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A modified reinforced clostridial medium for the isolation and enumeration of

Lactobacillus delbrueckii ssp. bulgaricus in a mixed culture

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DOI: 10.3168/jds.2019-17894

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 J. Dairy Sci. 103:5030–5042
https://doi.org/10.3168/jds.2019-17894
© American Dairy Science Association®, 2020.

A modified reinforced clostridial medium for the isolation and enumeration

of Lactobacillus delbrueckii ssp. bulgaricus in a mixed culture
Ayowole Oyeniran,1 Salam A. Ibrahim,1* Rabin Gyawali,1 Reza Tahergorabi,1 Tahl Zimmerman,1
and Albert Krastanov2
1
Department of Food and Nutritional Sciences, North Carolina Agricultural and Technical State University, Greensboro 27411
2
Department of Biotechnology, University of Food Technologies, 4002 Plovdiv, Bulgaria

ABSTRACT Key words: Lactobacillus bulgaricus, isolation, mixed

culture, selective media, bacterial enumeration
In this study, we modified reinforced clostridial
medium (RCM) to selectively enumerate and isolate
INTRODUCTION
Lactobacillus delbrueckii ssp. bulgaricus, a probiotic
and important starter culture in the dairy industry. Lactobacillus delbrueckii ssp. bulgaricus is an impor-
The disparity in the reported carbohydrate fermenta- tant and highly regarded species of lactic acid bacteria
tion pattern of L. delbrueckii ssp. bulgaricus was used due to its application in the production of several dairy
to develop a growth medium not only selective for L. products including yogurt, a fermented milk product
delbrueckii ssp. bulgaricus but significantly inhibitory that is well known for its health benefits (Zhu et al.,
to the growth of other lactic acid bacteria. A recently 2010). Yogurt starter cultures require a symbiotic blend
modified RCM (mRCM) was optimized for this study of L. delbrueckii ssp. bulgaricus and Streptococcus ther-
by the addition of 0.5% fructose, 0.5% dextrose, 1% mophilus in approximately equal amounts to obtain
maltose, and 0.25% sodium pyruvate while replacing the desired organoleptic properties that result from
lactose as a carbohydrate source. The cell recovery and the metabolic activity of both microorganisms during
bacterial counts of L. delbrueckii ssp. bulgaricus in test- their growth in milk. Consequently, the final quality of
ed products (pure L. delbrueckii ssp. bulgaricus strains, yogurt can suffer with regard to texture, acidity, and
starter culture, probiotic supplements, and yogurt) us- flavor if this proper bacterial balance is not achieved
ing our mRCM with sodium pyruvate (mRCM-PYR) (Lourens-Hattingh and Viljoen, 2001).
were significantly higher than in the recently modified The probiotic potential of L. delbrueckii ssp. bulgari-
RCM and the common de Man, Rogosa, and Sharpe cus has been mentioned in several studies (Singh et al.,
(MRS) culture medium. The growth of other lactic 1979; Mahmood, et al., 2015; Pescuma et al., 2015), and
acid bacteria (Streptococcus thermophilus, Lactobacillus as with any probiotic, it confers various health benefits
acidophilus, Lactobacillus rhamnosus, and Lactobacillus to the host when administered at an effective dose.
reuteri) and Bifidobacteria was retarded in this modi- Consequently, in order for L. delbrueckii ssp. bulgaricus
fied medium compared with their growth in MRS and to play its role as a probiotic, the requisite bacterial
mRCM. This result is a significant improvement in the population levels are critical. To support the quality
enumeration and differentiation of L. delbrueckii ssp. of yogurt, many countries have adopted standards for
bulgaricus in mRCM-PYR compared with the results these viable bacteria numbers and the ratio of L. del-
in MRS and mRCM where the high background growth brueckii ssp. bulgaricus to Streptococcus thermophilus.
of similar species interferes with the accuracy of bacte- For example, the Codex Alimentarius, a collection of
rial population counts. Our results thus suggest that internationally recognized standards for food produc-
mRCM-PYR could be recommended as a reliable al- tion, and most other national regulatory agencies in the
ternative growth medium for the selective enumeration US have established a minimum level of 1 × 107 cfu/g of
and isolation of L. delbrueckii ssp. bulgaricus in a mixed yogurt starter microorganisms. Other countries such as
culture. France, Switzerland, Italy, and Spain have also agreed
to follow the Codex Alimentarius recommendation of a
minimum level of yogurt starter microorganisms at the
Received November 11, 2019.
time of consumption. However, in the United Kingdom,
Accepted February 9, 2020. a minimum requirement of 10 million viable lactobacilli
*Corresponding author: ibrah001@​ncat​.edu per mL of yogurt at the time of sale was established

5030
 Oyeniran et al.: MODIFIED REINFORCED CLOSTRIDIAL MEDIUM 5031

(Davis and McLachlan, 1974; Robinson and Tamime, for L. delbrueckii ssp. bulgaricus very challenging. Nev-
1976; Hamann and Marth, 1984). ertheless, preliminary studies have demonstrated that
The current standard medium, de Man, Rogosa, and mRCM can be optimized to improve the recovery of
Sharpe (MRS), is unreliable as a selective method stressed bacterial cells and select for a broader range
for enumerating L. delbrueckii ssp. bulgaricus species of L. delbrueckii ssp. bulgaricus strains. Therefore, the
in mixed bacterial cultures as true counts are often objective of this study was to examine the modification
imprecise due to the presence of high background colo- of reinforced clostridial medium (RCM) for the isola-
nies of similar species. Other media proposed for the tion and enumeration of L. delbrueckii ssp. bulgaricus
selective enumeration of specific lactic acid bacteria from a mixed culture.
(Matalon and Sandine, 1986; Ghoddusi and Robinson,
1996; Yamani and Ibrahim, 1996; Tabasco et al., 2007; MATERIALS AND METHODS
Galat et al., 2016; Hayek et al., 2019) also struggle
to selectively enhance the growth of L. delbrueckii ssp. Source of L. delbrueckii ssp. bulgaricus
bulgaricus in the presence of other lactic acid bacteria.
A modified reinforced clostridial medium (mRCM) by A total of 32 sources of L. delbrueckii ssp. bulgaricus
Nwamaioha and Ibrahim (2018) recently showed re- strains were used in the study (Table 1). Eleven strains
markable results in the differentiation and enumeration of freeze-dried L. delbrueckii ssp. bulgaricus cultures
of 2 strains of L. delbrueckii ssp. bulgaricus in a mixed intended for industrial production of fermented milk
bacterial culture by giving distinct and large colonies. products were supplied by Albert Krastanov (Depart-
This culture medium supports the growth of lactic acid ment of Biotechnology, University of Food Technolo-
bacteria other than L. delbrueckii ssp. bulgaricus but gies, Plovdiv, Bulgaria). Six yogurt starter cultures and
still makes the isolation and enumeration steps required 3 probiotic supplements from Europe were selected. All

Table 1. Description of samples used

Product
No. code Sample Source Bacterial composition as labeled
1 S28 Pure industrial strain Bulgaria Lactobacillus bulgaricus
2 S6 Pure industrial strain Bulgaria L. bulgaricus
3 S19 Pure industrial strain Bulgaria L. bulgaricus
4 S8 Pure industrial strain Bulgaria L. bulgaricus
5 LB6 Pure industrial strain Bulgaria L. bulgaricus
6 S1 Pure industrial strain Bulgaria L. bulgaricus
7 LB9 Pure industrial strain Bulgaria L. bulgaricus
8 S22 Pure industrial strain Bulgaria L. bulgaricus
9 S9 Pure industrial strain Bulgaria L. bulgaricus
10 S7 Pure industrial strain Bulgaria L. bulgaricus
11 S5 Pure industrial strain Bulgaria L. bulgaricus
12 ST11 Starter culture Bulgaria Streptococcus thermophilus, L. bulgaricus, Lactobacillus
rhamnosus
13 ST12 Starter culture Bulgaria S. thermophilus, L. bulgaricus
14 ST13 Starter culture Bulgaria S. thermophilus, L. bulgaricus
15 ST14 Starter culture Bulgaria S. thermophilus, L. bulgaricus
16 ST15 Starter culture Canada S. thermophilus, L. bulgaricus, Lactobacillus acidophilus
17 ST16 Starter culture Bulgaria S. thermophilus, L. bulgaricus
18 SP17 Probiotic supplement Bulgaria S. thermophilus, L. bulgaricus, Lactobacillus reuteri
19 SP18 Probiotic supplement Bulgaria S. thermophilus, L. bulgaricus
20 SP19 Probiotic supplement Bulgaria S. thermophilus, L. bulgaricus, L. rhamnosus
21 SP8 Yogurt Spain S. thermophilus, L. bulgaricus, Bifidobacterium, L. reuteri
22 SP9 Yogurt Spain Live and active cultures
23 SP10 Yogurt Spain Live and active cultures
24 E1A Yogurt The Netherlands S. thermophilus, L. bulgaricus, L. acidophilus, Bifidobacterium
25 E2Z Yogurt The Netherlands Live and active culture
26 E3D Yogurt Spain Live and active culture
27 E4P Yogurt Bulgaria Live and active culture
28 L6N Yogurt United States S. thermophilus, L. bulgaricus, Bifidobacterium lactis, L.
acidophilus, L. rhamnosus
29 BY2 Yogurt Bulgaria L. bulgaricus, other live culture
30 BY4 Yogurt Bulgaria L. bulgaricus, other live culture
31 BY5 Yogurt Bulgaria L. bulgaricus, other live culture
32 BY6 Yogurt Bulgaria L. bulgaricus, other live culture

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samples were maintained at −20°C until further use. In Enumeration of L. delbrueckii ssp. bulgaricus
addition, 12 yogurt samples were obtained from Europe in Pure Industrial Strains, Yogurt Starter
and stored under refrigeration until further use. Culture, and Probiotic Supplements

For the enumeration of L. delbrueckii ssp. bulgaricus

Chemicals and Reagents
in these products, 0.1 g of freeze-dried samples was
All chemicals and reagents used in this study were measured and transferred into 3 mL of skim milk with
purchased from Fisher Scientific (Pittsburgh, PA). the addition of 10 µL of Tween 80 and 0.1 mL of yeast
extract to enhance the recovery of stressed bacterial
cells. This mix was fermented at 44°C for 7 h, at which
Lactobacilli MRS Agar Medium
point the skim milk curdled. The curdled milk was
A lactobacilli MRS agar medium was prepared by shaken, and 1 mL was transferred for serial dilution
dissolving 55 g of MRS broth (Neogen Co., Lansing, into a 9 mL of peptone solution. Aliquots (100 µL) of
MI) and 0.5 g of l-cysteine in 1 L of deionized distilled appropriate dilutions were plated in duplicates onto the
water (DDW), and the resultant solution was stirred 3 culture media (MRS, mRCM, and optimized RCM)
well until all particles were completely dissolved. Agar for the enumeration of L. delbrueckii ssp. bulgaricus.
powder (15 g; Neogen) was added, and the agar me- The inoculated plates were incubated for 72 h at 44°C
dium was sterilized at 121°C for 15 min and then cooled in anaerobe chambers, and the colonies were counted
in a water bath. using a Quebec colony counter (Fisher Scientific).

Modified Reinforced Clostridial Medium Enumeration of L. delbrueckii ssp. bulgaricus

in Commercial Yogurt Samples
Modified RCM was prepared according to the meth-
od of Nwamaioha and Ibrahim (2018) by completely Dilutions of yogurt samples were prepared by adding
dissolving 10 g of proteose peptone #3, 10 g of beef 10 g of each sample to a screw-capped bottle containing
extract, 5 g of yeast extract, 10 g of lactose, 5 g of 90 mL of deionized water and then properly shaking
sodium chloride, 3 g of sodium acetate, 2 g of K2HPO4, the mixture. This mixture was placed in an incubator
0.1 g of uracil, 0.25 g of calcium chloride, 0.2% Tween at 44°C for 15 min to repair weak and damaged cells.
80, and 0.5 g of L-cysteine in 1 L of DDW. This solu- Subsequent dilutions of up to 10−7 were made in a 0.1%
tion was adjusted to a final pH of 6.0 ± 0.2 using 6 M peptone solution and vortexed well between transfers.
HCl before the addition of 0.01% aniline blue and 15 g Aliquots (100 µL) of appropriate dilutions were then
of agar. This medium was then autoclaved at 121°C for plated in duplicate onto the 3 culture media (MRS,
15 min and cooled in a water bath. mRCM, and optimized RCM) for the enumeration of

Modified Reinforced Clostridial Medium-Pyruvate

Table 2. Composition of modified reinforced clostridial medium
A reinforced clostridial medium was optimized for (mRCM) and modified reinforced clostridial medium-pyruvate
selectivity and accurate enumeration of L. delbrueckii (mRCM-PYR)
ssp. bulgaricus by dissolving 10 g of peptone #3, 10 mRCM mRCM-PYR
g of beef extract, 5 g of yeast extract, 5 g of sodium
10 g of proteose peptone #3 10 g of proteose peptone #3
chloride, 3 g of sodium acetate, 2 g of K2HPO4, 0.1 g 10 g of beef extract 10 g of beef extract
of uracil, 0.25 g of calcium chloride, 5 g of dextrose, 5 5 g of yeast extract 5 g of yeast extract
g of fructose, 10 g of maltose, 2 g of sodium pyruvate, 5 g of sodium chloride 5 g of sodium chloride
0.25 g of calcium chloride 0.25 g of calcium chloride
0.2% Tween 80, and 0.5 g of l-cysteine in 1 L of DDW. 3 g of sodium acetate 3 g of sodium acetate
This solution was adjusted to a final pH of 6.0 ± 0.2 2 g of ammonium phosphate 2 g of ammonium phosphate
using 6 M HCl before the addition of 0.008% aniline dibasic dibasic
0.5 g of l-cysteine 0.5 g of l-cysteine
blue and 15 g of agar. The medium was autoclaved 0.1 g of uracil 0.1 g of uracil
at 121°C for 15 min and then cooled in a water bath. 0.01% aniline blue 0.008% aniline blue
All freshly prepared media in this study were poured 0.2% Tween 80 0.2% Tween 80
10 g of lactose1 5 g of dextrose1
into sterile Petri dishes and stored at 4°C until needed. 5 g of fructose1
The difference in the carbohydrate sources of mRCM 10 g of maltose1
and modified reinforced clostridial medium-pyruvate 2 g of sodium pyruvate1
(mRCM-PYR) is highlighted in Table 2. 1
Carbohydrate sources.

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L. delbrueckii ssp. bulgaricus. The inoculated plates RESULTS

were incubated for 72 h at 44°C in anaerobe (CO2/
N2) chambers (Labconco, Kansas City, MO), and the Preliminary Study
colonies were counted using a Quebec colony counter
In preliminary studies, the effect of different carbon
(Fisher Scientific).
sources on the morphology of L. delbrueckii ssp. bulgari-
cus colonies was evaluated. We observed that the use of
Enumeration of L. delbrueckii ssp. bulgaricus 2% sodium citrate and 1% sodium pyruvate as alterna-
in Traditional Bulgarian Yogurt tives to a proteose peptone diluent solution not only
produced more prominent and distinguishable colonies
To enumerate L. delbrueckii ssp. bulgaricus in tradi- in the previously modified RCM by Nwamaioha and
tional Bulgarian yogurt, 5 mL of samples was measured Ibrahim (2018) but also improved the bacterial count
and transferred into 45 mL of skim milk with 2 mL (data not shown). Our findings also showed that sodi-
of yeast extract (20%) added to repair damaged and um pyruvate retarded the growth of lactic acid bacteria
stressed bacterial cells. This mix was fermented at 44°C other than L. delbrueckii ssp. bulgaricus, which other-
for 7 h, at which point the skim milk curdled. The wise grew when 2% sodium citrate and 0.1% proteose
curdled milk was stirred, and 10 mL was sampled for peptone were used as diluent media. These observations
the enumeration of bacterial cells following the method would suggest that the inclusion of sodium pyruvate
outlined for the enumeration of L. delbrueckii ssp. bul- as a component in a growth medium could improve
garicus in yogurt samples. the recovery of injured or damaged bacterial cells.
Further tests employed various concentrations of indi-
vidual and combined treatments of fructose, maltose,
Statistical Analyses dextrose, lactose, sodium citrate, and sodium pyruvate
as components of mRCM. Initially, a combined treat-
The SAS version 9.4 (SAS Institute Inc., Cary, NC) ment of 0.5% dextrose, 0.5% fructose, 1% maltose, and
was used to analyze the experiment data obtained in 1% sodium pyruvate replacing 1% lactose as a carbon
this study. One-way ANOVA was used to determine source in mRCM appeared to be the most effective at
significant differences between the values. Significant selecting only for L. delbrueckii ssp. bulgaricus in the
differences (P < 0.05) between means of bacterial mixed bacterial culture. However, it became evident
counts (cfu/g) were compared using Tukey’s test. Bac- during the screening that a reduced concentration of
terial population counts were converted to log10 trans- sodium pyruvate would be needed to be effective in im-
formation before analysis. proving the selectivity of the modified medium. A 1%

Figure 1. The red arrow shows a rough-edged Lactobacillus bulgaricus colony in modified reinforced clostridial medium (mRCM; left image)
morph into a round and smooth colony in modified reinforced clostridial medium-pyruvate (mRCM-PYR) having a concentration of 1% sodium
pyruvate.

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sodium pyruvate concentration in this growth medium Cell Repair and Recovery
composition not only retarded other bacterial growth
but also converted the characteristic rough-edge mor- The L. delbrueckii ssp. bulgaricus colonies that were
phology of the surviving L. delbrueckii ssp. bulgaricus isolated from freeze-dried probiotic supplements on
colonies to a smooth one (Figure 1). Thus, a combina- MRS, mRCM, and mRCM-PYR growth media are
tion of 0.5% dextrose, 0.5% fructose, 1% maltose, and shown in Figure 2. The SP18 cell colonies (L. del-
0.2% sodium pyruvate as carbon sources in our growth brueckii ssp. bulgaricus circled in red) appear small and
medium proved to be the most effective at improving fragile in both the MRS and mRCM, suggesting that
the morphology and cell recovery of L. delbrueckii ssp. the cells were stressed or injured; however, this was not
bulgaricus colonies. the case in mRCM-PYR where the L. delbrueckii ssp.

Figure 2. Morphology of Lactobacillus bulgaricus colonies (circled in red) on de Man, Rogosa, and Sharpe (MRS) medium, modified rein-
forced clostridial medium (mRCM), and modified reinforced clostridial medium-pyruvate (mRCM-PYR). See Table 1 for sample description.

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bulgaricus cells appeared as large colonies. The result mRCM-PYR performed significantly better than MRS
for SP19 further highlighted the advantage of using and mRCM (P < 0.05) in 6 of the strains (S6, S19,
the mRCM-PYR growth media over the previously LB9, S9, S7, and S5) as measured by the bacterial pop-
modified RCM with regard to the repair of damaged ulations. No significant difference was observed in the
or stressed microbial cells. Nonlethal injury to bacterial bacterial counts recorded in mRCM and mRCM-PYR
cells can occur for several reasons. For example, the in 4 L. delbrueckii ssp. bulgaricus strains (S28, S8, S1,
destruction of bacterial cells as a result of exposure to and S22). However, both growth media performed sig-
low temperatures in the form of freeze drying, freezing/ nificantly better than MRS (P < 0.05) in these strains.
thawing, and prolonged storage at low temperatures The only case in which MRS performed noticeably bet-
is common in the industrial production of probiotic ter (P < 0.05) than mRCM-PYR and mRCM was with
supplements and starter cultures. L. delbrueckii ssp. bulgaricus strain LB6. The effect of
The cell colonies in mRCM were the smallest while the 3 media on the morphology of L. delbrueckii ssp.
the mRCM-PYR culture media facilitated the optimum bulgaricus colonies is shown in Figure 4.
growth and repair of L. delbrueckii ssp. bulgaricus cells,
resulting in larger colonies. Similar results were noted Selective Enumeration and Differentiation
with other starter cultures that were evaluated for the of L. delbrueckii ssp. bulgaricus in Mixed Cultures
viability of L. delbrueckii ssp. bulgaricus. For instance,
in the previously modified RCM growth media, the The bacterial counts of L. delbrueckii ssp. bulgaricus
colonies of L. delbrueckii ssp. bulgaricus in samples ST recovered from 21 products using MRS, mRCM, and
11 and ST 14 were tiny and very difficult to identify in mRCM-PYR are presented in Figure 5. The 21 products
the presence of other bacteria colonies. In contrast to consisted of a starter culture, probiotic supplements,
the mRCM, our optimized RCM produced visible and and yogurt and contained mixed bacterial cultures.
large colonies for the same samples, and the growth of The 3 media had similar effects on the bacterial counts
other bacteria was significantly inhibited (Figure 2). of 4 products (E1A, BY2, BY4, and SP8). No signifi-
cant difference was observed in the bacterial counts in
Selective Growth and Enumeration of Pure mRCM and mRCM-PYR for 6 products (E2Z, L6N,
L. delbrueckii ssp. bulgaricus Strains BY5, ST12, SP17, and SP19). However, both growth
media performed significantly better than MRS (P <
The bacterial counts of 11 pure L. delbrueckii ssp. 0.05) in these products. The mRCM-PYR performed
bulgaricus strains are presented in Figure 3. The better than MRS and mRCM (P < 0.05) in 10 prod-

Figure 3. Bacterial count of 11 pure Lactobacillus bulgaricus strains on de Man, Rogosa, and Sharpe (MRS) medium, modified reinforced
clostridial medium (mRCM), and modified reinforced clostridial medium-pyruvate (mRCM-PYR) at 44°C after 72 h of anaerobic incubation.
Error bars indicate SD for an experiment performed in duplicate.

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Figure 4. Morphology of pure Lactobacillus bulgaricus strains on de Man, Rogosa, and Sharpe (MRS) medium, modified reinforced clostridial
medium (mRCM), and modified reinforced clostridial medium-pyruvate (mRCM-PYR) after anaerobic incubation at 44°C for 72 h.

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ucts (E3D, BY6, SP9, ST11, ST14, SP10, ST13, ST15, DISCUSSION
ST16, and SP18). The bacterial counts of L. delbrueckii
ssp. bulgaricus in E4P were similar in mRCM-PYR and In a previous study by Nwamaioha and Ibrahim
MRS but considerably higher than those in mRCM (P (2018), a growth medium was modified to selectively
< 0.05). The results thus confirmed the effectiveness of enhance the isolation and enumeration of L. delbrueckii
our growth medium, mRCM-PYR, as a suitable alter- ssp. bulgaricus in dairy products. In the present study,
native to MRS and mRCM in the selective enumeration we further modified this medium for the superior selec-
and isolation of L. delbrueckii ssp. bulgaricus in mixed tivity and enumeration of L. delbrueckii ssp. bulgaricus
bacterial cultures. Figure 6 shows the morphology of by replacing lactose with 0.5% dextrose, 0.5% fruc-
L. delbrueckii ssp. bulgaricus in mixed cultures. The tose, 1% maltose, and 0.2% sodium pyruvate. Various
colonies of L. delbrueckii ssp. bulgaricus in mRCM-PYR carbohydrates that are the primary energy source for
were easy to identify and enumerate as the growth of bacteria in growth media have been used to selectively
other lactic acid bacteria was highly retarded. The enumerate lactic acid bacteria. For example, Tabasco et
identification of colonies was confirmed using direct al. (2007) supplemented MRS with fructose to enumer-
microscopic observation and the PCR method. ate L. delbrueckii ssp. bulgaricus. Another justification

Figure 4 (Continued). Morphology of pure Lactobacillus bulgaricus strains on de Man, Rogosa, and Sharpe (MRS) medium, modified
reinforced clostridial medium (mRCM), and modified reinforced clostridial medium-pyruvate (mRCM-PYR) after anaerobic incubation at 44°C
for 72 h.

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for the use of different carbon sources is the disparity reinforced clostridial Prussian blue (Ghoddusi and Rob-
in the reported carbohydrate use in L. delbrueckii ssp. inson, 1996), and bromocresol green whey agar (Yamani
bulgaricus. The inconsistencies recorded in the use of and Ibrahim, 1996) have not been able to selectively
fructose, mannose, and maltose among other sugars enhance the growth of L. delbrueckii ssp. bulgaricus in
in L. delbrueckii ssp. bulgaricus have been attributed a mixed culture. The mRCM-PYR performed well in
to strain type and composition of the growth medium the enumeration of L. delbrueckii ssp. bulgaricus in a
(Zourari et al., 1992; Chervaux et al., 2000; Yuksekdag mixed culture while retarding the growth of other lactic
and Aslim, 2008; Li et al., 2016). The mRCM-PYR acid bacteria. This is an improvement over the previous
was developed to optimally enumerate and select for study by Nwamaioha and Ibrahim (2018) where a lack
L. delbrueckii ssp. bulgaricus regardless of the source of significant differences in bacterial counts coupled
and strain type. We observed that the combination with the growth of other lactic acid bacteria was re-
of carbon sources was responsible for the selectivity corded in the tested products.
and high bacterial counts in mRCM-PYR. However, Further study may subject L. delbrueckii ssp. bul-
the substitution or elimination of any of the carbon garicus strains isolated in this study using mRCM-PYR
sources, particularly sodium pyruvate, did not yield to genotypic methods of identification with a view to
the same result. Pyruvate is an essential intermediate discovering relationships between the origins of various
in the metabolism of lactic acid bacteria and can be strains. The enzyme activity, secreted proteins, aroma
converted into several end products such as acetic acid, production, and stress resistance of the various strains
formic acid, and lactic acid. Higashio et al. (1977) at- should also be tested and compared. In addition, un-
tributed the stimulatory effect that S. thermophilus had derstanding of the pyruvate metabolism is critical to
on the growth of L. delbrueckii ssp. bulgaricus to the demonstrate the applications and health benefits of L.
production of formic and pyruvic acids. Polak-Berecka delbrueckii ssp. bulgaricus. This additional exploration
et al. (2010) recorded a better yield of cell biomass could lead to the identification and isolation of superior
when sodium pyruvate was combined with glucose as probiotic and industrial strains.
carbon sources. These results might help to explain the
importance of pyruvate in the cell repair and recovery CONCLUSIONS
observed in this study.
Many of the medium available in the literature such Our results demonstrated that mRCM-PYR modi-
as MRS (de Man et al., 1960), Lee’s agar (Lee et al., fied by the addition of sodium pyruvate, fructose,
1974), yogurt lactic agar (Matalon and Sandine, 1986), and maltose was effective in selectively isolating and

Figure 5. Bacterial count of Lactobacillus bulgaricus in mixed culture on de Man, Rogosa, and Sharpe (MRS) medium, modified reinforced
clostridial medium (mRCM), and modified reinforced clostridial medium-pyruvate (mRCM-PYR) at 44°C after 72 h of anaerobic incubation.
Error bars indicate SD for an experiment performed in duplicate.

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Figure 6. Morphology of Lactobacillus bulgaricus (circled) in mixed culture on de Man, Rogosa, and Sharpe (MRS) medium, modified re-
inforced clostridial medium (mRCM), and modified reinforced clostridial medium-pyruvate (mRCM-PYR) after anaerobic incubation at 44°C
for 72 h.

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 Oyeniran et al.: MODIFIED REINFORCED CLOSTRIDIAL MEDIUM 5040

Figure 6 (Continued). Morphology of Lactobacillus bulgaricus (circled) in mixed culture on de Man, Rogosa, and Sharpe (MRS) medium,
modified reinforced clostridial medium (mRCM), and modified reinforced clostridial medium-pyruvate (mRCM-PYR) after anaerobic incubation
at 44°C for 72 h.

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enumerating L. delbrueckii ssp. bulgaricus in a mixed L. delbrueckii ssp. bulgaricus. Thus, mRCM-PYR is
culture. The versatility of mRCM-PYR also makes it recommended as a growth medium for the isolation
highly suitable for the study and rapid cultivation of and enumeration of L. delbrueckii ssp. bulgaricus from

Figure 6 (Continued). Morphology of Lactobacillus bulgaricus (circled) in mixed culture on de Man, Rogosa, and Sharpe (MRS) medium,
modified reinforced clostridial medium (mRCM), and modified reinforced clostridial medium-pyruvate (mRCM-PYR) after anaerobic incubation
at 44°C for 72 h.

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different sources due to this medium’s inhibitory effect algal carcass. J. Dairy Sci. 99:4243–4250. https:​/​/​doi​.org/​10​.3168/​
jds​.2015​-10700.
on the growth of other LAB. Lourens-Hattingh, A., and B. C. Viljoen. 2001. Yogurt as probiotic
carrier food. Int. Dairy J. 11:1–17. https:​/​/​doi​.org/​10​.1016/​S0958​
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ACKNOWLEDGMENTS Mahmood, T., T. Masud, S. Ali, K. S. Abbasi, and M. Liaquat. 2015.
Optimization and partial characterization of bacteriocin produced
This publication was made possible by grant number by Lactobacillus bulgaricus-TLBFT06 isolated from Dahi. Pak. J.
NC.X-267-5-12-170-1 from the National Institute of Pharm. Sci. 28:549–555.
Matalon, M. E., and W. E. Sandine. 1986. Improved media for differ-
Food and Agriculture (NIFA, Kansas City, KS) and entiation of rods and cocci in yogurt. J. Dairy Sci. 69:2569–2576.
in part by NIZO Food Research BV (Ede, the Neth- https:​/​/​doi​.org/​10​.3168/​jds​.S0022​-0302(86)80702​-0.
erlands), Jarrow Formulas (Los Angeles, CA), and the Nwamaioha, N. O., and S. A. Ibrahim. 2018. A selective medium for
the enumeration and differentiation of Lactobacillus delbrueckii
Department of Family and Consumer Sciences and the ssp. bulgaricus. J. Dairy Sci. 101:4953–4961. https:​/​/​doi​.org/​10​
Agriculture Research Station at North Carolina Agri- .3168/​jds​.2017​-14155.
culture and Technical State University (Greensboro, Pescuma, M., E. M. Hébert, T. Haertlé, J.-M. Chobert, F. Mozzi,
and G. F. de Valdez. 2015. Lactobacillus delbrueckii ssp. bulgari-
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