Advances in Microbiology, 2020, 10, 39-51

https://www.scirp.org/journal/aim
ISSN Online: 2165-3410
ISSN Print: 2165-3402

Edible Growth Medium: A New Window for

Probiotic Research

Md. Nur Hossain1*, Annana Akter2, Sanjida Humayan1, Liton Chandra Mohanto1,
Shamima Begum2, Monzur Morshed Ahmed1
1
Institute of Food Science and Technology, Bangladesh Council of Scientific and Industrial Research,
Dhaka, Bangladesh
2
Department of Microbiology, Jagannath University, Dhaka, Bangladesh

How to cite this paper: Hossain, M.N., Abstract

Akter, A., Humayan, S., Mohanto, L.C.,
Begum, S. and Ahmed, M.M. (2020) Edible Conventional growth media for microbes contains lots of non-edible compo-
Growth Medium: A New Window for Pro- nents that are harmful to consume when the organism is ready to intake—like
biotic Research. Advances in Microbiology,
probiotics growing in chemical media (Lactobacillus grown in MRS media).
10, 39-51.
https://doi.org/10.4236/aim.2020.102004
The study was conducted to develop an edible and low-cost growth media
that supports the growth of probiotics Lactic Acid bacteria for the enhance-
Received: December 25, 2019 ment of probiotic research. The 04 isolates of Lactobacillus (L. plantarum, L.
Accepted: February 2, 2020
rhamnosus, L. ferciminis and L. bifarmentans) and 02 Bifidobacterium (B.
Published: February 5, 2020
infantis and B. Bifidum) were used for the evaluation of medium efficacy. To
Copyright © 2020 by author(s) and formulate the edible culture media, 04 vegetables and 02 pulses were used.
Scientific Research Publishing Inc. The media was formulated in different formulations. For Lactobacillus, maxi-
This work is licensed under the Creative
mum growth was observed at MM-2 media formulation that was about 15.62
Commons Attribution International
License (CC BY 4.0). log CFU/ml for L. plantarum and compared to MRS media that was 11.78 log
http://creativecommons.org/licenses/by/4.0/ CFU/ml. Bifidobaterium showed the highest viability at MW-1 edible media
Open Access formulation which was about 12.72 log CFU/ml whereas in Bifidobacteria se-
lective media the cell viability was 12.48 log CFU/ml. The edible media has no
toxic or unhealthy effect on mice while trailing in an animal model and shows
excellent results in encapsulation with alginate. In comparison with the per-
formance of traditional chemical media, the formulated media were found to
be cost-effective and safe for human consumption.

Keywords
Edible Media, Lactobacillus, Probiotic, Low Cost, Mass Culture

1. Introduction
A growth medium or culture medium is a solid, liquid or semi-solid designed to

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M. N. Hossain et al.

support the growth of microorganisms [1]. Culture media contains all the ele-
ments that most bacteria require for growth and are not selective, so they are
used for the general cultivation and maintenance of bacteria kept in laboratory
culture collections [2]. An undefined medium (also known as a basal or complex
medium) contains a carbon source such as glucose, various salts, a source of
amino acids and nitrogen like beef and yeast extract [3] [4].
Growth media provides minimized cell damage, suitable for screening and
maximum viability of cells. The cost of media is also an important consideration.
Commercially available media are not only costly but also non-consumable [5].
Peptones are the most widely used source of nitrogen in microbial media [6].
Some are made by cooking milk or meat products (beef, pork) in acid, but most
are made by incubating milk or meat with trypsin, pepsin, or other proteolytic
enzymes to digest the protein to a mixture of amino acids, peptides, and poly-
peptides [7] [8] [9]. On the other hand, these microbial growth media are highly
demanded in developing microbiological research. The most important applica-
tion of these media is in the production of commercially available fermented
food products, especially dairy products [10] [11]. Before starting the fermenta-
tion of food products, the required microbial cells are subjected to a pre-culture
cultivation [12]. Then the organisms are separated from media and introduced
into fermentation. For this reason, the constituents of the media are very impor-
tant as, if it contains any component that is harmful to human, it may come
across the fermented food through the starter culture. Lactobacillus are often
used as probiotics, in dairy food products that are grown in mainly De Man Ro-
gosa and Sharpe Agar (MRS agar) which contains manganese sulfate and mag-
nesium sulfate. These components can cause irritation in skin, respiratory sys-
tem and in long term repeat it may cause organ damage [13].
Probiotics are defined as live microorganisms that provide health benefits
when consumed [14]. Probiotics have to be alive when administered. These mi-
crobes are thought to help restore the natural balance of bacteria in the gut (in-
cluding stomach and intestines) when it has been disrupted by an illness or
treatment. But, these probiotics are not easy to produce on a large scale [1] [15].
It needs several steps in recovery stages such as centrifugation, filtration, formu-
lation, drying etc. which increase the production cost. Moreover, the viability of
bacteria is often reduced during these recovery steps [16] [17]. So, it was a little
attempt to prevent the loss of cell count.
Meanwhile, the numbers of viable micro-organisms are recommended for the
aptitude of probiotic foods and for this reason, the challenges of maintaining
viability and activity of probiotic cultures in foods to the end of shelf-life are two
important criteria that must be fulfilled to provide effective probiotic food
products for general consumption [18]. Dried concentrated probiotic cultures
are the most suitable form for inoculation into functional foods [19], given the
ease of storage, handling and transport, especially for shelf-stable functional
products. The challenges associated with the introduction and maintenance of

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 M. N. Hossain et al.

high numbers of viable probiotic cultures into foods include the form of the
probiotic inoculum used, process conditions, reconstitution conditions, ability
of the probiotic culture to grow and retain viability in the food environment and
maintenance of probiotic characteristics in the food product through to the time
of consumption [20] [21].
All these problems can be reduced if the probiotics are grown in human con-
sumable media [22]. This study was about to develop an edible media that can
support the growth of both probiotics and other non-fastidious bacteria at a low
cost. Probiotics, grown in this media, do not need to go through that conven-
tional method of recovery. Instead of these, viable cells with edible broth can be
directly inoculated into raw material. Moreover, this media has been formulated
with inexpensive vegetables as the source of carbohydrates and minerals and
pulses as the source of nitrogen [23].
Pulse 1 and pulse 2 are the very good source of protein (nitrogen source) and
carbohydrate (carbon source). Pluse 1 contains 32.98% carbohydrate and 33.33%
protein in dry conditions. Pulse 2 is a good source of protein, but it is also a
great source of carbohydrates [24]. It has 68.8% carbohydrate and 9.82% protein
in a dry state. These nitrogen and carbon sources were replaced with peptone,
beef extracts and glucose in chemically defined media [6].
Vegetables are a good source of minerals and vitamins need to grow bacteria.
These vegetables contain mostly protein, carbohydrate, vitamins (vitamin C,
thiamin, riboflavin, niacin, vitamin B6, folate) as well as minerals such as so-
dium, potassium, magnesium, phosphorus etc. [25]. A high content of potas-
sium present in veg 2 & veg 3. Veg 1 and veg 4 provide a large amount of phos-
phorus. Veg 2 and veg 3 are a great source of sodium and so on. All these ingre-
dients are available at the local fresh market.

2. Materials and Method

2.1. Materials
The microorganisms of Lactobacillus (L. plantarum, L. rhamnosus, L. ferciminis
and L. bifarmentans) and Bifidobacterium (B. infantis and B. Bifidum) were
taken from Industrial Microbiology Laboratory, Institute of Food Science and
Technology, Bangladesh council of Scientific and Industrial Research, Dhaka.
All the raw materials were collected from the local market. The dehydrated cul-
ture media and other chemicals were purchased from Himedia (India), Merck
(Germany) and Sigma (USA). The experiments were conducted in Industrial
Microbiology Laboratory, IFST, BCSIR, Dhaka from Jan/2018 to Dec/2018 re-
peatedly. The animal test was performed in animal house research section,
IFST.

2.2. Methods
2.2.1. Raw Material Processing
Fresh four vegetables were sliced and blanched at 85˚C for 15 minutes. The veg-

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M. N. Hossain et al.

etables and pulses were then dried at 72˚C using a hot air oven. All these ingre-
dients blended with electric blender separately and sieved to get a fine powder
with diameter of 180 - 330 µm.

2.2.2. Formulation of the Base Medium for Mass Culture

Respective amount of vegetable and pulses powder was mixed thoroughly to de-
velop a basal medium in comparison to synthetic commercial media composi-
tions. There are two types of formulations 1) with minerals (MM) and 2) with-
out minerals (MW) and the ingredients were added in different proportions to
formulate the edible medium.
The process is given below in a flow chart.

Base media powder + water

↓
Boiled at 100˚C for 25 - 30 mins
↓
Homogenized for 5 mins
↓
Filtrated
↓
Add mineral salts (optional)
↓
Mixed by homogenization and autoclave at 121˚C at 15 psi for 15 mins

Sodium acetate, dipotassium hydrogen phosphate, ammonium citrate miner-

als were used for media formulation. Sodium Acetate and Ammonium Citrate
inhibits Streptococci, molds, and other oral microbial flora and restrict swarm-
ing [26]. Dipotassium Hydrogen Phosphate is the buffering agent.

2.2.3. Inoculation of Probiotic Bacteria into Media for Cell Count

All the freshly cultured isolates were inoculated into McCartney bottle with dif-
ferent edible broth formulations and incubated in the anaerobic jar (Oxiod) at
37˚C for 24 hrs. MRS broth for Lactobacillus and Hichrome Bifidobacterium
broth for Bifidobacteria were used as a control. Each of the cultured media (1
ml) with probiotic bacteria was transferred to 9 ml of sterile Ringer’s solution
(solution of several salts dissolved in water for creating of isotonic solution) fol-
lowed by serial dilution, plated and incubated at 37˚C in anaerobic condition for
48 hrs. Then cells were counted and compared with edible broth and commer-
cial culture broth. The process was repeated for triplicate.

2.2.4. Measuring of Optical Density

All cultural broth after 24 hrs incubation was taken for the measurement of OD
by spectrophotometer at 600 nm wavelength.

2.2.5. Mass Culture

MW (medium without minerals) and MM (medium with minerals) formulated

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 M. N. Hossain et al.

media were prepared according to the composition as previously described. 1%

fresh culture of isolates inoculated into 1 L of edible broth and incubated at 37˚C
for 24 hrs anaerobically.

2.2.6. Determining the Growth Curve of Lactobacillus

The growth curve of L. plantarum was observed to determine the log, lag and the
stationary phase of the isolate. The overnight incubated fresh culture was inocu-
lated into MRS broth at 37˚C anerobacally. The cell count was performed every
hour with ringer’s solution as a diluent and plated onto MRS agar. The plates
were then incubated at 37˚C under the anaerobic condition for 24 hrs and then
the number of colonies was counted. The graph was plotted cell count against
time to observe the growth pattern.

2.2.7. Freeze Drying

The cultured isolates along with 7% skim milk and 0.3% sodium alginate were
added and the suspension was kept in −20˚C overnight. The freeze-drying
process was started to obtain a dry powder containing the probiotic bacteria us-
ing Alpha 1 - 2 LD Plus (Germany) freeze dryer. The bacterial cell was enume-
rated before and after the freeze-drying process and expressed as CFU/ml.

2.3. Animal Trials

Experimental protocols for animal trail approved by BCSIR institutional ethical
review committee and followed while performing the research with mice. Eight
healthy swiss albino male mice of 6 - 7 weeks of age were taken. They were di-
vided into 2 groups:
• A control group that contains 3 mice.
• A sample group that contains 5 mice.
The mice were kept at 25˚C temperature.

2.3.1. Feeding
Each mouse was served 20 gm of regular feed. The control group was served on-
ly regular feed (total 60 gm). The sample group was served (total 100 gm) regu-
lar feed mixed with 100 mg sample powder to each mice (total 500 mg). Each
gram of sample feed contained at least 8 log CFU of bacterial culture. This expe-
riment was carried out for 10 days straight. During this trial, all mice were ob-
served carefully by their locomotor behavior and physical parameters- stool,
urine, water consumption, body temperature.

2.3.2. Bodyweight Measurement

Body weights of all these mice were taken before starting feeding. After 10 days
trial, again their body weights were measured.

3. Results and Discussion

The nutritional value of dried raw materials was calculated from the nutritional
value of these ingredients according to USDA National Nutrient Data [25] in

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M. N. Hossain et al.

Table 1.

3.1. Viability of Lactobacillus Isolates in Edible Media

3.1.1. In the Case of Different Media Formulation
The isolates were cultured triplicate in MRS media and MM-1, MM-2, MM-3,
MM-4 formulations. All four isolates showed more growth in edible media
compared with MRS media selective for Lactobacillus. Among them, the viabili-
ty of the isolates was most high in MM-2 media formulation. Then the viability
has decreased linearly through the formulations of nutrients were increased.
This was most likely for the reason of increased osmotic stress exacted by the
carbon and nitrogen source of the media [27] [28]. All the isolates were not
grown equally in edible media. L. plantarum and L. bifermentum were showed a
high viability count. Among them, L. rhamnosus showed a slow growth and the
least viability in edible media (Figure 1).

3.1.2. In the Case of Comparison of Growth

MRS medium was designed to favor the luxuriant growth selectively for Lacto-
bacilli for lab study. Though the isolates grow at MM-2 formulation of edible
media, the viability was compared with standard culture maintained in MRS
broth [2] [29]. The results showed that edible media has a better ability to grow
the isolates than in MRS media. Log numbers were estimated and it was observed

Table 1. An estimation of the nutritional value of the ingredient used in this experiment.

Pulse 1 Pulse 2 Veg 1 Veg 2 Veg 3 Veg 4

Raw materials/Ingredients
(100 gm dw) (100 gm dw) (100 gm dw) (100 gm dw) (100 gm dw) (100 gm dw)

Carbohydrate, gm 32.98 68.8 81.26 79.16 62.12 72.5

Protein, gm 36.49 27.25 12.5 7.76 24.0 16.0

Sodium, mg 2.18 6.54 12.5 575 375.0 225.0

Potassium, mg 1964.78 738.28 4250.0 2666.6 3737.6 2125.0

Magnesium, mg 306.14 51.26 150.0 100.0 187.6 150.0

Phosphorus, mg 769.74 306.44 550.0 291.64 550.0 325.0

Figure 1. The log number of Lactobacillus in different media formulations.

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that all the four Lactobacillus have more growth in edible media than that of tra-
ditional MRS media. In the edible media, L. plantarum exihited the highest
growth followed by L. ferciminis. The other two isolates showed not so different.
Pathak and Singhal [30], replaced beef extracts with germinated lentil seed
powder in MRS media. The microbial growth of L. lactis, L. casei, L. plantarum had
a positive effect on the in modified MRS media as compared to MRS [29]. The av-
erage growth of Lactobacillus in MM-2 edible media was more than 15 log CFU/ml
where the average cell density in MRS media was 12.44 log CFU/ml (Figure 1).

3.2. Viability of Bifidobacterium Isolates in Edible Media

3.2.1. In the Case of Different Media Formulation
The isolates were cultured repeatedly in bifidobacterium selective broth and agar
compared with formulated ediblemedium MW-1, MW-2, MW-3, MW-4. The
two Bifidobacterium isolates showed more growth in edible media compared
with Bifidobacterium selective medium selective for Bifidobacterium. Among
them, the highest growth of the isolates was shown in MW-1 media formulation
and with the increment of media ingredients the culture density was decreased
followed by MW-4 (Figure 2).

3.2.2. In the Case of Comparison of Growth

Bifidobacterium selective medium was designed to favor the growth of Bifido-
bacteria for the lab study, though the isolates grew at MW-1 formulation of edi-
ble media at its highest. The viability was compared with standard culture main-
tained in Bifidobacterium selective media and the results showed that edible
media has a better ability to grow the isolates than the commercial one. Log
numbers of bacteria were estimated and observed that all the two isolates grew
well with small count difference. The B. infantis isolate showed a little increased
log number (12.56) in edible media than the log number (12.32) in bifidobacte-
rium selective media. Pathak and Martirosyan [5] worked on Bifidobacterium
strain 231, Bifidobacterium strain 234. They used their modified MRS media
for the culture of these two bacteria. In their study, they found that growth was
better in MRS media than in modified MRS media. The average growth of Bifi-
dobacterium in MW-1 edible media was 12.60 log CFU/ml where the viability

Figure 2. The log number of Bifidobacteria in different media formulation.

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M. N. Hossain et al.

in bifidobacteria media was 12.48 log CFU/ml (Figure 2). Arulanantham et al.
[6] carried out a study where they used cowpea, green gram, black gram and
soya meat (processed soya bean) in different formulations for the growth of E.
coli, Bacillus sp., Klebsiella sp., Staphyllococcus sp. and Pseudomonas sp. They
found that Staphyllococcus sp. grew well in all the protein formulations and
Klebsiella sp. grew least in the protein formulations tested. They concluded that
soya meat agar (soya meat + agar) was an effective alternative culture media
source next to nutrient agar to grow bacteria.

3.3. Measuring of OD for Lactobacillus and Bifidobacterium

Optical density (OD) or absorbance of a sample measured for estimating the
bacterial cells density in a broth midium. As visible light passes through a cell
suspension the light is scattered. The higher scatter indicates that more bacteria
or other material is present. The amount of light scatter can be measured in a
spectrophotometer. However, the absorbance of every isolate containing edible
broth was recorded at 600 nm wavelength as it was considered as standard be-
cause at this wavelength maximum absorption was observed. A High OD was
observed for Lactobacillus isolates at MM-2 media formulation than any other
media formulation indicating that at this media formulation bacterial cell was
most viable (Figure 3). Bifidobacterium showed the highest absorbance at MW-1
edible media formulation (Figure 4). Then the cell formulation has decreased

Figure 3. Optical density of Lactobacillus isolates in different media formulation.

Figure 4. Optical density of Bifidobacterium isolates in different media formulations.

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 M. N. Hossain et al.

with increasing media formulations. The specific growth rates of all four isolates
of Bifidobacterium decreased linearly as the concentration of dissolved solids
increased in the medium. The higher sugar concentration in the medium proba-
bly exerts severe osmotic stress on the bacterial cells [4] [20].

3.4. Growth Curve of Lactobacillus

The performed test evaluated the growth dynamics of the isolate over a period of
32 hours. A better understanding of the growth patterns gives the idea of opti-
mum timing for the maximum growth of cells when performing other tests on the
isolate. From the results, it can be determined that between the hours of 0 - 8 was
the lag phase. From hours 8 - 16 can be said to be the log phase where there is an
exponential growth of the cells. From 20 - 32 hours the cells are in the stationary
phase there is no significant net increase or decrease in cell number (Figure 5).

3.5. Survival of Lactobacillus before & after Freeze Drying

Freeze drying is a common method to incorporate probiotics into foods. How-
ever, during the processing, it affects the viability of cells. So, freeze-drying or-
ganisms are protected by adding cryoprotectants [20] [31]. In this experiment
skim milk and sodium alginate was used as cryoprotectants and the loss of
cells after freeze-drying was estimated. Cells were counted at 10−11 dilution in
both situations. Before drying the count was 14.15 log and after drying it was
decreased to 13.08 log count. Only 1 log of cell reduction was observed (Figure
6).

Figure 5. Growth curve of L. plantarum.

Figure 6. Survival of Lactobacillus in edible media before and after freeze-drying.

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M. N. Hossain et al.

3.6. Field Trials

At the end of 10 days trial, the mice were weighed and compared with initial
weight. During the trial, mice were kept in observation. It was seen that beha-
vior, movements, body temperature, water intake, urination and stool of sample
group members were as normal as control group members. The results showed
that a higher rate of weight has gained by sample group than that of the control
group (Table 2). The average weight gain in the control group was 5.67 gm
where the average weight increased in the sample group was 13.20 gm. These
results showed that the edible media with Lactobacillus culture in it does not
contain any toxic or unhealthy compound that is harmful to health. It is clearly
seen that the edible media is highly rich in nutrients that boosted the growth of
sample group mice.

3.7. Costing of Edible Media

Different type’s vegetables and grains were used to formulate the Edible media
which is way cheaper than the commercially available media like MRS, M17, NA
etc. which are continuously used for isolation, identification, enumeration of
microorganisms in the laboratory for routine work. The Edible media was fabri-
cated to replace these commercial media for daily experiments, which reduce the
disbursement a lot (Table 3).
No expensive chemical was used only available and low-priced vegetables
were used economical comparison showed this media is extremely economic

Table 2. Weight of the mice before & after feeding edible media with Lactobacillus.

Initial weight (gm) Final weight (gm) Average weight increased

Control group R1 23.8 27.9 4.934 gm

R2 21.9 28.1

R3 21.8 26.3

Sample group R1 19.8 33.8 12.50 gm

R2 21.5 34.1

R3 22.6 33.2

R4 22.8 34.7

R5 19.1 32.5

Table 3. Cost comparison of the edible media and other commercial media.

Name of the media (Manufacturer) Cost in BDT/500g

Bifidobacterium selective medium broth 23,000.00

MRS broth 9500.00

M17 broth 17,500.00

Nutrient broth 6500.00

Edible medium broth 1200.00

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moreover industrial use of this media is also possible.

4. Conclusion
Edible growth media are those that are used to grow probiotics and can be con-
sumed by a human with no harmful effects. Nowadays, only lactic acid bacteria
especially Lactobacillus and Bifidobacteria are used as probiotics. These study
results conducted that it is possible to formulate an edible media that can be
used commercially in food processing industries for the production of probio-
tics. For example, the starter culture of Lactobacillus for yogurt should contain 6
- 7 log CFU/ml cells. But this edible media showed 11.3 - 13.26 log CFU/ml cells.
It is clear that this media is more efficient in cell growth than conventional me-
dia. Industry requires that the organism grows faster, so that production time is
reduced which is economical. Similarly, low-cost media is always pursuing to
bring down production costs. Though it has formulated with the ingredients that
are available at the cheap local market and are very reasonable in price (which
are rich in nutrients required for microbes), this media can be a good alternative
of conventional chemically defined media. Moreover, this media contains lots of
vitamins, minerals and amino acids as it formulated with grains and vegetables.
It can be added extra nutrition in food. Because of being so nutritive, a high rate
of weight gain was observed in field trials. Another positive side of this media
is—it supported the growth of not only Lactobacillus and Bifidobacteria but also
gram-positive and gram-negative bacteria as comparing to conventional media.
So this media can be used rather than nutrient media for laboratory purposes. It
is recommended that more research curriculum should have carried out in the
future by using other possible ingredients. Moreover, other bacteria use in food
industries and also both fastidious, non-fastidious microbes for laboratory pur-
poses should be included in further research.

Ethical Approval and Consent to Participate

Experimental protocols for animal trail approved by BCSIR institutional ethical
review committee and followed while performing the research with mice.

Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this pa-
per.

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