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Save Nature to Survive

14(4): 299-301, 2019
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VIABILITY OF LACTIC CUL

LACTIC TURES BEFORE AND AFTER FREEZE
CULTURES
DRYING IN POMEGRANA
DRYING TE JUICE
POMEGRANATE

RITA NARAYANAN
Dept of Fod Processing Technology,
College of Food and Dairy Technology , Koduveli, Chennai-600052
e-mail: ritanarayanan@yahoo.com

KEYWORDS ABSTRACT
lactic culture viability in The purpose of this research was to study the viability of lactic cultures in pomegranate juice before and after
pomegrante juice freeze drying.Two dietary adjuncts of a combination of 50% pomegranate juice and 50% skim milk were
skim milk fermented separately with 1% culture of Lactobacillus plantarum and Lactobacillus casei and freeze-dried. The
fermented pomegranate viability of the freeze-dried strains was evaluated after 24 h on MRS agar. Results indicated a 2 log reduction in
juice the viability of lactic cultures in fermented combination of pomegranate juice and skim milk and also in
fermented skim milk (control) after freeze-drying . The viable count of L.casei after freeze drying was reported as
Received on : 8.04±0.1470log10cfu/ml and 8.030± 0.1483log10cfu/ml in control and pomegrante juice and skim milk mixed
05.07.2019 sample respectively . The viable count of L. plantarum after freeze drying was reported as 8.04±0.1470log10cfu/
ml and 8.02±0.0151log10cfu/ml in control and pomegrante juice and skim milk mixed sample respectively .
Accepted on : Survival of the lactic cultures were studied after freeze drying at intervals of 7 days, 15 days, 30 days and 90
08.11.2019 days of storage under refrigeration conditions. A higher percent of survival was noted upto 30 days in both the
lactic organisms in skim milk (control) and fermented combination of pomegranate juice with skim milk .
*Corresponding However there was a reduction in viability of both the cultures on the 90th day of enumeration.
author

membrane and DNA (Tripathi and Giri, 2014). Additionally,

INTRODUCTION the changes in the physical state of membrane lipids during
Fruit juices are a rich source of calcium and vitamin and could storage may result in severe loss of bacterial viability during
serve as a suitable media for cultivating starter cultures. storage (Fonseca et al., 2015).A number of cryoprotective
Considerable amount of total soluble solids, total sugars, agents such as proteins, sugars and carbohydrates have been
reducing sugars, anthocyanins, phenolics, ascorbic acid, used to minimize the bacterial inactivation after freeze drying
proteins and antioxidants are present in fruit juices. and subsequent storage (Carvalho et al., 2004). Freeze drying
Pomegranate (Punica granatum) is one of the important table has been a method of choice for the long term preservation of
bioactive materials. This dehydration method causes little
fruit and is known to have considerable health-promoting
shrinkage and results in a completely soluble product that is
properties such as antimicrobial, antiviral, antioxidant and
easily rehydrated. Moreover, lyophilization is frequently used
anti-mutagenic effects (Negi et al., 2003). Development of
to preserve lactic acid bacterial starter cultures involved in
foods that promote health and wellbeing is one of the key dairy and food fermentations (Lodato et al., 1999). Cell
research priorities of the food industry (Klaenhammer and immobilization in various carriers, including composite carrier
Kullen ,1999) matrix systems has recently attracted interest targeting to protect
It has been suggested that fruit juices could serve as suitable cultures from different types of environmental stress.
media for cultivating probiotic bacteria (Mattila-Sandholm et Methods of production of lactic starter juice powders should
al., 2002). Fermented fruit beverages are known for their be such that adequate numbers of viable bacteria are
health beneficial effects and nutritional properties. L.casei and maintained in the dried powder following manufacture, and
L.plantarum related species have been utilized widely in food throughout the shelf-life of powder. Both freeze-drying and
processing. L.casei is a common inhabitant of the human spray-drying can be used for manufacture of lactic starter fruit
intestinal tract and also found naturally in fermented vegetables, juice powders on a large scale (Wang et al., 2004). Keeping
milk and meat. L.plantarum also colonizes naturally in plant this in view the aim of this research was to investigate the
surfaces and is also able to successfully ferment fruit juices growth rate and substrate metabolism by the lactic cultures of
and vegetable juices (tomato, beetroot) with a high viability Lactobacillus plantarum and Lactobacillus casei in a medium
(Santo et al., 2011). containing 50% of pomegrante juice and 50% skim milk after
Freeze drying is considered as a suitable method for stabilizing fermentation and to evaluate their viability after freeze drying.
microorganisms that are greatly sensitive to high temperature
(Goderska, 2012; Fonseca et al., 2015). However, freezing MATERIALS AND METHODS
and subsequent sublimation of frozen water could be
attributed to cellular injuries including damage to cell Pomegranate (Punica granatum- Arakta variety) skim milk

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RITA NARAYANAN

powder with 5 per cent moisture and 95 per cent solubility difference in the viable count of L.casei before and after freeze
were purchased locally. Lactobacillus casei (NCDC- 142) and drying. There is almost a 2 log reduction in the viable count
Lactobacillus plantarum (NCDC- 21) were purchased from after freeze drying. The viable count of L.casei after freeze
the National Dairy Research Institute, Karnal, Haryana. drying was reported as 8.04±0.1470log10cfu/ml and 8.030±
Sterilized skim milk acted as control. The protocol for freeze 0.1483log10cfu/ml in control and pomegrante juice and skim
drying the cultures in omegranate substrate was done as per milk mixed sample respectively.This was in agrrement with
the modified procedure adopted by Ambuj and Athmaselvi the results of Jofre et al. (2015) on freeze dried samples of
(2016). Fifty percent of pomegranate juice was filter sterilized Lactobacillus casei/paracasei CTC1677 and L. casei/paracasei
and mixed with 50 per cent sterilized skim milk for the study CTC1678 . They reported survivability of ( ≥ 94%) and best
. The samples were inoculated separately with L.casei and performance on freeze drying Lactobacillus rhamnosus
L.plantarum and incubated overnight . The overnight cultures CTC1679, Lactobacillus casei/paracasei CTC1677 and L.
were then dispensed in sterile cryogenic vials and freeze casei/paracasei CTC1678 in skim milk alone or in
dried. supplementation with trehalose or lactose. The results are also
Enumeration of viable cultures after freeze drying concordant with the findings of Ambuj and Athmaselvi (2016)
on pomegranate juice samples containing L.rhamnosus treated
After freeze-drying, the freeze-dried powders were re-hydrated
at “40º C which had more regenerative power.
in skim milk powder and the cell suspensions were allowed to
stand for 10 min at room temperature, and subsequently plated Viability of Lactobacillus plantarum before and after freeze
on MRS agar. The number of viable cells were determined drying in various medium
before and after freeze-drying after incubation at 24 h at 37ºC. Results in table 2 shows that there is a highly significant
The viability of the freeze-dried cells were also determined at difference in the viable count of L. plantarum before and after
regular intervals for a period of 90 days. freeze drying. There is almost a 2 log reduction in the viable
The data obtained were analyzed statistically as per the count after freeze drying. The viable count of L. plantarum
standard procedure of Snedecor and Cochran (1980). after freeze drying was reported as 8.04±0.1470log10cfu/ml
and 8.02±0.0151log10cfu/ml in control and pomegrante
juice and skim milk mixed sample respectively. This differed
RESULTS AND DISCUSSION from the study of Sofia Carvalho et al. (2002) who found that
Viability of L.casei before and after freeze drying in various no significant differences in the viability of Lactobacillus
medium plantarum cells during freeze-drying in the presence of inositol,
sorbitol, fructose, trehalose, monosodium glutamate and
Results in table 1 shows that there is a highly significant
propyl gallate. This decrease of cell count is in line with the
Table 1: Total viable count#( log10 cfu/ml) of L. casei before and after results on freeze drying of. L.plantarum by Sawaminee et al
freeze drying in various medium @ (2012) who reported in their study , a decrease in cell viability
Lactobacillus casei# of approximately 40% during freeze drying leading to an
Control Combination of skim initial cell concentration in the instant dried powders to 6×107
milk and pomegranate CFU/m L. lactobacilli .
(50% each)
Before freeze drying 10.03±0.0141 10.02±0.0192 Viability of Freeze dried Lactobacillus casei during storage
After freeze drying 8.04±0.1470 8.030±0.1483 at refrigeration temperature.
t test ** ** Small case shows significant difference within treatments
@Average of six trials;#log10 cfu/ml;**Statistically highly significant (P ≤ 0.01)
Results in table3 shows that there is a highly significant
difference in the viable count of L.casei during storage. There
Table 2: Total viable count#( log10 cfu/ml) of Lactobacillus plantarum
before and after freeze drying in various medium @ was no difference in viability of cells till 30 days of storage.
Lactobacillus plantarum# However there was a significant difference in their viability on
Control Combination of skim the 90 th day.
milk and pomegranate No difference in the viability of cells between the treatments
(50% each) was noticed indicating that substitution of pomegranate juice
Before freeze drying 10.03±0.0141 10.01±0.022
for skim milk can also be considered to improve the functional
After freeze drying 8.04±0.1470 8.02±0.0151
t test ** **
property of dairy adjuncts.
@Average of six trials;#log10 cfu/ml;**Statistically highly significant (P ≤ 0.01) The findings corroborated with the work of Caroliny et al
Table 3: Total viable count# (log10 cfu/ml) of Lactobacillus casei during storage at refrigeration temperature @
Treatment Viability during storage in days
7th day 15th day 30th day 90th day F value
Control 8.033b ±0.180 7.983b±0.184 7.913b±0.215 6.408a±0.3380 **
31.07
Skim milk + 8.025 b ±0.180 7.983 b ±0.178 7.773 b ±0.062 6.460 a ±0.358 **
Pomegranate juice 24.04
t Test 0.9692NS 0.0573NS 0.6724 NS 0.9059NS
P Value
@Average of six trials;#log10 cfu/ml;**Statistically highly significant (P 0.01);NS – non significant (P> 0.05)

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Table 4:Total viable count#(log 10 cfu/ml) of Lactobacillus plantarum during storage at refrigeration temperature @
Treatment Viability during storage in days F value
7th day 15th day 30th day 90th day
Control 8.043b ±0.177 7.983 b ±0.18 7.873 b ±0.222 6.4600 a ±0.388 **
34.04
Skim milk+ 8.008 b ±0.185 7.967 b ±0.184 7.740 b ±0.062 6.511 a ±0.375 **
Pomegranate juice 23.02
t Test 0.8719 NS 0.9445 NS 0.4982 NS 0.9063 NS
P Value
@Average of six trials; #log10 cfu/ml; **Statistically highly significant (P ≤ 0.01); NS – non significant (P> 0.05)

(2020) who repoarted a small decrease (<1 log CFU/g) in Dantas Duarte Menezes, Erika Tayse da Cruz Almeida, Marcos dos
viable counts of L. casei L-26 after 15 days of storage under Santos Lima, Vanessa Bordin Viera, Estefânia Fernandes
refrigeration when the strain was freeze-dried with acerola, Garcia, Andrea Gómez-Zavaglia, Evandro Leite de Souza,, and Maria
Elieidy Gomes de Oliveira. 2020. Protective Effects of Tropical Fruit
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days of refrigerated storage onward. They reported a higher Carvalho, A. S., J. Siva, P. Ho, P. Teixeira, F. X. Malcata and P. Gibbs.
viable counts of L. casei L-26 freeze-dried with acerola (5.3 ± 2004. Relevant factors for the preparation of freeze dried lactic acid
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ACKNOWLEDGEMENTS (2): 627-633
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Malcata, Paul Gibbs. 2002. Survival of freeze-dried Lactobacillus
plantarum and Lactobacillus rhamnosus during storage in the presence
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