.20±0.04 82.05±0.00
0.5 100.00±0.02 94.71±0.27 91.93±0.02 86.65±0.09 82.94±0.65
Lp. plantarum HD48 2.5 100.00±0.02 96.25±0.10 91.25±0.01 86.61±0.35 83.36±0.10
11
(10 Log CFU/mL) 5.0 100.00±0.07 96.18±0.09 92.58±0.23 87.69±0.00 85.03±0.12
7.5 100.00±0.11 96.45±0.09 92.77±0.09 87.78±0.35 86.97±0.03
10 100.00±0.05 93.35±0.10 91.29±0.30 87.54±0.02 84.90±0.07
Lp. plantarum HD48 0 100.00±0.07 94.47±0.09 89.11±0.12 83.95±0.04 80.45±0.10
12
(10 Log CFU/mL) 0.5 100.00±0.09 94.30±0.10 91.91±0.02 85.71±0.13 82.49±0.01
2.5 100.00±0.01 94.52±0.23 92.54±0.01 85.66±0.05 83.39±0.00
22
5.0 100.00±0.19 95.64±0.01 92.62±0.00 85.65±0.15 83.94±0.05
7.5 100.00±0.12 96.12±0.16 93.01±0.04 89.75±0.17 86.19±0.00
10 100.00±0.06 92.55±0.03 92.26±0.10 88.08±0.20 75.96±0.06
Survivability (%)
Probiotic Lactobacillus Maltodextrin

strain concentration (%w/v)
0 100.00±0.05 92.80±0.02 90.67±0.02 85.20±0.13 82.13±0.09
0.5 100.00±0.02 96.52±0.33 90.99±0.06 85.99±0.16 80.75±0.08

Lp. plantarum HD48 2.5 100.00±0.14 95.69±0.20 92.32±0.12 88.89±0.03 81.95±0.14
(1010 Log CFU/mL) 5.0 100.00±0.09 92.50±0.35 90.78±0.01 90.04±0.14 83.28±0.17
7.5 100.00±0.05 96.18±0.14 92.47±0.05 90.46±0.05 85.59±0.05
10 100.00±0.01 94.21±0.07 96.51±0.57 84.18±0.01 83.23±0.02
0 100.00±0.01 88.95±0.55 91.55±0.04 84.73±0.02 81.94±0.02
0.5 100.00±0.07 94.93±0.33 92.40±0.05 86.62±0.00 82.09±0.83
Lp. plantarum HD48 2.5 100.00±0.01 95.33±0.17 90.88±0.03 85.80±0.28 83.04±0.11
11
(10 Log CFU/mL) 5.0 100.00±0.03 96.42±0.07 93.13±0.21 87.47±0.06 84.66±0.04
7.5 100.00±0.04 95.53±0.04 91.96±0.05 86.82±0.30 86.20±0.00
10 100.00±0.05 92.94±0.15 90.80±0.24 87.00±0.09 84.50±0.12
23
0 100.00±0.07 93.20±0.06 87.75±0.04 83.51±0.02 80.78±0.05
0.5 100.00±0.08 94.21±0.12 91.53±0.08 85.12±0.05 82.77±0.04
Lp. plantarum HD48 2.5 100.00±0.04 94.83±0.22 92.02±0.08 85.37±0.11 83.17±0.05
(1012 Log CFU/mL) 5.0 100.00±0.18 95.03±0.06 91.85±0.09 84.80±0.04 83.50±0.00

7.5 100.00±0.11 95.55±0.10 92.49±0.09 88.86±0.27 85.59±0.07
10 100.00±0.03 91.95±0.03 92.53±0.04 86.71±0.05 75.93±0.04
Effect of gum acacia on survivability of probiotic Lactiplantibacillus plantarum HD48 (55℃)
Survivability (%)
Probiotic Lactobacillus Gum acacia 0 min (Before Heat

0 100.00±0.08 93.02±0.03 90.99±0.05 82.03±0.14 74.85±0.04
0.5 100.00±0.05 93.58±0.06 91.26±0.15 82.12±0.13 75.65±0.04

Lp. plantarum HD48 2.5 100.00±0.09 93.94±0.06 91.82±0.04 83.79±0.01 76.00±0.08
(1010 Log CFU/mL) 5.0 100.00±0.09 94.62±0.05 92.45±0.02 84.65±0.04 76.95±0.00
7.5 100.00±0.01 94.98±0.12 92.47±0.05 84.75±0.03 77.72±0.00
10 100.00±0.04 95.13±0.05 93.49±0.02 85.88±0.03 81.20±0.05
0 100.00±0.03 93.19±0.12 90.57±0.04 83.94±0.12 77.22±0.04
0.5 100.00±0.00 94.29±0.16 91.66±0.14 84.49±0.04 77.22±0.04
Lp. plantarum HD48
2.5 100.00±0.01 94.05±0.12 91.08±0.22 86.07±0.06 78.50±0.08
(1011 Log CFU/mL)
5.0 100.00±0.05 94.64±0.09 90.63±0.05 86.17±0.03 79.23±0.01
7.5 100.00±0.09 94.82±0.12 92.26±0.00 86.63±0.02 80.11±0.00
24
10 100.00±0.00 95.01±0.07 93.06±0.04 88.12±0.06 81.87±0.02
0 100.00±0.05 92.42±0.06 89.64±0.10 85.21±0.14 79.20±0.04
0.5 100.00±0.02 92.98±0.04 92.14±0.02 85.74±0.13 80.30±0.04
Lp. plantarum HD48 2.5 100.00±0.01 93.24±0.05 92.66±0.06 87.03±0.01 80.48±0.08
(1012 Log CFU/mL) 5.0 100.00±0.15 93.31±0.00 92.49±0.05 86.82±0.04 80.42±0.01

7.5 100.00±0.02 93.41±0.09 91.75±0.05 88.02±0.03 82.09±0.00
10 100.00±0.02 94.91±0.04 93.62±0.05 88.07±0.03 80.87±0.05
Effect of gum acacia on heat tolerance of probiotic Lactiplantibacillus plantarum HD48 (60℃)
Survivability (%)

0 100.00±0.06 92.39±0.02 90.81±0.04 81.81±0.13 74.74±0.04
0.5 100.00±0.04 92.99±0.01 90.68±0.10 81.85±0.11 75.58±0.04

Lp. plantarum HD48 2.5 100.00±0.09 93.93±0.05 91.81±0.04 83.73±0.02 76.04±0.09
(1010 Log CFU/mL) 5.0 100.00±0.06 94.29±0.04 92.13±0.02 84.35±0.04 76.72±0.06
7.5 100.00±0.02 94.98±0.11 92.61±0.04 84.79±0.02 77.79±0.02
10 100.00±0.04 95.02±0.07 93.63±0.03 85.82±0.02 81.24±0.04
0 100.00±0.05 93.40±0.13 90.60±0.03 84.05±0.11 77.36±0.05
Lp. plantarum HD48
0.5 100.00±0.07 94.89±0.15 92.11±0.14 84.89±0.04 77.63±0.07
(1011 Log CFU/mL)
2.5 100.00±0.02 94.09±0.11 91.25±0.23 85.97±0.08 78.57±0.02
25
5.0 100.00±0.06 94.86±0.08 90.67±0.04 86.16±0.04 79.31±0.06
7.5 100.00±0.07 94.65±0.12 92.04±0.01 86.47±0.01 80.00±0.07
10 100.00±0.01 94.88±0.08 92.89±0.02 88.09±0.05 81.79±0.01
0 100.00±0.05 92.42±0.05 89.27±0.15 85.21±0.13 79.24±0.05
0.5 100.00±0.03 92.98±0.04 504.82±49.65 85.83±0.14 80.27±0.03
Lp. plantarum HD48 2.5 100.00±0.01 93.48±0.06 92.74±0.07 87.13±0.01 80.61±0.01
(1012 Log FU/mL) 5.0 100.00±0.14 93.23±0.00 92.29±0.04 86.67±0.04 80.32±0.14

7.5 100.00±0.02 93.41±0.09 91.67±0.04 87.98±0.02 82.09±0.02
10 100.00±0.04 94.83±0.04 93.34±0.07 87.80±0.00 80.77±0.04
Survivability (%)

0 100.00±0.06 92.52±0.03 90.89±0.04 81.92±0.12 74.84±0.04
0.5 100.00±0.04 93.17±0.00 90.89±0.09 82.03±0.10 75.84±0.04

Lp. plantarum HD48 2.5 100.00±0.09 93.98±0.05 91.81±0.05 83.76±0.02 76.06±0.09
10
(10 Log CFU/mL) 5.0 100.00±0.06 94.71±0.04 92.53±0.03 84.72±0.04 77.05±0.01
7.5 100.00±0.02 95.02±0.10 92.66±0.05 84.82±0.01 77.82±0.01
10 100.00±0.04 95.02±0.07 93.68±0.03 85.81±0.01 81.23±0.05
26
0 100.00±0.05 93.40±0.12 90.60±0.02 84.04±0.11 77.35±0.04
0.5 100.00±0.07 94.93±0.15 92.15±0.13 84.93±0.04 77.66±0.04
Lp. plantarum HD48 2.5 100.00±0.02 94.09±0.11 91.25±0.22 85.97±0.09 78.56±0.09
(1011 Log CFU/mL) 5.0 100.00±0.06 94.81±0.09 90.66±0.04 86.15±0.04 79.30±0.01
7.5 100.00±0.07 94.65±0.13 92.08±0.01 86.50±0.00 80.03±0.00
10 100.00±0.01 98.42±0.32 92.80±0.02 88.00±0.06 81.71±0.03
0 100.00±0.05 92.34±0.04 89.19±0.16 85.13±0.13 79.16±0.04
0.5 100.00±0.03 93.01±0.05 92.14±0.04 85.90±0.14 80.32±0.04
Lp. plantarum HD48
2.5 100.00±0.01 93.28±0.04 92.70±0.07 87.05±0.02 80.54±0.09
(1012 Log CFU/mL)
5.0 100.00±0.14 93.16±0.00 92.21±0.04 86.60±0.04 80.25±0.01
7.5 100.00±0.02 93.29±0.09 91.59±0.04 87.86±0.01 81.98±0.00
10 100.00±0.04 94.87±0.04 93.38±0.08 87.83±0.00 80.79±0.06
Effect of corn starch on survivability of probiotic Lactiplantibacillus plantarum HD48 (55℃)
Survivability (%)
Probiotic Lactobacillus Corn starch 0 min (Before Heat

Lp. plantarum HD48 0 100.00±0.03 94.43±0.27 90.25±0.05 84.93±0.20 81.77±0.06
(1010 Log CFU/mL) 0.5 100.00±0.01 96.31±0.26 91.64±0.11 86.72±0.00 81.85±0.00
2.5 100.00±0.09 96.65±0.18 93.10±0.17 89.35±0.04 82.41±0.15
5.0 100.00±0.09 97.14±0.06 92.84±0.01 90.77±0.02 85.00±0.05
7.5 100.00±0.01 94.98±0.04 90.11±0.05 89.77±0.09 83.96±0.12
27
10 100.00±0.04 92.89±0.03 91.05±0.04 86.52±0.04 84.09±0.10
0 100.00±0.05 93.60±0.00 90.55±0.03 85.45±0.00 82.27±0.04
0.5 100.00±0.10 94.66±0.30 92.01±0.00 86.44±0.14 82.91±0.68
Lp. plantarum HD48 2.5 100.00±0.06 95.93±0.17 91.90±0.05 88.59±0.16 83.48±0.11
(1011 Log CFU/mL) 5.0 100.00±0.01 97.04±0.03 96.05±0.08 89.41±0.09 84.20±0.07
7.5 100.00±0.09 96.13±0.03 92.98±0.08 90.41±0.07 82.45±0.05
10 100.00±0.02 94.61±0.10 92.30±0.33 89.18±0.07 86.24±0.04
0 100.00±0.04 93.42±0.00 89.02±0.10 83.63±0.05 80.75±0.04
0.5 100.00±0.09 95.08±0.01 91.57±0.02 86.04±0.09 82.65±0.02
Lp. plantarum HD48 2.5 100.00±0.02 95.65±0.14 92.59±0.06 86.67±0.13 83.60±0.01
(1012 Log CFU/mL) 5.0 100.00±0.15 97.41±0.05 95.12±0.03 87.77±0.04 84.90±0.11

7.5 100.00±0.02 97.14±0.11 94.40±0.04 90.55±0.10 84.00±0.02
10 100.00±0.02 93.54±0.13 91.55±0.00 87.61±0.27 79.08±0.31
Effect of corn starch on heat tolerance of probiotic Lactiplantibacillus plantarum HD48 (60℃)
Survivability (%)

0 100.00±0.02 92.05±0.06 88.92±0.04 82.15±0.04 80.33±0.05
Lp. plantarum HD48 0.5 100.00±0.04 95.79±0.26 90.60±0.04 84.53±0.04 80.86±0.06
(1010 Log CFU/mL) 2.5 100.00±0.11 95.45±0.32 92.29±0.06 89.62±0.06 80.63±0.05
5.0 100.00±0.00 98.00±0.03 93.11±0.10 90.76±0.10 85.48±0.08
28
7.5 100.00±0.06 91.84±0.06 89.62±0.04 89.72±0.04 82.16±0.11
10 100.00±0.01 92.43±0.03 90.10±0.09 85.55±0.09 82.88±0.18
0 100.00±0.05 93.86±0.06 90.88±0.02 85.59±0.02 82.34±0.12
0.5 100.00±0.00 94.96±0.36 91.89±0.11 86.59±0.11 80.93±0.37
Lp. plantarum HD48 2.5 100.00±0.04 95.69±0.22 91.79±0.09 87.66±0.09 82.64±0.00
(1011 Log CFU/mL) 5.0 100.00±0.07 98.11±0.04 93.78±0.05 90.98±0.04 86.38±0.27
7.5 100.00±0.07 95.64±0.01 91.64±0.05 88.90±0.05 81.89±0.00
10 100.00±0.06 93.73±0.16 89.54±0.06 88.23±0.06 82.19±0.00
0 100.00±0.02 92.95±0.08 88.67±0.04 83.48±0.04 82.57±0.03
0.5 100.00±0.07 94.91±0.06 91.39±0.03 85.84±0.03 83.06±0.02
Lp. plantarum HD48 2.5 100.00±0.00 95.35±0.19 93.16±0.11 86.23±0.11 83.66±0.03
(1012 Log CFU/mL) 5.0 100.00±0.13 97.99±0.04 92.87±0.12 88.77±0.02 84.80±0.11

7.5 100.00±0.00 95.65±0.05 93.34±0.08 90.31±0.08 83.36±0.08
10 100.00±0.03 93.05±0.09 90.44±0.12 85.19±0.12 78.15±0.21
Survivability (%)
Probiotic Lactobacillus 0 min (Before Heat 0 min (Before Heat

strain treatment) treatment)
0 100.00±0.01 93.52±0.21 89.45±0.10 84.10±0.25 80.86±0.12
Lp. plantarum HD48
0.5 100.00±0.03 95.42±0.16 90.79±0.21 86.31±0.05 80.99±0.10
29
2.5 100.00±0.12 97.33±0.14 92.59±0.25 89.52±0.05 82.16±0.20
5.0 100.00±0.10 97.83±0.08 92.44±0.04 90.42±0.03 84.39±0.12
(1010 Log CFU/mL)
7.5 100.00±0.04 94.77±0.09 90.08±0.08 89.99±0.09 83.72±0.17
10 100.00±0.08 91.49±0.06 89.77±0.02 80.38±0.04 82.65±0.04
0 100.00±0.05 93.45±0.11 91.06±0.06 85.28±0.10 82.48±0.01
0.5 100.00±0.04 95.39±0.35 92.75±0.04 87.11±0.19 83.27±0.76
Lp. plantarum HD48 (1011 2.5 100.00±0.00 95.91±0.22 92.17±0.03 88.57±0.21 83.45±0.17
Log CFU/mL) 5.0 100.00±0.03 96.76±0.08 92.90±0.12 89.66±0.05 83.59±0.15
7.5 100.00±0.02 95.30±0.00 91.95±0.14 87.56±0.34 81.66±0.02
10 100.00±0.06 93.82±0.15 91.38±0.39 88.36±0.02 81.60±0.01
0 100.00±0.03 93.34±0.08 89.24±0.14 83.66±0.10 80.72±0.09
0.5 100.00±0.07 94.91±0.06 91.59±0.05 85.38±0.20 82.65±0.00
Lp. plantarum HD48 2.5 100.00±0.04 95.16±0.19 92.02±0.12 85.73±0.03 82.80±0.10
(1012 Log CFU/mL) 5.0 100.00±0.07 95.60±0.20 93.68±0.14 89.44±0.05 83.93±0.05

7.5 100.00±0.01 94.32±0.04 93.66±0.05 83.46±0.05 82.06±0.25
10 100.00±0.09 92.59±0.08 90.86±0.02 83.24±0.11 78.17±0.25
Effect of maltodextrin on survivability of probiotic Lactiplantibacillus plantarum CRD7 (55℃)
Survivability (%)

30
0 100.00±0.05
82.75±0.02c 82.75±0.02c 80.75±0.02c 82.75±0.02c
0.5 100.00±0.04
89.13±0.05b 82.13±0.05b 80.13±0.05b 82.13±0.05b
Lp. plantarum CRD7 2.5 100.00±0.15
91.62±0.06c 81.62±0.06c 79.62±0.06c 78.62±0.06c
10
(10 Log CFU/mL) 5.0 100.00±0.12
88.69±0.03a 84.69±0.03a 79.69±0.03a 94.69±0.03a
7.5 100.00±0.03
89.83±0.09b 81.83±0.09b 76.83±0.09b 92.83±0.09b
10 100.00±0.00
88.04±0.02b 80.04±0.02b 79.04±0.02b 92.04±0.02b
0 100.00±0.04
93.45±0.00c 82.45±0.00c 81.45±0.00c 78.45±0.00c
0.5 100.00±0.05
88.56±0.04a 79.56±0.04a 77.56±0.04a 70.56±0.04a
91.65±0.01a 89.65±0.01a 81.65±0.01a 78.65±0.01a
11
(10 Log CFU/mL) 5.0 100.00±0.03
91.75±0.12a 86.75±0.12a 72.75±0.12a 69.75±0.12a
7.5 100.00±0.11
93.75±0.03b 83.75±0.03b 82.75±0.03b 79.75±0.03b
10 100.00±0.04
86.01±0.02b 78.01±0.02b 91.01±0.02b 88.01±0.02b
0 100.00±0.07
85.97±0.00c 85.97±0.00c 80.67±0.00c 78.97±0.00c
0.5 100.00±0.09
86.18±0.02a 83.18±0.02a 79.18±0.02a 74.18±0.02a
2.5 100.00±0.02
Lp. plantarum CRD7 90.61±0.01a 88.61±0.01a 84.61±0.01a 79.61±0.01a
5.0 100.00±0.21
(1012 Log CFU/mL) 78.82±0.17a 76.82±0.17a 74.82±0.17d 70.82±0.17a
7.5 100.00±0.16
82.75±0.02c 82.75±0.02c 80.75±0.02c 82.75±0.02c
10 100.00±0.03
89.13±0.05b 82.13±0.05b 80.13±0.05b 82.13±0.05b
Effect of maltodextrin on heat tolerance of probiotic Lactiplantibacillus plantarum CRD7 (60℃)
Survivability (%)

31
0 100.00±0.08
80.75±0.02c 80.75±0.02c 80.75±0.02c 82.75±0.02c
0.5 100.00±0.01
85.13±0.05b 82.13±0.05b 80.13±0.05b 82.13±0.05b
90.62±0.06c 88.62±0.06c 78.62±0.06c 78.62±0.06c
10
(10 Log CFU/mL) 5.0 100.00±0.08
86.69±0.03a 83.69±0.03a 77.69±0.03a 72.69±0.03a
7.5 100.00±0.04
87.83±0.09b 85.83±0.09b 79.83±0.09b 70.83±0.09b
10 100.00±0.01
88.04±0.02b 80.04±0.02b 79.04±0.02b 78.04±0.02b
0 100.00±0.00
92.67±0.00c 84.45±0.00c 81.45±0.00c 77.45±0.00c
0.5 100.00±0.02
85.56±0.04a 79.56±0.04a 77.56±0.04a 70.56±0.04a
89.65±0.01a 86.65±0.01a 81.65±0.01a 78.65±0.01a
11
(10 Log CFU/mL) 5.0 100.00±0.07
88.75±0.12a 80.75±0.12a 72.75±0.12a 69.75±0.12a
7.5 100.00±0.11
91.75±0.03b 82.75±0.03b 80.75±0.03b 76.75±0.03b
10 100.00±0.05
86.01±0.02b 75.01±0.02b 71.01±0.02b 69.01±0.02b
0 100.00±0.07
85.97±0.00c 85.97±0.00c 80.67±0.00c 78.97±0.00c
0.5 100.00±0.09
86.18±0.02a 83.18±0.02a 79.18±0.02a 74.18±0.02a
2.5 100.00±0.01
Lp. plantarum CRD7 90.61±0.01a 88.61±0.01a 84.61±0.01a 79.61±0.01a
5.0 100.00±0.19
(1012 Log CFU/mL) 79.82±0.17a 75.82±0.17a 73.82±0.17d 71.77±0.15a
7.5 100.00±0.12
80.75±0.02c 80.75±0.02c 80.75±0.02c 82.75±0.02c
10 100.00±0.06
85.13±0.05b 82.13±0.05b 80.13±0.05b 82.13±0.05b
Effect of maltodextrin on heat tolerance of probiotic Lactiplantibacillus plantarum CRD7 (65℃)
Survivability (%)
Probiotic Lactobacillus Maltodextrin 0 min 2.5 min 5.0 min 7.5 min 10 min
32
0 100.00±0.05 92.80±0.02 90.67±0.02 85.20±0.13 82.13±0.09
0.5 100.00±0.02 96.52±0.33 90.99±0.06 85.99±0.16 80.75±0.08

Lp. plantarum CRD7 2.5 100.00±0.14 95.69±0.20 92.32±0.12 88.89±0.03 81.95±0.14
10
(10 Log CFU/mL) 5.0 100.00±0.09 92.50±0.35 90.78±0.01 90.04±0.14 83.28±0.17
7.5 100.00±0.05 96.18±0.14 92.47±0.05 90.46±0.05 85.59±0.05
10 100.00±0.01 94.21±0.07 96.51±0.57 84.18±0.01 83.23±0.02
0 100.00±0.01 88.95±0.55 91.55±0.04 84.73±0.02 81.94±0.02
0.5 100.00±0.07 94.93±0.33 92.40±0.05 86.62±0.00 82.09±0.83
Lp. plantarum CRD7 2.5 100.00±0.01 95.33±0.17 90.88±0.03 85.80±0.28 83.04±0.11
(1011 Log CFU/mL) 5.0 100.00±0.03 96.42±0.07 93.13±0.21 87.47±0.06 84.66±0.04
7.5 100.00±0.04 95.53±0.04 91.96±0.05 86.82±0.30 86.20±0.00
10 100.00±0.05 92.94±0.15 90.80±0.24 87.00±0.09 84.50±0.12
0 100.00±0.07 93.20±0.06 87.75±0.04 83.51±0.02 80.78±0.05
0.5 100.00±0.08 94.21±0.12 91.53±0.08 85.12±0.05 82.77±0.04
Lp. plantarum CRD7 2.5 100.00±0.04 94.83±0.22 92.02±0.08 85.37±0.11 83.17±0.05
(1012 Log CFU/mL) 5.0 100.00±0.18 95.03±0.06 91.85±0.09 84.80±0.04 83.50±0.00

7.5 100.00±0.11 95.55±0.10 92.49±0.09 88.86±0.27 85.59±0.07
10 100.00±0.03 91.95±0.03 92.53±0.04 86.71±0.05 75.93±0.04
Effect of gum acacia on survivability of probiotic Lactiplantibacillus plantarum CRD7 (55℃)
33
Survivability (%)

0 100.00±0.08 93.02±0.03 90.99±0.05 82.03±0.14 74.85±0.04
0.5 100.00±0.05 93.58±0.06 91.26±0.15 82.12±0.13 75.65±0.04

Lp. plantarum CRD7 2.5 100.00±0.09 93.94±0.06 91.82±0.04 83.79±0.01 76.00±0.08
10
(10 Log CFU/mL) 5.0 100.00±0.09 94.62±0.05 92.45±0.02 84.65±0.04 76.95±0.00
7.5 100.00±0.01 94.98±0.12 92.47±0.05 84.75±0.03 77.72±0.00
10 100.00±0.04 95.13±0.05 93.49±0.02 85.88±0.03 81.20±0.05
0 100.00±0.03 93.19±0.12 90.57±0.04 83.94±0.12 77.22±0.04
0.5 100.00±0.00 94.29±0.16 91.66±0.14 84.49±0.04 77.22±0.04
Lp. plantarum CRD7 2.5 100.00±0.01 94.05±0.12 91.08±0.22 86.07±0.06 78.50±0.08
(1011 Log CFU/mL) 5.0 100.00±0.05 94.64±0.09 90.63±0.05 86.17±0.03 79.23±0.01
7.5 100.00±0.09 94.82±0.12 92.26±0.00 86.63±0.02 80.11±0.00
10 100.00±0.00 95.01±0.07 93.06±0.04 88.12±0.06 81.87±0.02
0 100.00±0.05 92.42±0.06 89.64±0.10 85.21±0.14 79.20±0.04
0.5 100.00±0.02 92.98±0.04 92.14±0.02 85.74±0.13 80.30±0.04
Lp. plantarum CRD7 2.5 100.00±0.01 93.24±0.05 92.66±0.06 87.03±0.01 80.48±0.08
(1012 Log CFU/mL) 5.0 100.00±0.15 93.31±0.00 92.49±0.05 86.82±0.04 80.42±0.01

7.5 100.00±0.02 93.41±0.09 91.75±0.05 88.02±0.03 82.09±0.00
10 100.00±0.02 94.91±0.04 93.62±0.05 88.07±0.03 80.87±0.05
34
Effect of gum acacia on heat tolerance of probiotic Lactiplantibacillus plantarum CRD7 (60℃)
Survivability (%)

0 100.00±0.06 92.39±0.02 90.81±0.04 81.81±0.13 74.74±0.04
0.5 100.00±0.04 92.99±0.01 90.68±0.10 81.85±0.11 75.58±0.04

Lp. plantarum CRD7 2.5 100.00±0.09 93.93±0.05 91.81±0.04 83.73±0.02 76.04±0.09
10
(10 Log CFU/mL) 5.0 100.00±0.06 94.29±0.04 92.13±0.02 84.35±0.04 76.72±0.06
7.5 100.00±0.02 94.98±0.11 92.61±0.04 84.79±0.02 77.79±0.02
10 100.00±0.04 95.02±0.07 93.63±0.03 85.82±0.02 81.24±0.04
0 100.00±0.05 93.40±0.13 90.60±0.03 84.05±0.11 77.36±0.05
0.5 100.00±0.07 94.89±0.15 92.11±0.14 84.89±0.04 77.63±0.07
Lp. plantarum CRD7 2.5 100.00±0.02 94.09±0.11 91.25±0.23 85.97±0.08 78.57±0.02
(1011 Log CFU/mL) 5.0 100.00±0.06 94.86±0.08 90.67±0.04 86.16±0.04 79.31±0.06
7.5 100.00±0.07 94.65±0.12 92.04±0.01 86.47±0.01 80.00±0.07
10 100.00±0.01 94.88±0.08 92.89±0.02 88.09±0.05 81.79±0.01
0 100.00±0.05 92.42±0.05 89.27±0.15 85.21±0.13 79.24±0.05
0.5 100.00±0.03 92.98±0.04 504.82±49.65 85.83±0.14 80.27±0.03
Lp. plantarum CRD7 2.5 100.00±0.01 93.48±0.06 92.74±0.07 87.13±0.01 80.61±0.01
(1012 Log FU/mL) 5.0 100.00±0.14 93.23±0.00 92.29±0.04 86.67±0.04 80.32±0.14

7.5 100.00±0.02 93.41±0.09 91.67±0.04 87.98±0.02 82.09±0.02
10 100.00±0.04 94.83±0.04 93.34±0.07 87.80±0.00 80.77±0.04
35
Effect of gum acacia on heat tolerance of probiotic Lactiplantibacillus plantarum CRD7 (65℃)
Survivability (%)

0 100.00±0.06 92.52±0.03 90.89±0.04 81.92±0.12 74.84±0.04
0.5 100.00±0.04 93.17±0.00 90.89±0.09 82.03±0.10 75.84±0.04

Lp. plantarum CRD7 2.5 100.00±0.09 93.98±0.05 91.81±0.05 83.76±0.02 76.06±0.09
10
(10 Log CFU/mL) 5.0 100.00±0.06 94.71±0.04 92.53±0.03 84.72±0.04 77.05±0.01
7.5 100.00±0.02 95.02±0.10 92.66±0.05 84.82±0.01 77.82±0.01
10 100.00±0.04 95.02±0.07 93.68±0.03 85.81±0.01 81.23±0.05
0 100.00±0.05 93.40±0.12 90.60±0.02 84.04±0.11 77.35±0.04
0.5 100.00±0.07 94.93±0.15 92.15±0.13 84.93±0.04 77.66±0.04
Lp. plantarum CRD7 2.5 100.00±0.02 94.09±0.11 91.25±0.22 85.97±0.09 78.56±0.09
(1011 Log CFU/mL) 5.0 100.00±0.06 94.81±0.09 90.66±0.04 86.15±0.04 79.30±0.01
7.5 100.00±0.07 94.65±0.13 92.08±0.01 86.50±0.00 80.03±0.00
10 100.00±0.01 98.42±0.32 92.80±0.02 88.00±0.06 81.71±0.03
0 100.00±0.05 92.34±0.04 89.19±0.16 85.13±0.13 79.16±0.04
0.5 100.00±0.03 93.01±0.05 92.14±0.04 85.90±0.14 80.32±0.04
Lp. plantarum CRD7 2.5 100.00±0.01 93.28±0.04 92.70±0.07 87.05±0.02 80.54±0.09
(1012 Log CFU/mL) 5.0 100.00±0.14 93.16±0.00 92.21±0.04 86.60±0.04 80.25±0.01

7.5 100.00±0.02 93.29±0.09 91.59±0.04 87.86±0.01 81.98±0.00
10 100.00±0.04 94.87±0.04 93.38±0.08 87.83±0.00 80.79±0.06
36
Effect of corn starch on survivability of probiotic Lactiplantibacillus plantarum CRD7 (55℃)
Survivability (%)

0 100.00±0.03 94.43±0.27 90.25±0.05 84.93±0.20 81.77±0.06
0.5 100.00±0.01 96.31±0.26 91.64±0.11 86.72±0.00 81.85±0.00

Lp. plantarum CRD7 2.5 100.00±0.09 96.65±0.18 93.10±0.17 89.35±0.04 82.41±0.15
10
(10 Log CFU/mL) 5.0 100.00±0.09 97.14±0.06 92.84±0.01 90.77±0.02 85.00±0.05
7.5 100.00±0.01 94.98±0.04 90.11±0.05 89.77±0.09 83.96±0.12
10 100.00±0.04 92.89±0.03 91.05±0.04 86.52±0.04 84.09±0.10
0 100.00±0.05 93.60±0.00 90.55±0.03 85.45±0.00 82.27±0.04
0.5 100.00±0.10 94.66±0.30 92.01±0.00 86.44±0.14 82.91±0.68
Lp. plantarum CRD7 (1011 2.5 100.00±0.06 95.93±0.17 91.90±0.05 88.59±0.16 83.48±0.11
Log CFU/mL) 5.0 100.00±0.01 97.04±0.03 96.05±0.08 89.41±0.09 84.20±0.07
7.5 100.00±0.09 96.13±0.03 92.98±0.08 90.41±0.07 82.45±0.05
10 100.00±0.02 94.61±0.10 92.30±0.33 89.18±0.07 86.24±0.04
Lp. plantarum CRD7 0 100.00±0.04 93.42±0.00 89.02±0.10 83.63±0.05 80.75±0.04
12
(10 Log CFU/mL) 0.5 100.00±0.09 95.08±0.01 91.57±0.02 86.04±0.09 82.65±0.02
2.5 100.00±0.02 95.65±0.14 92.59±0.06 86.67±0.13 83.60±0.01
5.0 100.00±0.15 97.41±0.05 95.12±0.03 87.77±0.04 84.90±0.11
7.5 100.00±0.02 97.14±0.11 94.40±0.04 90.55±0.10 84.00±0.02
37
10 100.00±0.02 93.54±0.13 91.55±0.00 87.61±0.27 79.08±0.31
Effect of corn starch on heat tolerance of probiotic Lactiplantibacillus plantarum CRD7 (60℃)
Survivability (%)

0 100.00±0.02 92.05±0.06 88.92±0.04 82.15±0.04 80.33±0.05
0.5 100.00±0.04 95.79±0.26 90.60±0.04 84.53±0.04 80.86±0.06

Lp. plantarum CRD7 2.5 100.00±0.11 95.45±0.32 92.29±0.06 89.62±0.06 80.63±0.05
10
(10 Log CFU/mL) 5.0 100.00±0.00 98.00±0.03 93.11±0.10 90.76±0.10 85.48±0.08
7.5 100.00±0.06 91.84±0.06 89.62±0.04 89.72±0.04 82.16±0.11
10 100.00±0.01 92.43±0.03 90.10±0.09 85.55±0.09 82.88±0.18
0 100.00±0.05 93.86±0.06 90.88±0.02 85.59±0.02 82.34±0.12
0.5 100.00±0.00 94.96±0.36 91.89±0.11 86.59±0.11 80.93±0.37
Lp. plantarum CRD7 2.5 100.00±0.04 95.69±0.22 91.79±0.09 87.66±0.09 82.64±0.00
(1011 Log CFU/mL) 5.0 100.00±0.07 98.11±0.04 93.78±0.05 90.98±0.04 86.38±0.27
7.5 100.00±0.07 95.64±0.01 91.64±0.05 88.90±0.05 81.89±0.00
10 100.00±0.06 93.73±0.16 89.54±0.06 88.23±0.06 82.19±0.00
0 100.00±0.02 92.95±0.08 88.67±0.04 83.48±0.04 82.57±0.03
Lp. plantarum CRD7 0.5 100.00±0.07 94.91±0.06 91.39±0.03 85.84±0.03 83.06±0.02
12
(10 Log CFU/mL) 2.5 100.00±0.00 95.35±0.19 93.16±0.11 86.23±0.11 83.66±0.03
5.0 100.00±0.13 97.99±0.04 92.87±0.12 88.77±0.02 84.80±0.11
38
7.5 100.00±0.00 95.65±0.05 93.34±0.08 90.31±0.08 83.36±0.08
10 100.00±0.03 93.05±0.09 90.44±0.12 85.19±0.12 78.15±0.21
Effect of corn starch on heat tolerance of probiotic Lactiplantibacillus plantarum CRD7 (65℃)
Survivability (%)
Probiotic Lactobacillus 0 min (Before Heat 0 min (Before Heat

0 100.00±0.01 93.52±0.21 89.45±0.10 84.10±0.25 80.86±0.12
0.5 100.00±0.03 95.42±0.16 90.79±0.21 86.31±0.05 80.99±0.10

Lp. plantarum CRD7 2.5 100.00±0.12 97.33±0.14 92.59±0.25 89.52±0.05 82.16±0.20
10
(10 Log CFU/mL) 5.0 100.00±0.10 97.83±0.08 92.44±0.04 90.42±0.03 84.39±0.12
7.5 100.00±0.04 94.77±0.09 90.08±0.08 89.99±0.09 83.72±0.17
10 100.00±0.08 91.49±0.06 89.77±0.02 80.38±0.04 82.65±0.04
0 100.00±0.05 93.45±0.11 91.06±0.06 85.28±0.10 82.48±0.01
0.5 100.00±0.04 95.39±0.35 92.75±0.04 87.11±0.19 83.27±0.76
Lp. plantarum CRD7 2.5 100.00±0.00 95.91±0.22 92.17±0.03 88.57±0.21 83.45±0.17
(1011 Log CFU/mL) 5.0 100.00±0.03 96.76±0.08 92.90±0.12 89.66±0.05 83.59±0.15
7.5 100.00±0.02 95.30±0.00 91.95±0.14 87.56±0.34 81.66±0.02
10 100.00±0.06 93.82±0.15 91.38±0.39 88.36±0.02 81.60±0.01
0 100.00±0.03 93.34±0.08 89.24±0.14 83.66±0.10 80.72±0.09
Lp. plantarum CRD7
0.5 100.00±0.07 94.91±0.06 91.59±0.05 85.38±0.20 82.65±0.00
39
2.5 100.00±0.04 95.16±0.19 92.02±0.12 85.73±0.03 82.80±0.10
5.0 100.00±0.07 95.60±0.20 93.68±0.14 89.44±0.05 83.93±0.05
(1012 Log CFU/mL)
7.5 100.00±0.01 94.32±0.04 93.66±0.05 83.46±0.05 82.06±0.25
10 100.00±0.09 92.59±0.08 90.86±0.02 83.24±0.11 78.17±0.25
Effect of maltodextrin on survivability of probiotic Lactiplantibacillus plantarum HD51 (55℃)
Survivability (%)

0 100.00±0.05 95.69±0.12 91.01±0.05 86.25±0.14 82.92±0.01
0.5 100.00±0.04 95.17±0.27 92.10±0.04 87.00±0.05 82.14±0.05

Lp. plantarum HD 51 2.5 100.00±0.15 94.77±0.10 92.46±0.15 89.28±0.00 83.06±0.03
10
(10 Log CFU/mL) 5.0 100.00±0.12 95.85±0.06 92.72±0.12 89.86±0.02 84.20±0.11
7.5 100.00±0.03 95.95±0.05 92.22±0.03 89.13±0.04 85.39±0.15
10 100.00±0.00 93.82±0.04 91.97±0.00 87.48±0.11 84.34±0.10
0 100.00±0.04 91.74±0.03 90.60±0.04 85.91±0.06 82.19±0.04
0.5 100.00±0.05 94.99±0.21 91.95±0.05 87.03±0.03 82.54±0.68
Lp. plantarum HD 51 2.5 100.00±0.00 96.08±0.10 92.43±0.00 88.64±0.10 83.07±0.11
11
(10 Log CFU/mL) 5.0 100.00±0.03 96.42±0.02 92.92±0.03 88.93±0.06 84.05±0.07
7.5 100.00±0.11 95.63±0.12 92.17±0.11 89.25±0.07 87.11±0.04
10 100.00±0.04 94.59±0.05 91.85±0.04 88.74±0.07 85.81±0.04
40
0 100.00±0.07 93.89±0.02 89.27±0.07 83.87±0.05 80.98±0.04
0.5 100.00±0.09 95.50±0.04 91.54±0.09 86.00±0.09 82.62±0.02
Lp. plantarum HD 51 2.5 100.00±0.02 95.67±0.09 92.24±0.02 86.34±0.13 83.29±0.01
(1012 Log CFU/mL) 5.0 100.00±0.21 95.79±0.01 92.57±0.21 87.34±0.04 84.48±0.11

7.5 100.00±0.16 95.02±0.11 91.20±0.16 88.63±0.12 82.89±0.05
10 100.00±0.03 92.19±0.13 91.46±0.03 88.69±0.09 75.69±0.07
Survivability (%)

0 100.00±0.08 91.97±0.06 91.74±0.05 84.54±0.19 81.12±0.07
0.5 100.00±0.01 95.53±0.36 91.06±0.09 85.67±0.06 81.86±0.04

Lp. plantarum HD 51 2.5 100.00±0.14 94.99±0.17 91.57±0.15 87.80±0.03 82.44±0.08
10
(10 Log CFU/mL) 5.0 100.00±0.08 91.65±0.33 92.32±0.08 89.34±0.11 82.82±0.11
7.5 100.00±0.04 95.61±0.09 92.91±0.00 91.71±0.02 84.95±0.09
10 100.00±0.01 93.72±0.14 95.36±0.57 84.06±0.12 82.67±0.04
0 100.00±0.00 89.28±0.50 90.39±0.04 84.20±0.04 81.05±0.00
Lp. plantarum HD 51 0.5 100.00±0.02 93.71±0.27 90.93±0.02 85.65±0.09 81.94±0.65
11
(10 Log CFU/mL) 2.5 100.00±0.02 95.25±0.10 89.25±0.01 84.61±0.35 82.36±0.10
5.0 100.00±0.07 95.18±0.09 89.58±0.23 85.69±0.00 84.03±0.12
41
7.5 100.00±0.11 95.45±0.09 91.77±0.09 86.78±0.35 85.97±0.03
10 100.00±0.05 92.35±0.10 90.29±0.30 86.54±0.02 81.90±0.07
0 100.00±0.07 93.47±0.09 88.11±0.12 82.95±0.04 81.45±0.10
0.5 100.00±0.09 93.30±0.10 90.91±0.02 85.71±0.13 81.49±0.01
Lp. plantarum HD 51 2.5 100.00±0.01 93.52±0.23 90.54±0.01 81.66±0.05 82.39±0.00
(1012 Log CFU/mL) 5.0 100.00±0.19 94.64±0.01 91.62±0.00 82.65±0.15 82.94±0.05

7.5 100.00±0.12 95.12±0.16 91.01±0.04 88.75±0.17 85.19±0.00
10 100.00±0.06 91.55±0.03 89.26±0.10 87.08±0.20 74.96±0.06
Survivability (%)
Probiotic Lactobacillus Maltodextrin

0 100.00±0.05 90.80±0.02 98.67±0.02 81.20±0.13 81.13±0.09
0.5 100.00±0.02 94.52±0.33 89.99±0.06 84.99±0.16 82.75±0.08

Lp. plantarum HD 51 2.5 100.00±0.14 93.69±0.20 91.32±0.12 87.89±0.03 82.95±0.14
10
(10 Log CFU/mL) 5.0 100.00±0.09 92.50±0.35 91.78±0.01 89.04±0.14 81.28±0.17
7.5 100.00±0.05 94.18±0.14 91.47±0.05 88.46±0.05 84.59±0.05
10 100.00±0.01 92.21±0.07 95.51±0.57 81.18±0.01 82.23±0.02
0 100.00±0.01 88.95±0.55 90.55±0.04 83.73±0.02 80.94±0.02
Lp. plantarum HD 51
0.5 100.00±0.07 92.93±0.33 91.40±0.05 84.62±0.00 81.09±0.83
42
2.5 100.00±0.01 93.33±0.17 91.88±0.03 81.80±0.28 82.04±0.11
5.0 100.00±0.03 94.42±0.07 92.13±0.21 82.47±0.06 81.66±0.04
(1011 Log CFU/mL)
7.5 100.00±0.04 93.53±0.04 90.96±0.05 81.82±0.30 82.20±0.00
10 100.00±0.05 90.94±0.15 90.80±0.24 86.00±0.09 82.50±0.12
0 100.00±0.07 90.20±0.06 88.75±0.04 81.51±0.02 81.78±0.05
0.5 100.00±0.08 90.21±0.12 89.53±0.08 82.12±0.05 82.77±0.04
Lp. plantarum HD 51 2.5 100.00±0.04 90.83±0.22 92.02±0.08 84.37±0.11 82.17±0.05
(1012 Log CFU/mL) 5.0 100.00±0.18 93.03±0.06 89.85±0.09 83.80±0.04 80.50±0.00

7.5 100.00±0.11 93.55±0.10 89.49±0.09 82.86±0.27 81.59±0.07
10 100.00±0.03 88.95±0.03 988.53±0.04 83.71±0.05 78.93±0.04
Effect of gum acacia on survivability of probiotic Lactiplantibacillus plantarum HD51 (55℃)
Survivability (%)

0 100.00±0.08 92.18±0.32 89.92±0.05 81.03±0.24 72.85±0.14
0.5 100.00±0.05 93.58±0.06 91.26±0.15 82.12±0.13 75.65±0.04

Lp. plantarum HD 51 2.5 100.00±0.09 93.94±0.06 91.82±0.04 83.79±0.01 76.00±0.08
10
(10 Log CFU/mL)
5.0 100.00±0.09 94.62±0.05 92.45±0.02 84.65±0.04 76.95±0.00
7.5 100.00±0.01 94.98±0.12 92.47±0.05 84.75±0.03 77.72±0.00
10 100.00±0.04 94.13±0.05 91.49±0.02 84.23±0.03 78.15±0.05
43
0 100.00±0.03 93.19±0.12 90.57±0.04 83.94±0.12 77.22±0.04
0.5 100.00±0.00 94.29±0.16 91.66±0.14 84.49±0.04 77.22±0.04
Lp. plantarum HD 51 2.5 100.00±0.01 94.05±0.12 91.08±0.22 86.07±0.06 78.50±0.08
11
(10 Log CFU/mL) 5.0 100.00±0.05 94.64±0.09 90.63±0.05 86.17±0.03 79.23±0.01
7.5 100.00±0.09 94.82±0.12 92.26±0.00 86.63±0.02 80.11±0.00
10 100.00±0.00 94.12±0.07 92.15±0.07 87.25±0.08 81.48±0.12
0 100.00±0.05 92.42±0.06 89.64±0.10 85.21±0.14 79.20±0.04
0.5 100.00±0.02 92.98±0.04 92.14±0.02 85.74±0.13 80.30±0.04
Lp. plantarum HD 51 2.5 100.00±0.01 93.24±0.05 92.66±0.06 87.03±0.01 80.48±0.08
(1012 Log CFU/mL) 5.0 100.00±0.15 93.31±0.00 92.49±0.05 86.82±0.04 80.42±0.01

7.5 100.00±0.02 93.56±0.09 91.75±0.05 88.02±0.03 82.09±0.00
10 100.00±0.02 92.26±0.04 90.26±0.05 91.26±0.73 81±0.15
Survivability (%)

0 100.00±0.06 91.26±0.02 90.78±0.24 80.26±0.13 72.15±0.04
Lp. plantarum HD 51 0.5 100.00±0.04 91.59±0.01 90.68±0.10 81.85±0.11 75.58±0.04
10
(10 Log CFU/mL) 2.5 100.00±0.09 94.93±0.05 91.81±0.04 82.73±0.02 76.04±0.09
5.0 100.00±0.06 94.29±0.04 92.13±0.02 84.35±0.04 76.72±0.06
44
7.5 100.00±0.02 94.98±0.11 92.61±0.04 84.79±0.02 77.79±0.02
10 100.00±0.04 94.56±0.07 92.65±0.03 91.82±0.02 79.24±0.04
0 100.00±0.05 93.40±0.13 90.60±0.03 84.05±0.11 77.36±0.05
0.5 100.00±0.07 94.89±0.15 92.11±0.14 84.89±0.04 77.63±0.07
Lp. plantarum HD 51 2.5 100.00±0.02 94.09±0.11 91.25±0.23 81.97±0.08 78.57±0.02
(1011 Log CFU/mL) 5.0 100.00±0.06 94.86±0.08 90.67±0.04 86.16±0.04 79.31±0.06
7.5 100.00±0.07 94.65±0.12 92.04±0.01 86.47±0.01 80.00±0.07
10 100.00±0.01 93.88±0.08 91.89±0.02 88.09±0.05 80.79±0.01
0 100.00±0.05 92.42±0.05 89.27±0.15 85.21±0.13 79.24±0.05
0.5 100.00±0.03 92.98±0.04 90.82±49.65 85.83±0.14 80.27±0.03
Lp. plantarum HD 51 2.5 100.00±0.01 93.48±0.06 92.74±0.07 87.13±0.01 80.61±0.01
(1012 Log FU/mL) 5.0 100.00±0.14 92.23±0.00 92.29±0.04 86.67±0.04 80.32±0.14

7.5 100.00±0.02 93.41±0.09 89.67±0.04 87.98±0.02 82.09±0.02
10 100.00±0.04 93.83±0.04 91.34±0.07 82.80±0.00 78.77±0.04
Survivability (%)

0 100.00±0.06 91.63±0.03 90.89±0.04 81.92±0.12 74.84±0.04
Lp. plantarum HD 51
0.5 100.00±0.04 92.26±0.00 90.89±0.09 82.03±0.10 75.84±0.04
45
2.5 100.00±0.09 92.21±0.05 91.81±0.05 83.76±0.02 76.06±0.09
5.0 100.00±0.06 93.69±0.04 92.53±0.03 84.72±0.04 77.05±0.01
(1010 Log CFU/mL)
7.5 100.00±0.02 94.26±0.10 92.66±0.05 84.82±0.01 77.82±0.01
10 100.00±0.04 94.29±0.07 92.89±0.03 84.82±0.01 80.33±0.05
0 100.00±0.05 93.40±0.12 90.60±0.02 84.04±0.11 77.35±0.04
0.5 100.00±0.07 94.93±0.15 92.15±0.13 84.93±0.04 77.66±0.04
Lp. plantarum HD 51 2.5 100.00±0.02 94.09±0.11 91.25±0.22 85.97±0.09 78.56±0.09
(1011 Log CFU/mL) 5.0 100.00±0.06 94.81±0.09 90.66±0.04 86.15±0.04 79.30±0.01
7.5 100.00±0.07 94.65±0.13 92.08±0.01 86.50±0.00 80.03±0.00
10 100.00±0.01 93.26±0.32 92.80±0.12 87.50±0.61 81.71±0.26
0 100.00±0.05 92.34±0.04 89.19±0.16 85.13±0.13 79.16±0.04
0.5 100.00±0.03 93.01±0.05 92.14±0.04 85.90±0.14 80.32±0.04
Lp. plantarum HD 51 2.5 100.00±0.01 93.28±0.04 92.70±0.07 87.05±0.02 80.54±0.09
(1012 Log CFU/mL) 5.0 100.00±0.14 93.16±0.00 92.21±0.04 86.60±0.04 80.25±0.01

7.5 100.00±0.02 93.29±0.09 91.59±0.04 87.86±0.01 81.98±0.00
10 100.00±0.04 93.75±0.04 92.15±0.24 87.46±0.12 79.79±0.15
Effect of corn starch on survivability of probiotic Lactiplantibacillus plantarum HD51 (55℃)
Survivability (%)

46
0 100.00±0.03 94.43±0.27 90.25±0.05 84.93±0.20 81.77±0.06
0.5 100.00±0.01 96.31±0.26 91.64±0.11 86.72±0.00 81.85±0.00

Lp. plantarum HD 51 2.5 100.00±0.09 96.65±0.18 93.10±0.17 89.35±0.04 82.41±0.15
10
(10 Log CFU/mL) 5.0 100.00±0.09 97.14±0.06 92.84±0.01 90.77±0.02 85.00±0.05
7.5 100.00±0.01 94.98±0.04 90.11±0.05 89.77±0.09 83.96±0.12
10 100.00±0.04 92.89±0.03 91.05±0.04 86.52±0.04 84.09±0.10
0 100.00±0.05 93.60±0.00 90.55±0.03 85.45±0.00 82.27±0.04
0.5 100.00±0.10 94.66±0.30 92.01±0.00 86.44±0.14 82.91±0.68
Lp. plantarum HD 51 2.5 100.00±0.06 95.93±0.17 91.90±0.05 88.59±0.16 83.48±0.11
(1011 Log CFU/mL) 5.0 100.00±0.01 97.04±0.03 96.05±0.08 89.41±0.09 84.20±0.07
7.5 100.00±0.09 96.13±0.03 92.98±0.08 90.41±0.07 82.45±0.05
10 100.00±0.02 94.61±0.10 92.30±0.33 89.18±0.07 86.24±0.04
0 100.00±0.04 93.42±0.00 89.02±0.10 83.63±0.05 80.75±0.04
0.5 100.00±0.09 95.08±0.01 91.57±0.02 86.04±0.09 82.65±0.02
Lp. plantarum HD 51 2.5 100.00±0.02 95.65±0.14 92.59±0.06 86.67±0.13 83.60±0.01
(1012 Log CFU/mL) 5.0 100.00±0.15 97.41±0.05 95.12±0.03 87.77±0.04 84.90±0.11

7.5 100.00±0.02 97.14±0.11 94.40±0.04 90.55±0.10 84.00±0.02
10 100.00±0.02 93.54±0.13 91.55±0.00 87.61±0.27 79.08±0.31
Survivability (%)

47
0 100.00±0.02 90.26±0.06 88.92±0.04 82.15±0.04 80.33±0.05
0.5 100.00±0.04 94.79±0.26 90.60±0.04 84.53±0.04 80.86±0.06

Lp. plantarum HD 51 2.5 100.00±0.11 94.45±0.32 92.29±0.06 89.62±0.06 80.63±0.05
10
(10 Log CFU/mL) 5.0 100.00±0.00 96.3±0.03 90.11±0.10 90.76±0.10 84.32±0.08
7.5 100.00±0.06 90.84±0.06 89.62±0.04 89.72±0.04 82.16±0.11
10 100.00±0.01 91.43±0.03 90.10±0.09 85.55±0.09 82.88±0.18
0 100.00±0.05 92.86±0.06 90.88±0.02 85.59±0.02 82.34±0.12
0.5 100.00±0.00 94.96±0.36 91.89±0.11 86.59±0.11 80.93±0.37
Lp. plantarum HD 51 2.5 100.00±0.04 92.69±0.22 91.79±0.09 87.66±0.09 82.64±0.00
(1011 Log CFU/mL) 5.0 100.00±0.07 97.11±0.04 93.78±0.05 89.98±0.04 85.38±0.27
7.5 100.00±0.07 94.64±0.01 91.64±0.05 88.90±0.05 81.89±0.00
10 100.00±0.06 92.73±0.16 89.54±0.06 88.23±0.06 82.19±0.00
0 100.00±0.02 91.95±0.08 88.67±0.04 83.48±0.04 82.57±0.03
0.5 100.00±0.07 92.91±0.06 91.39±0.03 85.84±0.03 83.06±0.02
Lp. plantarum HD 51 2.5 100.00±0.00 93.35±0.19 93.16±0.11 86.23±0.11 83.66±0.03
(1012 Log CFU/mL) 5.0 100.00±0.13 96.99±0.04 90.87±0.12 88.77±0.02 83.80±0.11

7.5 100.00±0.00 91.65±0.05 93.34±0.08 90.31±0.08 83.36±0.08
10 100.00±0.03 92.05±0.09 90.44±0.12 85.19±0.12 78.15±0.21
Survivability (%)
Probiotic Lactobacillus 0 min (Before Heat 0 min (Before Heat 2.5 min 5.0 min 7.5 min 10 min
48
0 100.00±0.01 93.61±0.21 89.45±0.10 83.10±0.25 80.86±0.12
0.5 100.00±0.03 94.26±0.16 90.79±0.21 85.31±0.05 80.99±0.10

Lp. plantarum HD 51 2.5 100.00±0.12 96.23±0.14 92.59±0.25 87.52±0.05 82.16±0.20
10
(10 Log CFU/mL) 5.0 100.00±0.10 96.53±0.08 91.45±0.04 91.42±0.03 83.39±0.12
7.5 100.00±0.04 93.14±0.09 90.08±0.08 88.99±0.09 83.72±0.17
10 100.00±0.08 90.48±0.06 8.45±0.02 81.38±0.04 82.65±0.04
0 100.00±0.05 92.45±0.11 91.06±0.06 84.28±0.10 82.48±0.01
0.5 100.00±0.04 94.26±0.35 91.75±0.04 85.11±0.19 83.27±0.76
Lp. plantarum HD 51 2.5 100.00±0.00 92.15±0.22 90.17±0.03 87.57±0.21 83.45±0.17
11
(10 Log CFU/mL) 5.0 100.00±0.03 95.56±0.08 90.90±0.12 88.66±0.05 82.59±0.15
7.5 100.00±0.02 94.21±0.00 89.95±0.14 86.56±0.34 81.66±0.02
10 100.00±0.06 92.82±0.15 90.38±0.39 87.36±0.02 81.60±0.01
0 100.00±0.03 92.31±0.08 88.24±0.14 8.66±0.10 80.72±0.09
0.5 100.00±0.07 93.59±0.06 90.59±0.05 85.38±0.20 82.65±0.00
Lp. plantarum HD 51 2.5 100.00±0.04 94.21±0.19 91.02±0.12 85.73±0.03 82.80±0.10
(1012 Log CFU/mL) 5.0 100.00±0.07 96.21±0.20 92.68±0.14 89.44±0.05 81.93±0.05

7.5 100.00±0.01 92.21±0.04 92.66±0.05 83.46±0.05 82.06±0.25
10 100.00±0.09 91.56±0.08 91.86±0.02 83.24±0.11 78.17±0.25
49
Heat tolerance determination of selected probiotic Lactiplantibacillus plantarum HD51 to sub lethal heat stress
50
RESULTS AND DISCUSSIONS
For spray drying matrix purposes, polysaccharides, proteins, and combinations thereof have
been explored as carrier agents. Polysaccharides with prebiotic properties such as
maltodextrin, gum acacia and corn starch have been used to protect probiotic bacteria during
spray drying and under storage conditions (Bhagwat et al., 2020). Maltodextrin is a
polysaccharide produced by the acidic or enzymatic hydrolysis of starch. It has a nutritional
value of only 4 cal/g. Maltodextrins have been widely used in the food industry as they
provide different benefits such as texture improvement, reduction in floury taste, sweetness
modifying agents, controlling non-enzymatic browning, decreasing the freezing point of
mixtures, and as carrier materials (Jobbehdar et al., 2021). Using a protein-carbohydrate
mixture result in better survival of probiotics during spray drying. Yoha et al., (2020) found
that Lactobacillus plantarum microencapsulated with fructo-oligosaccharides (FOS) by
spray drying, improved the encapsulation efficiency and preserved 96% of viability.
The thermal tolerance of L. rhamnosus GG, L. rhamnosus E800 and L. salivarius UCC 500
were compared in reconstituted skim milk (RSM, 20% w/v), supplemented with yeast
extract (0.5% w/v). Sucrose was included for studies involving L. rhamnosus GG, as it
utilizes lactose poorly (Goldin et al., 1992). Two 50-ml volumes of RSM, contained in 100-
mL bottles and agitated by magnetic stirrer bars, were placed in a water bath at the
appropriate test temperatures of 37 oC (control and to obtain initial counts), 55, 58, 59, 60
and 61oC. One bottle was used to monitor the temperature, while, after temperature
equilibration, a 1% (v/v) inoculum of an overnight culture (~17 h) of either L. salivarius
UCC 500, L. rhamnosus E800 or L. rhamnosus GG was added to the second bottle. At
appropriate intervals (between 30 s and 4 min), 1-mL aliquots were removed from the test
bottle, serially diluted in MRD and pour plated in MRS agar. Survivors were enumerated
after 3 days of anaerobic incubation at 37 oC. Tests were conducted in duplicate and mean
log survivor counts were plotted as a function of heating time for each test temperature. At
each temperature, a best fit straight line was obtained by regression analysis, and D-values,
which represent the time (min) required to kill 90% of cells were determined by taking the
absolute value of the inverse of the slope of this line (Stumbo, 1965).
EFFECT OF CARRIER MATERIALS ON SURVIVABILITY DURING HEAT

CHALLENGE EXPERIMENT
Results of viable counts and percent survival rate have been presented in above tables. In
heat challenge experiment of Lp. planatum CRD7, Lp. planatum HD48 and Lp. planatum
51 | P a g e
HD51 viable counts of the selected strains varied from 8.57±0.24 to 9.67±0.27 log CFU/
mL. After exposure to different temperatures of 55, 60 & 65 oC at varied exposure time at
different concentrations (0, 0.5, 2.5, 5.0, 7.5 and 10% (w/v) of maltodextrin. Minimal unit
reduction have been observed in viability of probiotic culture of Lp. plantarum CRD7 (0.48
Log CFU/ mL) during exposure to elevated temperatures of 55, 60 and 65 oC. The maximum
viable counts have been observed in Lp. plantarum CRD7 and (9.67±0.27 Log CFU/mL)
Lp. plantarum HD51 compared to Lp. plantarum HD48 with highest survivability rate of
94.27±0.24 percent when maltodextrin @2. 5 percent (w/v) in case of CRD7 whereas
highest survivability rate of 96.18±0.24 percent was used as protectant at 65 oC. However,
least difference in the percent survivable rate was observed with increased concentrations of
maltodextrin up to 10% at varied exposure i.e., 55, 60, 65 oC compared to corn starch and
gum acacia and thus maltodextrin along with L. plantarum HD51 @7.5% (w/v) was
optimization of spray drying parameters for the production of probiotic DVS starters.
Heat tolerance determination of selected probiotic Lactiplantibacillus plantarum HD
51 to sub-lethal heat stress
In this study heat treatment experiments were performed in 45% RSM by the
addition maltodextrin in varied concentrations at 55,60 and 65°C with varied time intervals
to evaluate the thermotolerance of L. plantarum HD51. Reconstituted skimmed milk (RSM)
is one of the most effective composite protectants in of LAB. It shows superior protective
performance on maintaining the survival of LAB than a vast number of protectants
composed of single and multiple components (Khem et al., 2016). Skim milk can prevent
cellular injury by stabilizing the cell membrane, forming a structure that is easy to
rehydrate and protect the cells with proteins that provide a protective coating (Liao et al,
2017). However, some authors believe that the protective effect of some compounds is
masked by the protective effect of milk components (Carvalho et al., 2002). It was common
to add carrier materials to medium that can improve cellular protection during
drying/heating and subsequent storage (Carvalho et al., 2004; Ferreira et al., 2005). Thus,
among the carrier materials maltodextrin was used to enhance the survivability of L.
plantarum HD51 during sub lethal heat stress during heat challenge experiment.
Maltodextrin in particular has been used as a carrier material in many studies that showed
high solubility in water and is why it is mainly used in the high heat treatment process
(Fazaeli et al., 2012). Using maltodextrin alone as the wall material gave the lowest
52 | P a g e
efficiency probably because of its lack of emulsification and low film forming capacity as
reported by Mahdavi et al., (2016).
Fig. 3(a) depicts the survival of cells by addition of maltodextrin during heat stress.
The viability of L. plantarum HD51 at 55℃ by 0.5% concentration was 9.80±0.27 Log
CFU/mL with 3.82% reduction at 2.5 min compared to control (0 min), and on with 5.0 min
it was 9.39±0.04 Log CFU/mL with 7.85% reduction. The higher log reduction of 17.86%
with a viable count of 8.37±0.05 Log CFU/mL was shown by 10 min followed by 5.0
(7.85%) and 7.5 min (12.95%) with a viability of 9.39±0.04 and 8.87±0.05 Log CFU/g.
Similarly, the viability of L. plantarum HD51 at 55℃ by 2.5% concentration was 9.88±0.10
Log CFU/mL with 3.23% reduction at 2.5 min compared to control (0 min), and on with 5.0
min it was 9.44±0.15 Log CFU/mL with 7.54% reduction. The higher log reduction of 16.94
% with a viable count of 8.48±0.03 Log CFU/mL was shown by 10 min followed by 5.0
(7.85%) and 7.5 min (12.95%) with a viability of 9.44±0.15 and 9.12±0.00 Log CFU/mL.
Whereas, in 5.0% concentration it showed 9.84±0.06 Log CFU/mL with 3.14% reduction at
2.5 min compared to control (0 min), and on with 5.0 min it was 9.42±0.12 Log CFU/mL
with 7.28% reduction. The higher log reduction of 15.74 % with a viable count of 8.56±0.11
Log CFU/mL was shown by 10 min followed by 5.0 (7.28%) and 7.5 min (10.13%) with a
viability of 9.42±0.12and 9.13±0.02 Log CFU/mL. At 7.5% concentration the results
observed was 9.81±0.05 Log CFU/mL with 3.63 % reduction at 2.5 min compared to control
(0 min), and on with 5.0 min it was 9.31±0.03 Log CFU/mL with 8.54% reduction. The
higher log reduction of 13.55% with a viable count of 8.80±0.15 Log CFU/mL was shown
by 10 min followed by 5.0 (8.54%) and 7.5 min (9.82%) with a viability of 9.31±0.03 and
9.18±0.04 Log CFU/mL. Whereas in case of 10% concentration the results obtained were
9.41±0.04 Log CFU/mL with 6.18 % reduction at 2.5 min compared to control (0 min), and
on with 5.0 min it was 9.22±0.01 Log CFU/g with 8.07 % reduction. The higher log
reduction of 15.65% with a viable count of 8.46±0.10 Log CFU/g was shown by 10 min
followed by 5.0 (8.54%) and 7.5 min (12.56%) with a viability of 9.22±0.02 and 8.77±0.11
Log CFU/mL. Similar results have been observed in case of higher concentration of cells in
L. plantarum HD51 at 55℃ (Fig 3.d and Fig 3.g). Among all the concentrations, the L.
plantarum HD51 strain, when pre-treated to sub-lethal heat stress at 55 ℃ had shown
significantly higher survivability (p < 0.05) at 2.5 min with maltodextrin 2.5% concentration
during heat stress compared to others.
53 | P a g e
As per the results obtained, L. plantarum HD51 at 55°C in general showed better
survival at 2.5 min time interval compared to control. Our results were in compliance with
Gouesbet et al. (2002). When lactobacilli cells were subjected to heat pre-treatment above
50°C before the lethal temperature challenge, the viability of the cells increased. Reddy et
al. (2009) discovered that maltodextrin in combination with skim milk with a dilute
suspension of cells protected the probiotic properties of the organisms with better
survivability. Furthermore, supplementing the cells in skim milk with protective agents may
significantly improve the viability during the heating and storage processes. This cellular
protection is affected by the ingredients used, the strain tested, and the drying process
(Carvalho et al., 2004; Silva et al., 2011; Ananta et al., 2005).
Fig 3(b) depicts the survival of cells by addition of maltodextrin during heat stress.
The viability of L. plantarum HD51 at 60℃ by 0.5% concentration was 9.82±0.36 Log
CFU/g with 4.10% reduction at 2.5 min compared to control (0 min), and on with 5.0 min it
was 9.33±0.09 Log CFU/mL with 8.88% reduction. The higher log reduction of 19.14%
with a viable count of 8.28±0.04 Log CFU/Ml was shown by 10 min followed by 5.0
(8.88%) and 7.5 min (13.28%) with a viability of 9.33±0.09 and 8.88±0.06 Log CFU/g.
Similarly, the viability of L. plantarum HD51 at 60℃ by 2.5% concentration was
9.89±0.17 Log CFU/mL with 3.32% reduction at 2.5 min compared to control (0 min), and
on with 5.0 min it was 9.47±0.15 Log CFU/g with 7.42% reduction. The higher log
reduction of 17.59% with a viable count of 8.43±0.08 Log CFU/mL was shown by 10 min
Log CFU/mL. Whereas, in 5.0% concentration it showed 9.75±0.33 Log CFU/mL with 4.41
% reduction at 2.5 min compared to control (0 min), and on with 5.0 min it was 9.32±0.08
Log CFU/g with 8.62% reduction. The higher log reduction of 16.17 % with a viable count
of 8.55±0.11 Log CFU/mL was shown by 10 min followed by 5.0 (8.62%) and 7.5 min
(9.60%) with a viability of 9.47±0.15 and 9.22±0.11 Log CFU/mL. At 7.5% concentration
the results observed was 9.63±0.09 Log CFU/g with 5.40% reduction at 2.5 min compared
to control (0 min), and on with 5.0 min it was 9.45±0.01 Log CFU/g with 7.17% reduction.
The higher log reduction of 14.04% with a viable count of 8.75±0.09 Log CFU/g was shown
by 10 min followed by 5.0 (7.17%) and 7.5 min (9.33%) with a viability of 9.45±0.01 and
9.23±0.02 Log CFU/g. Whereas in case of 10% concentration the results obtained were
on with 5.0 min it was 9.41 ±0.57 Log CFU/mL with 6.27 % reduction. The higher log
54 | P a g e
followed by 5.0 (6.27%) and 7.5 min (14.94%) with a viability of 9.41 ±0.57 and 8.54±0.12
L. plantarum HD51 at 60℃ (Fig 3(e) and Fig 3(h).
Fig. 3 (c) depicts the viability of L. plantarum HD51 at 65℃ by 0.5% concentration
was 9.56±0.33 Log CFU/g with 4.10% reduction at 2.5 min compared to control (0 min),
and on with 5.0 min it was 9.29±0.06 Log CFU/mL with 8.88% reduction. The higher log
Log CFU/mL. Similarly, the viability of L. plantarum HD51 at 2.5% concentration was
on with 5.0 min it was 9.44±0.12 Log CFU/g with 7.42% reduction. The higher log
Log CFU/g. Whereas, in 5.0% concentration it showed 9.43±0.35 Log CFU/mL with 4.41 %
reduction at 2.5 min compared to control (0 min), and on with 5.0 min it was 9.26±0.01 Log
CFU/mL with 8.62% reduction. The higher log reduction of 16.17 % with a viable count of
8.49±0.17 Log CFU/g was shown by 10 min followed by 5.0 (8.62%) and 7.5 min (9.60%)
with a viability of 9.26±0.01 and 9.22±0.11 Log CFU/mL. At 7.5% concentration the results
observed was 9.41±0.14 Log CFU/g with 5.40% reduction at 2.5 min compared to control (0
min), and on with 5.0 min it was 9.40±0.05 Log CFU/mL with 7.17% reduction. The higher
log reduction of 14.04% with a viable count of 8.70±0.05 Log CFU/mL was shown by 10
min followed by 5.0 (7.17%) and 7.5 min (9.33%) with a viability of 9.40±0.05 and
9.20±0.05 Log CFU/mL. Whereas in case of 10% concentration the results obtained were
on with 5.0 min it was 9.38±0.57 Log CFU/mL with 6.38 % reduction. The higher log
followed by 5.0 (6.38%) and 7.5 min (15.76%) with a viability of 9.38±0.57and 8.44±0.01
L. plantarum HD51 at 65℃ (Fig 3.f and 3.j).
According to Desmond et al. (2002), the preliminary adaptation of L. casei N and L.
plantarum 8329 cultures to heat stress had increased the survivability by enhancing cell
resistance to higher temperatures. This protection conferred by thermal stress is probably
55 | P a g e
due to the synthesis of specific stress proteins, known as heat shock proteins (Silva et al.,
2005). In our study, the heat tolerance of L. plantarum HD51 could be enhanced by a
sublethal level of heat stress, which was similar to the results reported by Kang et al. (2015).
Moreover, the results suggested that a heat tolerance response could be induced when
exposed to a wide range of sublethal temperatures. This effect is mainly due to heat stress
resulting in significant protein denaturation, cell wall, and nucleic acid molecule damage
(Salar-Bezhadi et al., 2013). Additionally, LAB can produce a series of heat-induced
proteins called heat shock proteins (HSPs) when exposed to high temperatures, which
contribute to stabilizing the cytoskeleton and repairing damaged proteins (Sottile et al.,
2018).
Various mechanisms are proposed to explain the effects of heat adaptation on the
viability of bacteria. One theory hypothesized that the increased saturation and length of
fatty acids after heat adaptation can maintain the activity of intrinsic proteins and the
fluidity of the membrane (Russell and Fukunaga, 1990). Another theory proposes the
production of heat shock proteins being responsible for the positive effect of heat
adaptation because heat shock proteins are related to the precise folding of nascent
polypeptides, assembly of protein complexes, degradation and translocation of proteins
(Bukau and Horwich, 1998; De Angelis and Gobbetti, 2004). The experimental observation
about the effects of heat adaptation on cell survivability was affected by factors such as
growth media of bacteria, culture age, and shock media, and heat adaptation in a complex
medium can improve the thermal tolerance of bacteria better than in a buffer (Teixeira et
al., 1994).
In this study, the enhanced thermotolerance of L. plantarum HD51 grown at the
increased temperatures was in agreement with the findings in previous reports Cebrian et
al. (2019). At the high temperature, certain essential cellular activities might be
compromised to an overly high degree that cannot be countered by cellular response
system, leading to variations in cell survival after heat treatment (Wang et al., 2020). It is
well known that prokaryotic cells could activate heat shock proteins toward heat stress (De
Angelis and Gobbetti 2004). Although the specific cellular response mechanism varies
among species and strains, the conserved chaperones such as GroES, GroEL, DnaK, and
DnaJ families are often identified in LAB cells after heat shock (De Angelis and Gobbetti
2004; Sanders et al. 1999).
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DETERMINATION OF HEAT SHOCK PROTEINS DUE TO SUBLETHAL STRESSES BY
SDS-PAGE
Heat shock proteins are responsible for providing heat tolerance of the bacteria. Hence,
determination of heat shock proteins by SDS-PAGE was done according to Moyes et al. (2009).
Briefly, Bacterial cultures subjected to sub lethal stresses as mentioned in Table 5.1 was centrifuged
at 10000×g for 10 min. The pellet obtained was washed three times using phosphate buffer saline
(PBS pH 7.0). The cell pellet was taken to a prechilled sterilized mortar and pestle along with acid-
washed glass beads (210–300 μm, Sigma) and was crushed in 1–2 mL PBS. This was centrifuged at
3000×g for 10 min, and the supernatant was transferred to fresh Eppendorf and stored at 4 °C for
further analysis. SDS-PAGE analysis of extracted intracellular proteins was carried out in a 10%
polyacrylamide gel gradient in standard Tris buffer (pH 8.30). For separating gel 30% acrylamide
solution, 1.5 M Tris/HCl (pH 8.8) and for stacking gel 1.0 M Tris/HCl (pH 6.8) was used along with
10% w/v sodium dodecyl sulphate (SDS), 10% w/v ammonium persulphate (APS) and 10 μL (v/v)
N,N,N′,N’-Tetramethylethane-1,2-diamine (TEMED). Broad range protein marker (PageRuler™
prestained protein ladder, 10–180 kDa) for each sample was loaded into each well and gel was run at
60–70 V for 5–6 h.
Silver staining of the gel was performed by keeping the gel overnight in a fixative solution (50%
v/v methanol, 12% v/v acetic acid, formaldehyde 0.075% v/v) and the gel was washed with 50, 30%
ethanol, and water subsequently for 20 min each. The gel was sensitized (0.02% w/v sodium
thiosulphate) for 60 sec and washed three times using water. Impregnator solution (0.2% w/v silver
nitrate) was added to the gel and kept in the dark conditions for 20 min. Finally, developer solution
was added to view the bands. Once the bands appeared, a stopping solution (50% v/v methanol and
12% v/v acetic acid) was added to stop the reaction. Then the gel was visualized under electronic gel
documentation equipment and results was noted.
Determination of heat shock proteins due to sublethal stresses by SDS-PAGE
The free cells of L. plantarum HD51 was subjected to different temperatures to study
the effect of heat stress on protein expression. A significance difference (p < 0.05) was
observed when cells were subjected to higher temperatures of 55,60 and 65 ℃. The
intracellular proteins from heat-stressed culture was determined by SDS PAGE analysis.
57 | P a g e
Determination of heat shock proteins in maltodextrin (7.5%w/v) in L. plantarum HD51 – SDS

PAGE
Determination of heat shock proteins in corn starch (5.0%w/v) in L. plantarum HD51 – SDS
PAGE
58 | P a g e
Determination of heat shock proteins in maltodextrin (10.0%w/v) in L. plantarum HD51 –

SDS PAGE
The above shows the presence of differentially expressed proteins in SDS PAGE gel, where
some of the protein bands in the gel were increased, indicating the higher expression of
proteins. At around 20 kDa (GrpE), an increase in protein content was observed in 55℃
59 | P a g e
compared to control. Also, differences in protein content were observed approximately at
40-55 kDa, 60 kDa, and 70 kDa representing the possible overexpression of DnaJ, GroEL,
and DnaK in all heat-stressed cells. There was also an increase in protein concentration
around 100 kDa, revealing the possibility of ClpB, ClpA, ClpX proteins. It appears to be a
strategy utilized mainly by lactobacillus or bacterial pathogens to general stress proteins in
response to heat stresses (Gandhi and Chikindas, 2007; Guzzo et al., 2000). Under stress,
this biochemical reaction reflects cells adaptation through modifying protein synthesis.
(Fiocco et al., 2007). It is crucial for a cells survival to produce heat shock proteins (HSP) in
order to maintain metabolic activity and repair cell damage during heat stress. The HSP,
mainly chaperones and proteases, are responsible for refolding and degradation. GroEL (60
kDa) is a group 1 chaperonin that protects folding proteins from premature cytosolic
breakdown and abnormal interactions. (Jain et al., 2017). DnaK (homologue to Hsp70) is
the most constitutively expressed and stress-inducible chaperones in the cytosol. DnaK,
together with its co-chaperone DnaJ (Homologue to Hsp40) and regulator GrpE
(Homologue to Hsp20), helps in de novo folding of proteins with the ribosome-bound
chaperone Trigger factor (TF) (Calloni et al., 2012). ClpB, ClpA, and ClpX, on the other
hand, are Hsp100 family members that operate as both chaperones and proteases. (Sangpuii
et al., 2018).
 Differences in protein content were observed approximately at 20, 40–55 kDa, 60
kDa, and 70 kDa representing the possible overexpression of DnaJ, GroEL, and
DnaK in all heat-stressed cells.
 There was also an increase in protein concentration around 100 kDa, revealing the
possibility of ClpB, ClpA, ClpX proteins.
 GroEL (60 kDa) is a group 1 chaperonin that protects folding proteins from
premature cytosolic breakdown and abnormal interactions (Jain et al., 2017).
 DnaK (homologue to Hsp70) is the most constitutively expressed and stress-
inducible chaperones in the cytosol. DnaK, together with its co-chaperone DnaJ
(Homologue to Hsp40) and regulator GrpE (Homologue to Hsp20), helps in de novo
folding of proteins with the ribosome-bound chaperone Trigger factor (TF) (Calloni
et al., 2012).
 The small Hsp20 has emerged as a key mediator of protection against apoptosis,
remodeling and ischemia/reperfusion injury.
60 | P a g e
 ClpB, ClpA, and ClpX, on the other hand, are Hsp100 family members that operate
as both chaperones and proteases. (Sangpuii et al., 2018).
SLURRY PREPARATION AND OPTIMIZATION OF SPRAY DRYING PARAMETERS
After optimization of stress parameters for better survivability during spray drying process the feed
or slurry preparation, optimization of spray drying parameters was performed according to
Chandralekha et al., (2011). Response surface methodology (RSM) was used to estimate the
effects of spray-drying conditions on survival ratio (SR) of selected probiotic strain, color change (Δ
DE), moisture content (MC), and overall sensory acceptability (SA) of dried culture during spray
drying. Central composite rotatable design (CCRD) was used for designing the experimental data.
The following experiment of design as per Table 5.2 was used to optimization of spray drying
parameter.
Experimental design for optimization of spray drying parameters and selection of carrier
materials to enhance survivability of Lactiplantibacillus plantarum HD51
Carrier Control Concentration (% Spray dryer Spray dryer inlet temp
material (%w/v) w/v) outlet temp (°C) (°C)
Maltodextrin 0 0, 2.5, 5.0 ,7.5 and 10 55, 60, 65 and 70 170,180,190 and 200
Gum acacia 0 0, 2.5, 5.0 ,7.5 and 10 55, 60, 65 and 70 170,180,190 and 200
Corn starch 0 0, 2.5, 5.0 ,7.5 and 10 55, 60, 65 and 70 170,180,190 and 200
Briefly, selected strain was subjected for biomass production in whey based medium in pilot
scale at fermenter followed by centrifugation (8000 ×g for 10min at 4°C). Pellet was washed twice
with PBS and feed for spray drying was prepared by adding varied quantities of carrier materials
(maltodextrin, acacia gum, and corn starch) as mentioned in Table 5.2 along with microbial cells
(1010 CFU/mL) in 1000 mL of 45% reconstituted sterilized skim milk as mentioned in the below
Table 5.2. The contents was mixed using a tabletop magnetic stirrer to get a uniform solution and
subjected to spray drying as given below flowchart.
61 | P a g e
The selected cultures was grown under optimized growth conditions in whey
based media at 37°C/16-20 h
Centrifugation (8,000 rpm/10 min at 4°C) and washing with phosphate buffer
saline
slurry or feed preparation in sterile 45% reconstituted skimmed milk RSM

supplemented optimized carrier material level
Optimization of of spray drying parameters

(Varied inlet temp. 170°C, 180°C,190°C and 200°C) and outlet temperatures
(55°C, 60°C,65°C and 70°C)
Spray dried DVS probiotic was packed in aluminium laminates, LDPE, EVOH
and assessed for technofunctional, sensory and textural attributes
A pilot-scale spray dryer was used for the spray drying process. At first, distilled water was fed to
the spray dryer until the steady-state and thermal equilibrium conditions attained and then probiotic-
containing feed solution was pumped into the drying chamber using a peristaltic pump and
atomization was performed using a two-fluid nozzle. Throughout the experiments, the atomizer
pressure and the feed solids concentration was kept constant. Different trails was taken for
optimization of inlet air temperature and outlet air temperature (55 -70°C) by feeding the sample in
sterile 45%TS reconstituted skim milk during spray drying. Spray dried probiotic DVS powder with
higher survivability after spray drying was selected for preparation of DVS powder. The spray-dried
probiotic powder was collected from the cyclone base and packed in low density polyethylene
(LDPE), high density polyethylene (HDPE) and ethyl vinyl alcohol (EVOH) at -20°C, 4°C & 30°C
for further quality analysis.
The yield of spray-dried DVS powder was calculated according to Nunes et al., (2018) by the
following equation.
Dry cell powder yield (%, w/w) = (Pw/ Ts) × 100
Where ‘Pw’- the cell powder weight collected after spray drying, and
‘Ts’ - the total soluble solid content in the slurry.
Flowchart for preparation of DVS powder by spray dryer
62 | P a g e
Optimization of spray drying parameters for RSM

Unknown variables (with selected range) :
• Carrier materials (0,2.5,5.0,7.5 and 10%)
 Inlet temperature (170,180 and 190)
 Outlet temperature(55,60 and 65%)
Responses studied
• Survivability rate (%)
• Moisture (%)
• Water activity (aw)
• Hygroscopicity (%)
• Bulk density (g/cm3)
• Tap density (g/cm3)
• Browning index (▲E)
• Wettability (sec)
• Solubility index (%)
63 | P a g e
The technique suggested 20 formulations after computation
64 | P a g e
65 | P a g e
66 | P a g e
67 | P a g e
Experimental design, statistical analysis and optimization

 Response surface methodology (RSM) was used to investigate the main effects of spray
drying process variables on the Survivability rate, Moisture, Water activity ,Hygroscopicity,
Bulk density, Tap density, Browning index, Wettability, Solubility indexduring production
of DVS powder.
 Inlet drying temperature (x1,) and outlet drying temperature (x2,) were selected as
independent variable.
68 | P a g e
 Atomization pressure (x3) , feed total solids (45%TS) and feed flow rate (2 ml/min) kept
constant.
 Experimental data were fitted to a second-order polynomial model and regression
coefficients were obtained for each response.
 Significant terms in the models were found by analysis of variance (ANOVA) and
significance was judged by the F-statistic calculated from the data.
 Model adequacies were checked by R 2 , adj-R 2 , pre-R 2 , Adeq. Precision, PRESS and
C.V. (lack of fit > 0.1) After model fitting, residual analyses including the examination of
diagnostic plots and calculation of case statistics were conducted to validate assumptions
used in ANOVA.
 Design Expert Ver. 7.0.0 (Stat-Ease, 2005) was used to fit response surfaces and optimize
the drying process
FLOW CHART FOR PREPARATION OF DAHI FOR DVS INOCULUM OPTIMIZATION
69 | P a g e
Optimization of DVS inoculum levels by preparation of probiotic dahi
SALIENT FINDINGS
 Optimization of spray drying parameters i.e. (Inlet and outlet temperatures)
 Response surface methodology (RSM) was used to investigate the main effects of spray
drying process variables on the Survivability rate, Moisture, Water activity, Hygroscopicity,
70 | P a g e
Bulk density, Tap density, Browning index, Wettability, Solubility indexduring production
of DVS powder.
 Lp. plantarum HD51 was selected for spray drying of powder in 45% RSM with different
carrier materials with varied concentrations.
 Lp. plantarum HD51 against Maltodextrin showed better survivability at 7.5 (% w/v)
compared to 0.5, 2.5, 5.0 and 10.0 (% w/v).
 Lp. plantarum HD51 against Corn starch showed better survivability at 5.0 (% w/v)
 Lp. plantarum HD51 against Gum acacia showed better survivability at 10.0 (% w/v)
 Storage study of DVS powder was analyzed for a storage period of 90 days in which no any
significant difference was found among three different packaging materials in -20,4 and
30℃ w.r.t techno-functional, sensory and textural attributes.
71 | P a g e

Results of Heat Challenge Experiment and Related Work

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Direct vat set (DVS) probiotic dairy starters can be produced to address the issue of starter

Probiotic strains Negative staining Grams staining Catalase

TECHNO-FUNCTIONAL ATTRIBUTES OF PROBIOTIC DAHI PREPARED BY

Techno-functional attributes of probiotic dahi prepared with lactobacillus strains

Techno-functional attributes of probiotic dahi prepared with lactobacillus strains

Hardness or firmness, is the most important characteristics in determining texture of dairy

Textural attributes of probiotic dahi prepared with lactobacillus strains

Consistency Cohesiveness Work of

Sensory scores of probiotic dahi prepared with lactobacillus strains.

Texture profile of probiotic dahi prepared with lactobacillus strains

L. plantarum CRD7 (1010) L. plantarum CRD7 (1011) L. plantarum CRD7 (1012)

1 11.37±0.0 10.48±0.0 11.38±0.0

2 11.23±0.0 10.51±0.0 10.82±0.0

0 11.18±0.0 11.37±0.0 12.11±0.0

1 11.37±0.0 10.76±0.0 11.73±0.0

2 11.23±0.0 10.46±0.0 11.09±0.0

0 11.18±0.0 11.37±0.0 12.12±0.0

1 11.37±0.0 10.80±0.2 11.73±0.0

2 11.23±0.0 10.46±0.0 11.18±0.0

1 11.37±0.0 10.76±0.0 11.73±0.0

2 11.23±0.0 10.37±0.0 11.34±0.2

0 11.18±0.0 11.13±0.1 12.20±0.1

1 11.37±0.0 10.77±0.1 11.72±0.1

2 11.23±0.0 10.37±0.1 11.37±0.1

3 11.16±0.0 10.05±0.0 10.94±0.1

L. plantarum CRD7 (1010) L. plantarum CRD7 (1011) L. plantarum CRD7 (1012)

L. plantarum CRD7 (1010) L. plantarum CRD7 (1011) L. plantarum CRD7 (1012)

Probiotic viable count (Log CFU/g or mL)

5.0 7.0 10.01±0.03c 10.01±0.03c 9.88±0.02a 9.66±0.02ab 8.21±0.02ab 8.11±0.02ab

1.0 4.5 12.15±0.02b 12.12±0.02b 11.62±0.01b 10.62±0.02b 10.02±0.02b 9.08±0.02b

5.0 7.0 12.08±0.01a 12.09±0.01a 11.23±0.01c 10.61±0.01b 9.78±0.01b 9.10±0.01b

Salt Survivability (%)

HD48 (9 Log CFU/mL) 1.0 4.5 89.13±0.05b 82.13±0.05b 80.13±0.05b 82.13±0.05b

1.0 4.5 88.04±0.02b 80.04±0.02b 79.04±0.02b 92.04±0.02b

2.5 6.5 93.75±0.03b 83.75±0.03b 82.75±0.03b 79.75±0.03b

1.0 4.5 86.18±0.02a 83.18±0.02a 79.18±0.02a 74.18±0.02a

5.0 7.0 78.82±0.17a 76.82±0.17a 74.82±0.17d 70.82±0.17a

Probiotic viable count (Log CFU/g or mL)

HD48 (12 Log

Salt Survivability (%)

HD48 (9 Log CFU/mL)

1.0 4.5 86.18±0.02a 83.18±0.02a 79.18±0.02a 74.18±0.02a

Probiotic viable count (Log CFU/g or mL)

HD48 (10 Log

Salt Survivability (%)

HD48 (9 Log CFU/mL)

1.0 4.5 83.18±0.02a 81.18±0.02a 77.18±0.02a 75.18±0.02a

5.5 100.00 81.75±0.02c 81.75±0.02c 81.75±0.02c 81.75±0.02c

Lp. plantarum HD 51 6.0 100.00 88.13±0.05b 80.13±0.05b 81.13±0.05b 80.13±0.05b

(109 Log CFU/mL) 6.5 100.00 90.62±0.06c 80.62±0.06c 72.62±0.06c 74.62±0.06c

7.0 100.00 87.69±0.03a 83.69±0.03a 71.69±0.03a 91.69±0.03a

5.5 100.00 88.83±0.09b 80.83±0.09b 78.83±0.09b 92.83±0.09b

5.5 100.00 81.75±0.02c 80.75±0.02c 8.75±0.02c 81.75±0.02c

Lp. plantarum HD 51 6.0 100.00 82.13±0.05b 82.13±0.05b 80.13±0.05b 81.13±0.05b

(109 Log CFU/mL) 6.5 100.00 91.62±0.06c 88.62±0.06c 74.62±0.06c 74.62±0.06c

7.0 100.00 81.69±0.03a 83.69±0.03a 72.69±0.03a 71.69±0.03a

5.5 100.00 84.83±0.09b 85.83±0.09b 71.83±0.09b 70.83±0.09b

5.5 100.00 82.35±0.02c 79.75±0.02c 75.75±0.02c 73.75±0.02c

Lp. plantarum HD 51 6.0 100.00 84.13±0.05b 81.13±0.05b 79.13±0.05b 77.13±0.05b

(109 Log CFU/mL) 6.5 100.00 90.52±0.06c 85.62±0.06c 80.62±0.06c 74.62±0.06c

7.0 100.00 85.69±0.03a 82.69±0.03a 71.69±0.03a 70.69±0.03a

5.5 100.00 86.83±0.09b 84.83±0.09b 75.83±0.09b 74.83±0.09b