 *Corresponding author.

Email: alsulabo@up.edu.ph

eISSN: 2550-2166 / © 2020 The Authors. Published by Rynnye Lyan Resources

  Food Research 4 (4) : 964 - 975 (August 2020)
  Journal homepage: http://www.myfoodresearch.com

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Storage stability of freeze-dried Lactobacillus plantarum S20 starter culture as

affected by various formulations of drying medium, and its fermentation

characteristics in mung bean (Vigna radiata L.) slurry

*Sulabo, A.S.L., Villasanta, M.E.L., Hermo, K.G., Lascano, R.A., Collado, L.S. and

Babaran, G.M.O.

Julian A. Banzon Hall, Institute of Food Science and Technology, College of Agriculture and Food Science,

University of the Philippines Los Baños, Laguna, Philippines, 4031

Article history:

Received: 6 November 2019

Received in revised form: 6

February 2020

Accepted:  9 February 2020

Available Online: 28

February 2020

Keywords:

Lactobacillus plantarum,

Freeze drying,

Mung bean,

Trehalose,

Sucrose

DOI:
https://doi.org/10.26656/fr.2017.4(4).361

Abstract

Development of an active starter culture is essential in the production of safe and high-

quality fermented foods. Freeze-drying is an effective microbial cell immobilization

technology intended to produce active and stable starter cultures for long-term storage. To

determine the influence of various drying medium formulations with mung bean powder

and disaccharides on the viability of L. plantarum S20 during freeze-drying and

subsequent storage at 6±2°C and 30±1°C, three treatments of drying medium consisting of

mung bean powder without disaccharides (MBP), MBP with 5% (w/v) trehalose

(MBP+T), and MBP with 5% (w/v) sucrose (MBP+T) were evaluated. Results showed

that L. plantarum S20 viability after freeze-drying ranged from 9.56 to 10.03 log (CFU/g)

with viability loss ranging from 0.20 to 0.51 log (CFU/g), with MBP being the least

effective (p<0.05) in minimizing viability loss. Supplementing MBP with either 5%

sucrose or 5% trehalose improved (p<0.05) survival of freeze-dried L. plantarum S20; and

both disaccharides exhibit similar (p>0.05) cryoprotective effects on L. plantarum S20

during freeze-drying and subsequent storage. Storage at 6±2°C for 180 days resulted in

significant decreases of 2.15, 2.17, and 3.27 log (CFU/g) in L. plantarum S20 population

in MBP+T, MBP+S, and MBP, respectively. A more drastic decline [>6.0 Log (CFU/g)]

in L. plantarum S20 population was observed in freeze-dried culture stored at 30±1°C for

60 days. Fermentation characteristics of freeze-dried L. plantarum S20 in mung bean

slurry were also determined. Freeze-dried L. plantarum S20 in MBP with 5% (w/v)

sucrose was able to acidify mung bean slurry from pH 6.8 to pH 3.8 after 8 hrs with

maximum LAB population of 9.2 log (CFU/mL). Acidification of mung bean slurry by

fresh culture (non-freeze-dried) on the other hand was relatively slower where pH dropped

from 6.8 to pH 3.72 after 24 hrs. Current work suggested that lyophilized L. plantarum

S20 could be used as a starter culture for mung bean-based fermentations but further

research on this area is needed.

1. Introduction

Fermentation is an old but useful form of food

preservation, and it is valuable in developing functional

foods. It is commonly used in food processing to

enhance the safety, nutritional content, flavor, and

texture of food. The most popular fermented foods are

those achieved by lactic acid bacteria (LAB) (Rezac et

al., 2018), which is a diverse group of Gram-positive

bacteria capable of converting carbohydrates into lactic

acid as the major end product (Paul et al., 2017). A

variety of LAB species, particularly those that belong in

the genus Lactobacillus, can be found in a variety of

fresh produce, fermented foods, and mammalian

digestive tracts. The species Lactobacillus plantarum is

the most studied Lactobacillus due to its ability to thrive

in a wide range of environmental niches. Different

strains of L. plantarum have emerged as functional LAB

exhibiting a long history of safe use.  It is considered as

“generally recognized as safe” (GRAS) and has qualified

presumption of safety (Ray and Joshi, 2014; Ricci et al.,

2017). The versatility of L. plantarum makes it a good

functional organism to be used in a wide range of

applications in the food industry, especially as a starter

culture.

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The use of starter cultures plays an essential role in

controlled fermentations to ensure the safety and quality

of commercially produced fermented foods. A starter

culture is composed of either a single species or a

mixture of microorganisms that are added directly into a

food matrix to start the fermentation process, and to

obtain a desired and consistent quality of the finished

product (Durso and Hutkins, 2003). The production of

starter cultures aims to ensure long-term delivery of

viable and stable cultures, which can be achieved

through the application of preservation technologies

(Carvalho, 2003a). Freeze-drying is a cell

immobilization technology that can be used to preserve

active and stable starter cultures. It has been established

in the food industry as the best method of preserving

bacterial cultures in a powdered/crystalline form for long

-term storage. However, freeze-drying can result in cell

injury that could lead to cell death. Thus, selecting

appropriate drying medium as a protective matrix can

produce a stable final formulation of freeze-dried cell

culture with minimal viability loss. Several studies have

investigated the use of various protective agents during

feeze-drying consisting of additives (Carvalho et al.,

2002), disaccharides, and skim milk (Carvalho et al.,

2003b; Jalali et al., 2012; Mendoza et al., 2013; Gisela et

al., 2014; Jofré et al., 2015) in order to minimize

viability loss of LAB during cryopreservation. In

addition, drying media also serve as carriers of freeze-

dried starter cultures for fermentation applications. The

use of skim milk supplemented with disaccharides as

drying medium for dairy starter cultures has been

successful in minimizing bacterial cell death during

freeze-drying and subsequent storage (Carvalho et al.,

2004). Food-grade plant-based materials were also

utilized as components of freeze-drying media for

probiotic bacteria and non-dairy starter cultures and have

shown to reduce microbial viability loss. A number of

these plant-based materials include plant protein powder

(Savedboworn et al., 2017), fruit powders (Trachoo et

al., 2008; Nualkaekul et al., 2012), soybean flour

(Trachoo et al., 2008), plant extracts and fibers

(Hongpattarakere et al., 2012). Starchy materials like

flours of rice, cassava, yam, millet, maize, and sorghum

have been utilized as cryoprotective drying media for

non-dairy starter cultures for gari and beer fermentations

(Okafor et al., 1999; N’Guessan et al., 2016).

Mahidsanan et al. (2017) also reported that a novel

soybean flour as an inexpensive cryoprotectant option

showed enhanced protection on Bacillus subtilis SB-

MYP-1 starter culture for Thu-nao (fermented soybean)

processing.

Mung bean (Vigna radiata L.) or munggo, locally

known in the Philippines, is a pulse crop with high

protein content (21-31%) and considered as a poor man’s

meat (Anwar et al., 2007). Mung bean is also known for

its high carbohydrates content (64.2%) with small

portions of dietary fiber (14.6%), ash (3.9%) and some

traces of vitamins and minerals (BAFS, 2012). In food

processing applications, mung bean has established its

popularity due to its good quality starch, which

comprises a majority of total carbohydrates. Starch in

mung bean comprises more than 50% of the total seed

weight (Li et al., 2011). It has the excellent gelling

capability, pasting property, and cohesiveness, becoming

it a better choice for film-forming biopolymers and

starch noodles (Kim et al., 2007; Li et al., 2015;

Rompothi et al., 2017). Fermentation of mung bean with

L. plantarum strains was associated with the release of

angiotensin I-converting enzyme (ACE) inhibitory

peptides, which are drugs used to lower blood pressure.

Fermentation of pea with L. plantarum 299 has produced

bioactive metabolites that facilitated the release of

angiotensin I-converting enzyme (ACE) inhibitory

peptides during in vitro gastrointestinal process (Torino

et al., 2013). L. plantarum B1-6-fermented mung bean

milk was shown to produce significantly higher ACE

inhibitory activity (67.5%) at the end of fermentation

(Wu et al., 2015). In addition, Wu et al. (2015) also

demonstrated that mung bean milk could be a carrier

matrix of probiotics. Thus, it is interesting to explore the

potential of utilizing of mung bean as starter culture

drying medium and fermentation substrate for L.

plantarum.

Freeze-drying at commercial scale is an expensive

dehydration technology. However, it is still the most

used method at commercial scale for the production of

starter cultures. Thus, utilizing freeze-drying media

formulated with low-cost ingredients like mung bean

should be able to reduce a portion of the cost. To date, no

studies have been conducted in utilizing mung bean as a

drying medium component that will also serve as a

carrier matrix for L. plantarum starter culture. This study

aimed to evaluate the influence of various protective

agents formulated with mung bean powder and

disaccharides on the freeze- and post-freeze-drying

survival of L. plantarum S20 starter, which can be used

in mung bean-based fermentation. Results of the study

will provide baseline data necessary to produce starter

culture by utilizing cheap food-grade materials like

mung bean as a component of drying medium for the

production of novel functional non-dairy fermented

foods.

2. Materials and methods

2.1. Preparation of starter culture

2.1.1 Cultivation of Lactobacillus plantarum S20

Lactobacillus plantarum S20 was acquired from the

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Philippine National Collection of Microorganisms of the

University of the Philippines Los Baños College,

Laguna. It was isolated from Nem chua, a fermented

meat product of Vietnam with 98% homology with L.

plantarum WCFS1 (Nguyen et al., 2010). It was isolated

using streak plating technique and identified by its 16s

rDNA sequence. L. plantarum S20 was maintained in 7.0

mL deMann Rogosa Sharpe (MRS) broth (HiMedia,

Mumbai, India) at 4±1°C and sub-cultured every 2

weeks.

2.1.2 Preparation of mung bean powder

Whole green mung bean seeds (NSIC MG-14) were

purchased from the Bureau of Plant Industries-National

Crop Research and Development Center of Los Baños,

Laguna. The seeds are characterized to be dull-green and

can be planted and harvested during the wet and dry

season. Seeds were dried at 70°C for 2 hrs (moisture

content= 9.33±0.40%) and were then pulverized with the

fabricated stainless steel pin mill machine (50 Kg

capacity; Alameda, Tondo, Manila, Philippines) and

screened through 80-mesh sieve. Mung bean powder was

then packed and vacuum-sealed in Polyethylene bags

and stored at room temperature until use.

2.1.3 Preparation of drying medium

A total of three treatments of drying medium

formulated with mung bean base mixture and two types

of disaccharides (sucrose and trehalose) were prepared.

The treatments include mung bean with 5% (w/v)

trehalose (MBP+T), mung bean with 5% (w/v) sucrose

(MBP+S), and mung bean powder only (MBP). A

mixture of mung bean powder and sterile 0.85% (w/v)

NaCl solution was used as base mixture ingredients of

the freeze-drying medium. The proportion used in

preparing the base mixture of drying medium was based

on the optimized cryoprotectant formulation using skim

milk described by Gisela et al. (2014) with some

modifications. About 25 g of mung bean powder was

diluted with 0.85% (w/v) NaCl solution to 100 mL. The

mixture was then transferred to a sterile media bottle and

pasteurized at 71±1°C for 66±1 mins in order to

inactivate pathogenic bacteria and reduce population

spoilage microorganisms. The hot mixture was then

assigned to one of the three treatments: MBP, MBP+T,

and MBP+S. Hot mixture assigned to MBP with

disaccharides was prepared by supplementing it with

either trehalose (Hayashibara, Japan) or sucrose (Ajax-

FineChem Univar, Australia) at 5% (w/v) inclusion rate.

All treatments were then allowed to cool down at room

temperature.

2.1.4 Preparation and freeze-drying of starter

culture

The preparation of starter culture was based on the

procedures described by Yao et al. (2009) with major

modifications. About 1 mL of overnight (16-18 hrs at

37°C) MRS-cultured L. plantarum S20 was propagated

in fresh 100 mL MRS broth at 37°C for 18 hrs. The L.

plantarum S20 cells were then harvested by

centrifugation at 4300 x g at 4°C for 15 mins and washed

with sterile 0.85% NaCl solution twice. Washed cells

were then re-suspended in 5 mL of sterile 0.85% NaCl

solution. The resulting cell suspension was then

aseptically transferred onto a media bottle containing one

of the previously prepared freeze-drying media. All

mixtures were then aseptically transferred onto sterile

petri dishes and were immediately frozen for 24 hrs at -

20°C. All treatments were then lyophilized at a vacuum

pressure of 0.20 Torr (ULVAC DF-01H, ULVAC

Corp.), and secondary drying temperature set at 40°C for

72 hrs. Preparing a freeze-drying medium devoid of any

of the cryoprotectants and L. plantarum S20 was used as

a negative control.

2.1.5 Packaging and storage of freeze-dried starter

culture

Under a sterile laminar flow cabinet, freeze-dried

cultures were pulverized aseptically using a sterile

spatula and transferred onto pre-sterilized amber glass

vials. Sample vials were immediately sealed with sterile

rubber stopper and stored either at 6±2°C for 180 days in

the chiller or at 30±°C for 60 days in the incubator.

2.2. Evaluation of fermentation performance of the

starter culture on mung bean slurry

2.2.1 The mung bean slurry preparation

Whole green mung bean seeds (NSIC MG-14) were

soaked in water for 9-10 hrs. Soaked beans were

manually de-hulled and a 200 g portion was transferred

onto a clean stainless steel stockpot. About 1.5 L of

sterile distilled water was added into the beans and the

mixture was heated to boiling at approx. 100°C.

Immediately after boiling, the heat was adjusted to the

medium low setting and the mixture was heated to

approx. 100°C for another 20 mins or until beans

become soft. The mung bean mixture was then

transferred into a blender and an additional 500 mL of

sterile distilled water was used to wash the remaining

mixture. The mixture was then homogenized for 2 mins

and transferred into a clean stainless stockpot. The

mixture was then added with 500 mL sterile distilled

water and was brought to boil. About 250 mL portion of

mung bean slurry was immediately transferred into a

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sterile screw-capped media bottle glass container and

was supplemented with sucrose at 5% (w/v) inclusion

rate. The mixture was then transferred to the boiling

water bath and was allowed to be pasteurized at 71±1°C

for 66 mins and was allowed to cool at room

temperature.

2.2.2 Fermentation of mung bean slurring using

freeze-dried Lactobacillus plantarum S20 culture

Three fermentation treatments containing 250 mL

mung bean slurry were prepared for fermentation

experiment. The first treatment of mung bean slurry was

supplemented with fresh L. plantarum S20 culture. This

was done by harvesting L. plantarum S20 cells from 10

mL overnight (37°C for 18 hrs) culture of L. plantarum

S20 in MRS broth, using the procedure described in the

preparation of starter culture for freeze-drying (Section

2.1.4). Harvested cells from 10 mL of overnight culture

(approx. 1010 CFU/mL) in MRS broth were re-suspended

in 9 mL 0.85% NaCl. About 5 mL of the cell suspension

was added to previously prepared mung bean slurry to

contain initial LAB population of 7 log (CFU/mL). The

second fermentation treatment of mung bean slurry was

supplemented with freeze-dried L. plantarum S20

culture. This was done by rehydrating 1 g of 2-day

chilled freeze-dried L. plantarum S20 culture (approx.

1010 CFU/g) in 9 mL 0.85% NaCl solution for 15 min.

About 5 mL of the rehydrated cells were transferred onto

250 mL pasteurized mung bean slurry to contain initial

LAB population of 7 log (CFU/mL). Lastly, the third

fermentation treatment of mung bean slurry was not

supplemented with L. plantarum S20 culture and served

as the control of the experiment.

2.2.3. Sample collection from the fermentation

mixture

During fermentation, sample aliquots for LAB

viability test, pH, and total soluble solids (TSS)

measurements were aseptically drawn from the

fermenting mung bean slurry at 0, 4, 8, 16, 24, and 32 h

of fermentation at 37°C.

2.3. Analytical procedures and LAB viability test

2.3.1 Freeze-drying and storage viability of

L.actobacillus plantarum S20

The ability of L. plantarum S20 to survive freeze-

drying and storage conditions in mung bean powder with

either trehalose or sucrose was determined using

standard spread plating technique. To determine the

influence of cryoprotectants on L. plantarum S20

viability in mung bean powder during freeze-drying,

about 1 g of freeze-dried culture was aseptically drawn

from each freeze-drying medium treatment before and

after the freeze-drying process. To assess the stability of

freeze-dried L. plantarum S20 during storage, the same

amount of freeze-dried culture was aseptically drawn at

0, 60, 120, and 180 days of 6±2°C storage and at 0, 20,

40, and 60 days of 30±1°C storage. Each sample

obtained was serially diluted in 0.1% peptone (HiMedia

Sa, Mumbai, India). About 0.1 mL sample aliquot from

the diluted sample was spread plated onto MRS agar and

overlaid with 5.0 mL molten MRS agar. Plates were then

incubated at 37°C for 48 hrs. Viable cell count was

reported as Log (CFU/g).

2.3.2 Enumeration of LAB population during

fermentation

Population of lactic acid bacteria during

fermentation of mung bean slurry with L. plantarum S20

was enumerated using a standard pour plating technique

on MRS agar. About 1 mL sample aliquot drawn from

fermenting mung bean slurry (section 2.2.3) was serially

diluted in 0.1% peptone. About 1 mL of the diluted

sample was pour plated with molten MRS agar. Plates

were then incubated at 37°C for 48 hrs. Viable LAB

counts were reported as log CFU/mL.

2.3.3 Water activity of freeze-dried starter during

storage

About 2 g of sample was obtained from freeze-dried

L. plantarum S20 before and after 180 and 60 d storage

at 6±2°C and 30±1°C, respectively. Water activities of

freeze-dried samples were then measured using a water

activity meter (LabSwift-aw meter; Novasina, Hamilton,

NJ, USA) at room temperature.

2.3.4 Total soluble solids and pH during mung bean

slurry fermentation

About 20 mL of sample was used for TSS and pH

measurement. The total soluble solids content of samples

was measured using a handheld refractometer (Atago,

Japan) and the results were expressed as °Brix. The pH

of samples was measured using a hand-held pH meter

fitted with spear-shaped probe (PH60S; Apera

Instruments) calibrated with pH 7.00 and pH 4.00 buffer

solution. All readings for each parameter were done in

duplicate for each sample.

2.4 Statistical analysis

Analysis of variance (ANOVA) with repeated

measures was used to determine treatments significantly

differ from each other, using 5% level of significance. A

pairwise comparison using Tukey’s HSD (honestly

significant difference) was used to compare means using

5% level of significance. The analysis was carried out

using SPSS Statistics (Version 25; IBM, USA). Both

freeze-drying and storage stability experiments were

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independently replicated three times with two

independent samples used in each replication.

Fermentation experiment, on the other hand, was

replicated four times with one independent sample used

in each replication.

3. Results and discussion

3.1 Effect of protective agents on Lactobacillus

plantarum S20 during freeze-drying

The population L. plantarum S20 in mung bean

powder with or without cryoprotectants after freeze-

drying is shown in Figure 1. It is shown that viability

after freeze-drying ranged from 9.56 to 10.03 log (CFU/

g) with viability loss ranging from 0.20 to 0.51 log

(CFU/g). Addition of either sucrose or trehalose at 5%

(wt/v) exhibit improved protection on L. plantarum S20

during freeze-drying where higher (p<0.05) level of

surviving L. plantarum population remained after freeze-

drying was observed.

Viability loss observed in the current study was

expected due to cell injury caused by the freezing and

dehydration process. Several studies reported that

cellular damage during freeze-drying was largely

associated with cell membrane damage due to ice crystal

formation that leads to high osmolarity of the cell

(Telang et al., 2003), macromolecule denaturation

(Thammavongs et al., 1996), and the removal of water

(Allison et al., 1999; De Paz et al., 2002). Viability loss

in L. plantarum S20 population observed in this study

was almost similar to the values reported by

Hongpattarakere et al. (2012). They reported that freeze-

drying of L. plantarum suspended in MRS growth

medium added with and without mung bean fiber has

resulted in 0.25 and 0.51 log (CFU/g) viability loss. In

the present work, all treatments were exposed at -20°C

for 24 hrs freezing temperature prior to freeze-drying.

This freezing temperature may have resulted in bigger

ice crystals that resulted in cell injury. Hongpattarakere

et al. (2012) and Perry (1998) noted that slow freezing

occurring at temperatures above −30°C may result in

poor survival due to loss of water from the cell while

increasing the effects of solute concentration. Exposure

of L. plantarum culture to extremely low freezing

temperature of -196°C have shown to have the

significantly lower freeze-drying viability loss of 0.18

log (CFU/g) as compared to -80 and -20°C with cell

reduction of 0.57 and 0.38 log (CFU/g), respectively

(Hongpattarakere et al., 2012).

Current results showed that using mung bean powder

only as drying medium exhibit significantly lower

viability compared to those formulated with either

sucrose or trehalose. The surviving L. plantarum S20 in

mung bean drying medium after freeze-drying was 9.56

log (CFU/g). This survival level is equivalent to approx.

31% of the initial population level before freeze-drying.

This survival rate was slightly better compared to the

survival rate (23%) of L. plantarum TISTR 2075 when

15% plant protein was used as a protective and drying

medium (Savedboworn et al., 2017). In the current study,

the use of ground mung bean may have served as inert

bulking agent forming viscous layer that provided some

protection on L. plantarum S20 cells during the freeze-

drying process. Carvalho et al. (2004) noted that some

high molecular weight substances present in the drying

medium like polysaccharides and proteins have no

capability to penetrate the cell wall or interact with the

cell wall or membrane. Their protective effect lies on

their ability to be adsorbed on the surfaces of

microorganisms forming viscous layer, which could

cause partial removal of water from the cell to the

surroundings. This would inhibit ice crystal growth while

keeping the structure of amorphous ice in the close

proximity of the cell (Hubálek, 1996). Consequently, this

will decrease the amount of ice crystals inside the cell,

hence could prevent cell death.

The addition of either trehalose or sucrose at 5% (w/

v) in the current study appeared to have increased the

resistance of L. plantarum S20 to freeze-drying. Previous

studies showed that these two disaccharides were

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Figure 1. Population of L. plantarum S20 in various freeze-

drying media before and after lyophilization. Data presented

as mean Log (CFU/g) from 6 independent samples. Drying

medium treatments: MBP+T= mung bean powder with 5%

trehalose; MBP+S= mung bean powder with 5% sucrose;

MBC= mung bean powder only. abValues within each

treatment not sharing the same letters are different from each

other (p<0.05) analyzed using Tukey’s HSD post hoc test;
XYZValues across treatments not sharing the same letters are

different from each other (p<0.05) analyzed using Tukey’s

HSD post hoc test; Error bars represent standard error of the

mean.

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effective as protective agents for cryopreservation of

lactic acid bacteria (Chavarri et al., 1988; Carvalho et

al., 2002; Carvalho et al., 2003b; Gisela et al., 2014).

Cryoprotectants in freeze-drying serve as antifreeze

agents, which minimize cell death by lowering the

melting point of freeze-drying media (Polge et al., 1949).

This would eventually freeze the medium surrounding

the cell faster than the intracellular components, hence,

preventing cell death. Carvalho et al. (2004) noted that

trehalose and sucrose have the ability to decrease the

transition temperature of dry membranes by replacing

water between the lipid head groups. This phenomenon

prevents phase transition, and its accompanying leakage

upon rehydration (Leslie et al., 1995; Castro et al.,

1997). An earlier study on cryopreservation of

biomaterials suggested that trehalose was more effective

cryoprotectant than other sugars (Crowe et al., 1996). On

the contrary, current work did not observe a significant

difference between the cryoprotective ability of sucrose

and trehalose. Current observation is supported by a

previous study conducted by Strasser et al. (2009) where

trehalose and sucrose demonstrated similar

cryoprotective effects on L. plantarum strain IFA No.

278 during freeze-drying. In addition, Strasser et al.

(2009) also reported that these two cryoprotectants were

the most effective among the cryoprotectants tested in

enhancing the survival of L. plantarum strain IFA No.

278 during lyophilization.

3.2 Stability of lyophilized Lactobacillus plantarum S20

during storage

Post-freeze-drying survival of L. plantarum S20

during chilled and ambient temperature storage is shown

in Figure 2. It is shown that storage of freeze-dried

cultures for either 180 days at 6±2°C or 60 days at 30±1°

C resulted in a significant decrease on the viability of

lyophilized L. plantarum S20 cells. At the end of 180 d

chilled storage period, viable L. plantarum population in

MBP+T, MBP+S, and MBP decreased (p<0.05) by 2.15,

2.17, and 3.27 log (CFU/g), respectively (Figure 2A).

Meanwhile, L. plantarum S20 did survive poorly during

storage at a much higher temperature (Figure 2B).

Ambient storage for 60 d resulted in >4 log CFU/g

decrease in viability of L. plantarum S20. Results also

show that for both storage temperatures, the addition of

either 5% sucrose or 5% trehalose did improve survival

of L. plantarum S20 during storage with lower

population reduction as compared to drying medium

containing ground mung bean only. No difference

(p>0.05) in viability was observed in L. plantarum S20

population in MBP+T and MBP+S during ambient and

chilled storage indicating that both cryoprotectants have

similar protective effects on L. plantarum S20 survival at

post-freeze-drying conditions. In addition, MBP exhibit

lowest (p<0.05) survival after 180 d (cell reduction 3.78

log CFU/g) and 60 days (5.48 log CFU/g) storage at

chilled and ambient storage, respectively. It appears that

storage of freeze-dried cultures with mung bean powder

and cryoprotectants at 6±2°C for 60 and 120 d manage to

preserve L. plantarum S20 survival >8.0 Log (CFU/g)

with only <0.70 and <1.70 log (CFU/g) reduction in cell

population, respectively.

The drastic decline in L. plantarum S20 population

during chilled and ambient storage could be attributed to

relatively high water activities of the freeze-dried

samples. Table 1 shows the water activity values of

freeze-dried L. plantarum S20 in ground mung bean

using different cryoprotectants before and after 180 and

60 d of storage at 6±2°C and 30±1°C, respectively. It is

also shown that water activities slightly increase

(p<0.05) after storage at 6±2°C and 30±1°C. Generally,

dehydrated microorganisms survive better at much lower

water activity. Stability of dried LAB upon storage is

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Figure 2. Population of lyophilized L. plantarum S20 culture in various freeze-drying media during storage at 6±2°C for 180

days (A) and at 30±1°C for 30 (B). Data presented as mean Log (CFU/g) from 6 independent samples. Drying medium

treatments: Open diamond = MBP+T (mung bean powder with 5% trehalose); Open circle= MBP+S (mung bean powder with

5% sucrose); Closed triangle with dash line = MBP (mung bean powder only).

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shown to be optimal at 0.1 to 0.2 aw (Champagne et al.,

1996). In the current study, the slightly higher water

activity of the dried cultures may have largely affected

the viability of the L. plantarum cells during storage.

Previous studies indicated that lipid oxidation of the

membrane fatty acid was predominantly responsible for

cell death during storage (Teixeira et al., 1996;

Kurtmann et al., 2009). Thus, decreasing water activity

and oxygen in the packaging have shown to stabilize

freeze-dried cultures (Kurtmann et al., 2009). In

addition, the temperature can also influence the rate and

magnitude of lipid oxidation. Current work observed

higher mortality in L. plantarum population during

storage at ambient temperature. A number of studies

have shown that storage of freeze-dried cultures at higher

temperatures resulted in a higher loss in viability a

shorter shelf life. For instance, storage of vacuum-

packaged freeze-dried P. aglomerans stored at 4°C for

90 d showed no more than 0.5 log viability loss, while

those that were stored at 25°C resulted in 3 log decrease

in viability after 28 d (Costa et al., 2002). Moreover,

previous work of Champagne et al. (1996) showed that

viability loss of a freeze-dried culture containing

Bifidobacrium longum at 20°C were 100x greater than

those that were stored at 4°C. Similar observation was

reported by Dhewa et al. (2014) where a considerable

reduction in viability of L. plantarum was observed in

freeze-dried symbiotic formulation stored at 25°C for 90

d while those that were stored at 4°C exhibit higher L.

plantarum survival. Selmer-Olsen et al. (1999)

recommended 0-5°C storage temperature for freeze-dried

cultures because low temperatures maintain lower

metabolic rate, thus increases storage stability.

3.3 Fermentation characteristics of freeze-dried

Lactobacillus plantarum S20

Changes in lactic acid bacteria (LAB) population,

pH, and total soluble sugar (TSS) of mung bean slurry

during 32 hrs of aerobic fermentation at 37°C are

presented in Figure 3. Results show that LAB population

changes in mung bean slurry with fresh and freeze-dried

starter culture showed a similar trend (Figure 3A). It

appears that LAB in both treatments entered exponential

growth phase any time between 0 and 4th hr of

fermentation due to the added L. plantarum S20

inoculum.  Exponential growth phase for mung bean

slurry with freeze-dried L. plantarum S20 extended up to

8 hrs of fermentation where about 2.0 log cycle increases

in LAB population was observed, from an initial LAB

population of approx. 7.0 log (CFU/mL) to 9.30 log

(CFU/mL). It is evident that the maximum LAB

population in mung bean with freeze-dried culture was

reached faster compared to mung bean slurry with fresh

culture. The LAB population in mung bean slurry with

freeze-dried culture peaked after 8 hrs with cell

population of 9.30 log (CFU/mL), while LAB in mung

bean with fresh culture of L. plantarum S20 peaked after

16 and 18 hrs with cell population 9.07 and 9.12 log

(CFU/g). The significant increases in LAB population in

both treatments caused a significant drop in mung bean

slurry pH and TSS. Highest pH drop (from pH 6.78 to

pH 3.82) was observed after 8 hrs of fermentation in

mung bean slurry inoculated with freeze-dried starter,

while pH drop in treatment with fresh starter culture was

slower from pH 6.81 to pH 3.72, which extended up to

16 and 24 hrs. It is observed that TSS in mung bean

slurry with either fresh or freeze-dried L. plantarum S20

starter decreased (p<0.05) exponentially over time from

0 to 16 hrs and leveled off until the end of fermentation

(Figure 3C).

Higher (p<0.05) drop in TSS was observed in mung

bean slurry with a freeze-dried starter. The LAB growth

curve trend corresponding to pH and TSS drop in mung

bean slurry with L. plantarum S20 indicates sugar

utilization of L. plantarum inoculum added in the mung

bean slurry. This trend was not observed in the control

where natural fermentation was used. The LAB

population present in the natural microflora of mung

bean was only observed after 4 hrs of fermentation and

exponential growth phase was extended up to 16 hrs

where the maximum population of 7.15 log (CFU/mL)

was reached. This population level is significantly lower

than the maximum population achieved by mung bean

slurry with L. plantarum S20. This trend also

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Chilled storage (6±2°C) Ambient storage (30±1°C)
Drying medium

0 days 180 days 0 days 60 days

Mung bean with 5% (w/v) trehalose 0.409±0.013aX 0.458±0.008aY 0.413±0.238aX 0.438±0.253aY

Mung bean with 5% (w/v) sucrose 0.396±0.010aX 0.461±0.004aY 0.401±0.231aX 0.421±0.243aY

Mung bean 0.429±0.009aX 0.472±0.006aY 0.412±0.238aX 0.441±0.255aY

Table 1. Water activity of freeze-dried cultures throughout storage at 6±2°C for 180 days and at 30±1°C for 60 days.

Data presented as mean ± standard error of the mean from 3 independent samples (n=3).
abValues within the same column not sharing the same superscript are different (p<0.05) analyzed using Tukey’s HSD post hoc

test.
XYValues within the same row and within the same storage temperature not sharing the same superscript are different (p<0.05)

analyzed using Tukey’s HSD post hoc test.

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corresponds to higher minimum pH achieved by mung

bean slurry without starter culture as compared to mung

bean inoculated with fresh or freeze-dried L. plantarum

S20 starter. Interestingly, unlike the other treatments,

TSS gradually increased in mung bean slurry without a

starter culture. This can be attributed to biological

activities by some LAB species naturally present in

mung bean that survived pasteurization. For instance,

heat-resistant, spore-forming, and lactic acid-producing

Bacillus spp. (e.g., Bacillus subtilis) was reported to be

the most predominant fermenting species in soybean and

mung bean (Shrestha et al., 2013; John and Olusegun,

2016). Bacillus species can produce a variety of enzymes

capable of hydrolyzing macromolecules like starch into

soluble sugars like oligosaccharides and simple sugars

(Neway, 1989; Shrestha et al., 2013), and this may have

contributed to the increasing trend in TSS observed in

the current study. This additional step could have slowed

down the conversion of soluble sugars (sucrose, glucose,

etc.) into lactic acid, hence resulted in a much slower pH

drop.

Previous work by Wu et al. (2015) on L. plantarum

fermentation of mung bean milk showed slightly

different results due to different mung bean preparation,

sucrose concentration, and L. plantarum strain used.

They reported that L. plantarum B1-6 fermentation mung

bean beverage (without okara) supplemented with 5.55%

sucrose and 5.19% inoculum concentration reached

optimum acidity and highest LAB population of 8.96 log

(CFU/mL) after 4.86 h of fermentation at 35.4°C.

Previous studies have demonstrated that mung bean

could be used as a fermentation substrate for L.

plantarum. L. plantarum has been found to dominate

early stages of a 4-stage of fermentation of mung bean as

a base material for lu-doh-huang (Taiwanese herbal

medicine) (Chao et al., 2013). In addition, a number of

studies have documented the use of L. plantarum as a

starter culture for mung bean-based fermentations (Wu et

al., 2015; Landete et al., 2015; Gan et al., 2017; Świeca

et al., 2018). Current work showed that the use of freeze-

dried L. plantarum S20 starter was able to rapidly

decrease pH of fermenting mung bean below pH 4.0

corresponding to maximum population of 9.3 log after 8

hrs. This indicates that freeze-dried L. plantarum S20 has

successfully started the fermentation process. Previous

studies documented the ability of rapid acidification of

freeze-dried L. plantarum starter for cassava

fermentation has been previously documented (Yao et

al., 2009). An important characteristic of potential starter

strains is their ability to acidify their environment

rapidly, which could inhibit the growth of contaminating

spoilage and pathogenic microorganisms (Smulders et

al., 1986). Leroy and De Vuyst (2004) reported that the

direct addition of selected starter culture to raw materials

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Figure 3. Changes in lactic acid bacteria population (A), pH (B), and total soluble sugars (TSS) (C) during 32 hrs mung bean

slurry fermentation at 37°C with lyophilized culture, fresh, and without culture. Data presented as means from 4 independent

samples. Error bars represent standard error of the mean (n=4).

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resulted in dominance by the best-adapted strains.

Furthermore, it is worth noting that freeze-dried L.

plantarum S20 starter culture developed in the current

study showed faster substrate acidification and LAB

growth compared to MRS-grown living cells. This

finding was inconsistent to previous reports, which

showed that freeze-drying could impair metabolic

activities of microorganisms upon rehydration (Meng et

al., 2008; Mahidsanan et al., 2017) and could decrease

growth and acidification activity (Fonseca et al., 2000;

Santivarangkna et al., 2007). In the current study,

sublethal stress due to freezing and freeze-drying process

may have a role in rapid acidification and growth

observed when freeze-dried L. plantarum S20 culture

was used to ferment mung bean slurry. Previous work on

the pre-inoculation treatment of Lactobacillus sakei

starter culture with sublethal stress (cold shock, heat

shock, salt shock) resulted in increased growth and acid

production during sausage fermentation (Hüfner and

Hertel, 2008). However, the extent of damage to

microbial cells exerted by sublethal stresses applied to L.

sakei is not as damaging compared to freeze-drying.

Since current work has limited scope, current findings on

this area warrant further study.

4. Conclusion

Results of the current study were able to demonstrate

that mung bean powder formulated with either trehalose

or sucrose could be used as a freeze-drying medium for

L. plantarum S20. Results indicate that drying medium

formulation and storage temperature greatly affected L.

plantarum S20 viability during freeze-drying and

subsequent storage. Supplementing of mung bean

powder with any of the two disaccharides at 5% (w/v)

has improved tolerance of L. plantarum S20 to freeze-

drying. In addition, no difference was observed between

sucrose and trehalose in terms of their cryoprotective

ability on L. plantarum S20 during freeze-drying and

subsequent storage. Low temperature storage at 6±2°C

performed better than ambient storage (30±1°C) in

minimizing viability loss of L. plantarum S20. However,

drying medium formulations used in the study was not

able to maintain the stability of L. plantarum S20

throughout storage. A significant decline of 2.0 to >4.0

log (CFU/g) in L. plantarum S20 population was

observed in all drying medium treatments during cold

and ambient storage. In future research, it will be useful

to optimize the formulation of freeze-drying medium

preparations, packaging conditions, and freeze-drying

parameters to achieve higher recovery and stability for L.

plantarum S20. In addition to mung powder and

disaccharides, the incorporation of additives must be

considered to further improve the shelf stability of freeze

-dried culture. When freeze-dried L. plantarum S20 was

used as a starter culture in mung bean slurry, it was able

to acidify the slurry from pH 6.8 to pH 3.8 after 8 hrs

with maximum LAB population of 9.2 log (CFU/mL).

Acidification of mung bean slurry by fresh culture (non-

freeze-dried) on the other hand appeared to be slower

than the freeze-dried culture. This observation

contradicts previous findings. While freeze-dried L.

plantarum S20 culture developed in the current study

could be used as a starter culture for mung bean-based

fermentations, its ability to perform better than non-

freeze-dried culture by rapidly growing and acidifying

mung bean slurry warrant further research.

Conflict of Interest

Authors declare no conflict of interest.

Acknowledgments

The University of the Philippines Los Baños Office

of the Vice-Chancellor for Research and Extension

funded this study. Special thanks to Dr. Francisco

Elegado of the UPLB National Institute of Molecular

Biology and Biotechnology for providing us with pure L.

plantarum S20 culture obtained from their previous

research.

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