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The aim of the study was to characterize the Bacillus subtilis ATCC 6051a strain, in order to establish its probiotic utility in piglet nutrition. The strain was assayed morphologically, culturally, biochemically, for hemolytic activity and enzymatically (amylase and protease). The identification and analysis of the biochemical characteristics was performed by catalase assay, API 50 CHB Biomerieux strips, apiweb API 50 CHB V 4.0 soft (B. subtilis very good identification, 99.4% ID) and ABIS online. The hemolytic activity was assayed on blood agar medium. The growth activity of strain was evaluated in two ways: static incubation (30 C, 24 h, 1.36 x 10 8 CFU/ml) and under constant agitation (30 C, 24 h, 150 rpm, (1.6 x 10 9 CFU/ml). The strain is a Gram-positive and rod-shaped bacteria, arranged in short chains or in small irregular pairs with ability to produce spores on nutrient medium. The endospores were central, paracentral and subterminal, which did not deform the vegetative cell. The strain growth was aerobic and was non-hemolytic. The enzymatic process was observed by appearance of distinct zones around strain colonies. In conclusion, the results suggested that the strain present probiotic traits and can be further assessed for other probiotic characters (resistance to pH 2.0, resistance to bile acids and salts, antibacterial activity, induction of local immune response etc.).
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DOI: 10.5937/FFR1802203D UDK 636.4.087.8:579.86
Original research paper
PRELIMINARY CHARACTERISATION OF
BACILLUS SUBTILIS
STRAIN
USE AS A DIETARY PROBIOTIC BIO-ADDITIVE IN WEANING PIGLET
Mihaela Dumitru*1,2, Ionuț Sorescu1, Mihaela Habeanu1, Cristina Tabuc1, Lavinia Idriceanu1,
Stefana Jurcoane2,3
1 National Research Development Institute for Biology and Animal Nutrition (IBNA), Bucharest, No. 1,
Balotesti, Ilfov, 077015, Romania
2 University of Agronomic Sciences and Veterinary Medicine of Bucharest, 59, Marasti Blvd, District 1,
Bucharest, Romania
3 Academy of Romanian Scientists, Bucharest, Romania
*Corresponding author:
E-mail address: mihaela.dumitru22@yahoo.com
ABSTRACT: The aim of the study was to characterize the Bacillus subtilis ATCC 6051a strain, in
order to establish its probiotic utility in piglet nutrition. The strain was assayed morphologically,
culturally, biochemically, for hemolytic activity and enzymatically (amylase and protease). The
identification and analysis of the biochemical characteristics was performed by catalase assay, API 50
CHB Biomerieux strips, apiweb API 50 CHB V 4.0 soft (B. subtilis very good identification, 99.4% ID)
and ABIS online. The hemolytic activity was assayed on blood agar medium. The growth activity of
strain was evaluated in two ways: static incubation (30 C, 24 h, 1.36 x 108 CFU/ml) and under
constant agitation (30 C, 24 h, 150 rpm, (1.6 x 109 CFU/ml). The strain is a Gram - positive and rod-
shaped bacteria, arranged in short chains or in small irregular pairs with ability to produce spores on
nutrient medium. The endospores were central, paracentral and subterminal, which did not deform the
vegetative cell. The strain growth was aerobic and was non hemolytic. The enzymatic process was
observed by appearance of distinct zones around strain colonies. In conclusion, the results suggested
that the strain present probiotic traits and can be further assessed for other probiotic characters
(resistance to pH 2.0, resistance to bile acids and salts, antibacterial activity, induction of local immune
response etc.).
Key words: API 50 CHB, hemolytic activity, enzymatic screening
INTRODUCTION
The Bacillus species constitutes an interes-
ting group of probiotic bacteria for humans
(Ritter et al., 2018) and animals as direct
fed microbials (DFM) (Lese et al., 2007).
DFM or probiotics are defined as life
microorganisms which, when administered
in adequate amounts, confer a health be-
nefit on the host (FAO/WHO, 2001).
Microorganisms used in animal feed as pro-
biotic products may contain one or more
bacterial strains. In the European Union
(EU) microorganisms added as feed sup-
plementation are bacterial strains, often
Gram-positive belonging to the following
genus: Bacillus (B. cereus var. toyoi, B.
licheniformis, B. subtilis), Enterococcus (E.
faecium), Lactobacillus (L. acidophilus, L.
casei, L. farciminis, L. plantarum, L. rha-
mnosus), Pediococcus (P. acidilactici),
Streptococcus (S. infantarius); some others
probiotics are microscopic fungi such as
Mihaela Dumitru et al., Preliminary characterisation of Bacillus subtilis strain use as a dietary probiotic bio-additive in weaning
piglet, Food and Feed Research, 45 (2), 203-211, 2018
strains of yeast belonging to the Saccha-
romyces cerevisiae species and Kluyvero-
myces (Markowiak and Śliżewska, 2018;
Yang et al., 2015a; Bajagai et al., 2016).
The most important sources for enzyme
production are microorganisms. Selection of
the right organism plays a key role in high
yield of enzymes (Vishwanatha et al., 2010).
Supplementation with probiotic as bio-
additive in animal livestock suggested the
most desirable alternative for intestinal
microbiota by increased intestinal immunity,
improved resistance to disease, reduced
number of pathogens bacteria and improved
animal health (Markowiak and Śliżewska,
2018; Meng et al., 2010; Yirga, 2015).
Probiotic used in piglets feed based on
Bacillus subtilis (B. subtilis) improved para-
meters such as: weight gains, feed con-
version, meat quality (Link and Kovac,
2006), animal health by modifying micro-
biota and pig’s performance (Kaewtapee et
al., 2017). These microorganisms have
demonstrated high probiotic potential; they
have the ability of sporulation, thereby ma-
king them stable during thermal treatment of
feed (high temperature and pressure),
resistance during the enzymatic digestion
along to the gastrointestinal tract (Cutting,
2011). B. subtilis is a strain which grows
efficiently with low-cost carbon and nitrogen
sources. Their enzymatic capacity is very
efficient breaking down a great variety of
proteins, carbohydrates and lipids from
animal and vegetable origin, into their
constituent units (Zaid, 2018).
The objective of this study, was to assess
the B. subtilis ATCC 6051a strain, to
describe morphological, cultural, and
biochemical characteristics, hemolytic ability
and enzymatic production (amylase and
protease screening), as a preliminary
investigation of probiotic potential in order to
use it in piglet nutrition.
MATERIAL AND METHODS
Characterization of bacterial strain,
growth media and enumeration of spore
counts
Morphological and cultural properties of B.
subtilis ATCC 6051a strain was investigated
according to the methods described in Ber-
gey’s Manual of Systematic Bacteriology
(1957). Bacteria B. subtilis ATCC 6051a
was grown in nutrient broth (Merck) and on
nutrient agar (Merck), 90 mm in Petri
dishes, to evaluate the cultural traits. Serial
dilution (1:10, in 0.85% saline) was done
(10-5 - 10-10-fold), for CFU/ml in broth
culture, incubated static (30 C, 24-48 h)
and under agitation (30 C, 24 h, 150 rpm).
An aliquot of 1 ml from each dilution was
homogenized and spread on nutrient agar
plate. At least three replicas were done for
each dilution. The strain was stored at -
80C with 20% sterile glycerol and
deposited in the Collection of National
Research Development Institute for Biology
and Animal Nutrition Balotești (INCDBNA),
Romania, under the code IBNA 74. The
research was carried out at Laboratory of
Biotechnology of (INCDBNA), Romania.
Biochemical test
The strain was tested for biochemical
characters (catalase assay, API 50 CHB
Biomerieux strips) and identified by API 50
CHB V4.0 and ABIS online soft.
The catalase test
Analysis of catalase test was done
according to the protocol described by
Dumitru et al. (2017).
The API 50 CHB test
API 50 CHB strips were used for evaluated
the carbohydrate acidification of B. subtilis
ATCC 6051a according to the manu-
facturer’s protocol (BioMerieux). The API 50
CHB consists of 50 microtubes used to
study fermentation of substrates belonging
to the carbohydrate family and its
derivatives. The density of the suspensions
used for API test was adjusted to 2.0
McFarland standard turbidity. The strips are
read after 24 h incubation, with a final
interpretation after 48 h, at 37 C, in aerobic
conditions. The obtained results are inter-
preted using database system API 50 CHB
V4.0 and ABIS online software (Stoica and
Sorescu, 2017).
Hemolysis production
Blood agar plates [Trypticase soy agar
(TSA, Sanimed) containing 5% (w/v)] sheep
blood, were used to test hemolysis activity.
The strain was streaked on blood agar
plates and incubated at 37 °C, for 24h. In
Mihaela Dumitru et al., Preliminary characterisation of Bacillus subtilis strain use as a dietary probiotic bio-additive in weaning
piglet, Food and Feed Research, 45 (2), 203-211, 2018
this test, a greenish zone around bacteria
indicates α-hemolysis, a clear zone β-
hemolysis, and no change γ-hemolysis (i.e.,
no hemolysis) (Jeon et al., 2018).
SCREENING OF AMYLASE PRODUCING
BACTERIA
Bacterial strain was screened for amylolytic
properties by starch hydrolysis test, on
starch (1%, 2%, 3% w/v) agar plate. The
culture medium was sterilized by
autoclaving at 121 °C, for 15 min. The strain
was streaked on the starch agar plate,
followed by incubated at 37 °C, for 24 h.
After incubation, 1% iodine solution (Lugol
solution from Gram’s staining) was flooded
on the starch agar plate. A clear zone of
hydrolysis on starch (after addition of
iodine), around bacterial growth, is an
indication of amylase production (Singh et
al., 2015).
SCREENING OF PROTEASE
PRODUCING BACTERIA
Bacillus subtilis ATCC 6051a was screened
for proteolytic activity. The bacteria strain
was inoculated on the agar plates
containing casein (1% w/v) and milk powder
(1% w/v), incubated at 37 C, for 48 h. The
plates were flooded with 25% TCA
(trichloroacetic acid) solution and incubated
for 15 min., at 45 C (Siddalingeshwara et
al., 2010). Protease synthesis was observed
by a zone of clearance on agar plate.
RESULTS AND DISCUSSION
Morphological and biochemical
characterization
Colony morphology was determined on
nutrient agar after 24 h incubation at 30 °C,
under aerobic conditions. Grown colonies
were opaque, whitish with rough matte sur-
face, irregular edged and diameter 1.2-5
mm (Figure 1).
After growth in the nutrient medium, the
tested strain at microscopic observation
appeared as Gram positive rods shaped,
arranged in diploid form, in short chains or
in small irregular pairs (Figure 2).
Bacillus subtilis ATCC 6051a produced oval
endospores located central, paracentral or
subterminal positions without distorting the
vegetative cell.
Bacilli present the ability of sporulation,
making them stabile to survive at low pH of
gastrointestinal tract (GIT) and during
thermal processing and storage of feed
(Elshaghabee et al., 2017). This statement
is reinforced by Merchant et al. (2011)
which affirmed that Bacillus spp. can be
used as DFM in animal nutrition because
the pH in the small intestine is 6 to 7, which
is optimal for spores to germinate, grow and
produce enzymes and, also, to resist of the
enzymatic degradation and stomach’s acidic
condition.
The strain was catalase positive, formed
gas bubbles after addition of 3% solution
H2O2. Hosoi et al. (2000) reported that B.
subtilis can stimulate the growth and
viability of Lactobacillus spp., maybe
through the production of catalase. In
addition, the spores resistant of B. subtilis to
acid and oxygen may influence the intestinal
microbiota and affect the microbial com-
munity from piglet feces. These data show
the strong interaction between the B. subtilis
and Lactobacillus.
Results obtained from the API 50 CHB tests
indicated that used test was able to confirm
tested strain B. subtilis ATCC 6051a around
99.4% ID (very good percentage iden-
tification) and ABIS online (~90.7% simi-
larity). The fermentation capacity of car-
bohydrate was observed by the discolo-
ration of the basal medium, from red to
yellow, as positive answer (Figure 3).
The results by API 50 CHB were registered
as final interpretation after 48 h, at 37 C
(Table 1).
B. subtilis ATCC 6051a fermented D-gly-
cerol, salicin, D-cellobiose, D-maltose, L-
arabinose, D-ribose, D-melibiose, D-xylose,
D-saccharose (sucrose), D-trehalose, D-
raffinose, D-glucose, starch, D-fructose,
glycogen, D-mannose, gentibiose, D-
turanose, inositol, D-mannitol, D-sorbitol,
methyl-αD-glucopyranoside, amygdalin,
arbutin and esculin.
The strain did not ferment of D-arabinose,
D-lactose, L-xylose, D-adonitol, methyl-βD-
xylopyranoside, D-melezitose, D-galactose,
xylitol, L-sorbose, L-rhamnose, dulcitol, D-
lyxose, D-tagatose, D-fucose, L-fucose,
methyl-αD-mannopyranoside, D-arabitol, L-
Mihaela Dumitru et al., Preliminary characterisation of Bacillus subtilis strain use as a dietary probiotic bio-additive in weaning
piglet, Food and Feed Research, 45 (2), 203-211, 2018
arabitol, N-acetylglucosamine, potassium
gluconate, potassium 2-ketogluconate and
potassium 5-ketogluconate.
After incubation, the change in colour of API
50 CHB medium from red to yellow, rep-
resents a positive result corresponds to the
substrates acidification (Aruwa and Olatope,
2015).
Hemolysis production
The hemolytic evaluation was assayed on
Trypticase soy agar supplemented with 5%
sheep blood (TSA, Sanimed) and it is based
on the ability of strain to lyse blood cells of
culture medium.
Figure 1. Cultural aspect on agar plate for
Bacillus subtilis ATCC 6051a
Figure 2. Microscopic observation of Bacillus
subtilis ATCC 6051a strain (1000x)
Figure 3. API 50 CHB strips inoculated with
Bacillus subtilis ATCC 6051a
Figure 4. Haemolysis assay of Bacillus subtilis
6051a, at 37C, 24 h
Figure 5. Bacterial growth (A: static incubation; B: shaking incubation)
8,13
9,2
7
7,5
8
8,5
9
9,5
10
A B
CFU/ml
Mihaela Dumitru et al., Preliminary characterisation of Bacillus subtilis strain use as a dietary probiotic bio-additive in weaning
piglet, Food and Feed Research, 45 (2), 203-211, 2018
Table 1.
The results obtained with API 50 CHB for B. subtillis ATCC 6051a
Glycerol
Erythritol
Salicin
D-cellobiose
+
+
D-arabinose
D-maltose
+
L-arabinose
D-lactose
-
D-ribose
D-melibiose
+
D-xylose
D-saccharose (sucrose)
+
L-xylose
D-trehalose
+
D-adonitol
Inulin
+/?
Methyl-βD-xylopyranoside
D-melezitose
-
D-galactose
D-raffinose
+
D-glucose
Starch
+
D-fructose
Glycogen
+
D-mannose
Xylitol
-
L-sorbose
Gentibiose
+
L-rhamnose
D-turanose
+
Dulcitol
D-lyxose
-
Inositol
D-tagatose
-
D-mannitol
D-fucose
-
D-sorbitol
L-fucose
-
Methyl-αD-mannopyranoside
D-arabitol
-
Methyl-αD-glucopyranoside
L-arabitol
-
N-acetylglucosamine
Potassium gluconate
-
Amygdalin
Potassium 2-ketogluconate
-
Arbutin
Potassium 5-ketogluconate
-
Esculin
,,-” Negative test; ,,+ ” Positive test; ,,?” Weakly positive
The safety of B. subtilis ATCC 6051a to be
used as a potential probiotic in piglets’
nutrition was confirmed by non-hemolytic
activity on 5% sheep blood agar plate -
hemolysis) (Figure 4). Similar observation
for Bacillus spp. was reported by Soro-
kulova et al. (2008).
Non-hemolytic activity is one of the main
criteria needed to be satisfied by a probiotic
organism which confirms its non-patho-
genicity (Jung and Chang, 2012).
Bacillus strains are known as potential pro-
biotics which could promote animals’ health
by direct consumption of high concentra-
tions of viable number of cells (Guo et al.,
2006; Abdhul et al., 2015). For a strain to be
selected as a possible probiotic, it should
not form halo of degradation around the de-
veloped colony.
It can be noticed that, if a strain involved a
clear zone around colonies on blood sheep
agar that indicate a complete hydrolysis (β-
hemolysis), the strain must be eliminated to
be used as a probiotic in animal nutrition.
Non-hemolysis (γ-hemolysis) and α hemo-
lysis (a green zone around colony) are con-
sidered to be safe (Seker, 2010).
Growth of the bacterial strain
The growth of B. subtilis was monitored
after 24 h incubation at 30 C under static
conditions and under a constant agitation
(150 rpm). After static incubation number
cells of B. subtilis were amounted 1.36 x 108
(CFU/ml), while it is in case of agitation
were amounted 1.6 x 109 (CFU/ml). The ex-
perimental results, given in Figure 5, sho-
wed that agitation is a better parameter for
growing bacteria, compared with the static
incubation. The result was expressed as lo-
garithm of colony forming units/ml.
Screening of amylase enzyme
Productivity of the amylase, as qualitative
assay, was investigated on starch agar
plate (1-3% w/v starch). Production of this
enzyme was studied after 24 h of incubation
at 30 C and pH 7. A clear zone of starch
hydrolysis, surrounding bacterial growth, re-
presents the capacity of tested strain to
synthesize enzyme amylase (Figure 6).
According to Singh et al. (2015), the Ba-
cillus spp. is a group with strong properties
to produces amylase. In their study, Mishra
and Behera (2018) presented similar data of
amylase hydrolysis on starch agar plate with
a clear zone around Bacillus spp.
Mihaela Dumitru et al., Preliminary characterisation of Bacillus subtilis strain use as a dietary probiotic bio-additive in weaning
piglet, Food and Feed Research, 45 (2), 203-211, 2018
This qualitative method for evaluation of
amylase production by B. subtilis ATCC
6051a strain provides information about the
substrate on which it acts and is used for
selection of the right diet for animals.
Figure 6. Screening of hydrolysis amylase of Bacillus subtilis ATCC 6051a
Figure 7. Screening of hydrolysis protease of Bacillus subtilis ATCC 6051a
Generally, Bacillus spp. are sources of
extracellular hydrolytic enzymes, which may
help the digestive process and utilization of
nutrients from feed (Davis et al., 2008).
Weaning is a difficult period for piglets due
to their incomplete development of the
enzymatic system (Habeanu et al., 2017).
The change from highly digestible liquid milk
from sows to a less-digestible, more com-
plex solid feed has also critical conse-
quences on piglet performance and the
physiology of their GIT (Campbell et al.,
2013).
Feeding exogenous microbial enzymes
could aid digestion of complex matrix of
non-milk-based ingredients present in the
piglet’s weaning diet and could bridge the
gap until the piglet’s endogenous enzyme
secretory capacity for α-amylase and pro-
teases has had time to develop. Dietary
supplementation with Bacillus spp. has
been reported to improve growth perfor-
mance, immune status, intestinal microbiota
and nutrient digestibility of non-starch poly-
saccharides (NSP) from corndiets piglets
(Lei and Kim, 2014), due to exogenous
enzymes secreted into the host intestine or
to endogenous enzymes available into the
bacterial cells and released when they are
lysed by the effect of the acidic environment
of hosts’ stomach (Ortiz et al., 2015).
Screening of protease enzyme
Protease and amylase production was
identified on the nutrient agar supplemented
with starch and milk powder, by observing
the zone of hydrolysis around the colony or
growth (Nagaraju and Divakar, 2012).
The cleared zone around colony on the agar
plate medium represents the enzymatic
potential of B. subtilis ATCC 6051a (Figure
7). Lee et al. (2012) reported that addition of
extracellular microbial enzymes with pro-
biotic properties can enhance feed diges-
tibility, for example the exogenous pro-
teases in feed can be an option to reduce
dietary protein levels maintaining high ani-
mal performance.
2%
1%
3%
Mihaela Dumitru et al., Preliminary characterisation of Bacillus subtilis strain use as a dietary probiotic bio-additive in weaning
piglet, Food and Feed Research, 45 (2), 203-211, 2018
CONCLUSIONS
The results suggested that the Bacillus
subtilis ATCC 6051a strain presents the
capacity to secret amylase and protease
enzymes. The strain showed no hemolytic
activity - hemolysis) on TSA medium
confirming that it is not pathogenic. Further
experiments will be performed to study
other probiotic features such as: resistance
to pH 2.0, resistance to bile acids and salts,
antibacterial activity, induction of local
immune response etc. Bacillus subtilis
ATCC 6051a represents a source of
amylase and protease and their probiotic
potential will be researched in animal
nutrition as a source of feed additive.
ACKNOWLEDGEMENTS
This study was funded by Romanian
Ministry of Research and Innovation through
Program 1 Development National Re-
search-Development, Sub-program 1.2
Institutional Performance - Projects funding
excellence in R & D, Contract no. 17 PFE/
17.10.2018 and Grant PN 18200103.
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Mihaela Dumitru et al., Preliminary characterisation of Bacillus subtilis strain use as a dietary probiotic bio-additive in weaning
piglet, Food and Feed Research, 45 (2), 203-211, 2018
ПРЕЛИМИНАРНА КАРАКТЕРИЗАЦИЈА ВРСТЕ
BACILLUS SUBTILIS
КАО
ПРОБИОТИЧКОГ ДОДАТКА ИСХРАНИ ПРАСАДИ
Михаела Думитру*1,2, Ионут Сореску1, Михаела Хабеану1, Кристина Табук1, Лавиниа
Идрицеану1, Стефана Јуркоане 2,3
1 Национални истраживачки развојни институт за биологију и исхрану животиња (ИБНА),
Букурешт бр. 1, Балотешти, 077015, Румунија
2 Универзитет пољопривредних наука и ветеринарске медицине из Букурешта, 59,
Марасти булевар, Букурешт, Румунија
3 Румунска академија наука, Букурешт, Румунија
Сажетак: Циљ овог истраживања био је карактеризација бактеријске врсте Bacillus
subtilis ATCC 6051a, како би се утврдила могућност њене пробиотичке примене у исхрани
прасади. У овом истраживању анализиране су морфолошке, културалне и биохемијске
карактеристике тест соја, као и његова хемолитичка и ензимска активност (амилаза и протеаза).
Идентификација и анализа биохемијских карактеристика тест соја је спроведена применом
каталаза теста, API идентификациониог система (API 50 CHB Biomerieux) и примену apiveb API
50 CHB V 4.0 софтвера (B. subtilis, врло висок ниво идентификације, 99,4% ID) уз ABIS online
подршку. Хемолитичка активност тест соја је спроведена на крвном агару. Способност раста
тест соја је испитана у статичким условима (30 °C, 24 h, 1.36 x 108 CFU/ml) и условима
константне агитације (30 ° C, 24 h, 150 rpm, (1,6 k 109 CFU/ml ). Резултати су показали да је тест
сој Грам-позитивна штапићаста бактерија, са штапићима распоређеним у виду кратких ланаца
или неправилним паровима са способношћу формирања спора на хранљивом медијуму.
Ендоспоре су локализоване парацентрално и суптерминално не узрокујући деформа-
цију вегетативне ћелије. Тестирани сој расте аеробно и не показује хемолитичку активност.
Ензимска активност тестираног соја је утврђивана као појава просветљених зона око саме
бактеријске колоније. На основу добијених резултата може се закључити да испитивани сој има
пробиотичке особине и може се надаље користити за испитивање других пробиотичких особина
(отпорност при pH 2.0, отпорност на жучне киселине и соли, антибактеријска активност,
индукција локалног имунолошког одговора итд.).
Кључне речи: API 50 CHB, хемолитичка активност, ензиматски скрининг
Received: 29 October 2018
Received in revised form: 12 December 2018
Accepted: 19 December 2018
... Significant differences are distinguished according to alphabetical superscripts, where means not sharing a common superscript are significantly different. lack of hemolytic activity in red blood cells (Dumitru et al., 2019). This strain is also Generally Recognized as Safe (GRAS) by the FDA, making it an attractive probiotic culture for live trials (Kabisch et al., 2013). ...
... However, according to the results obtained in the current study and in a previous study, this serotype is not safe for in ovo inoculation in broiler hatching eggs (Triplett et al., 2018). One key characteristic of this specific B. subtilis serotype (ATCC 6051), is the production of antimicrobial peptides as well as amylase and protease enzymes (Dumitru et al., 2019). When added to poultry feed, these enzymes are known to improve nutrient availability and absorption, thus inducing improvements in growth performance (Amerah et al., 2017;Alagawany et al., 2018). ...
... Bacillus species have become of great interest for the industry due to their ability to produce high quantities of enzymes and antimicrobial peptides (Abriouel et al., 2011;Sumi et al., 2015;Dumitru et al., 2019). In previous studies evaluating the inclusion of non-specified serotype of B. subtilis in the feed, reductions were obtained in Salmonella (Shivaramaiah et al., 2011) and Clostridium, which are two of the main pathogens of concern in the poultry industry (Sen et al., 2012). ...
Article
Full-text available
Recent research has tried to maximize broiler chick health and performance by utilizing commercial in-feed probiotics to inoculate fertile hatching eggs, and thus expose birds earlier to beneficial bacteria. However, the in ovo inoculation of a specific serotype of Bacillus subtilis was detrimental for broiler hatchability. Therefore, the objective of this study was to determine if other B. subtilis serotypes negatively affect hatchability or if it is associated with a specific serotype. It was also of interest to determine if the B. subtilis serotype influence chick performance and intestinal microflora. On d18 of incubation, 1886 fertile broiler eggs were in ovo inoculated with the following treatments (T): T1=Marek's vaccine (MV), T2= MV + B. subtilis (ATCC 6051), T3=MV + B. subtilis (ATCC 8473), and T4= MV + B. subtilis (ATCC 9466). It should be noted that in a previous study, T2 was detrimental to hatchability. Inoculated eggs were transferred to 3 hatchers/T. At hatch, chicks were weighed, feather sexed, and hatch residue analysis was conducted. Male chicks were randomly assigned to 40 raised wire cage so that there were 10 birds/cage. On d 0, 7, 14, and 21 of the grow-out, chicks and feed were weighed to calculate performance data. On these days, the ileum and ceca were aseptically collected to enumerate total aerobes and coliforms. No differences were observed for percentage of mid dead embryos, cracked eggs, and cull chicks (P>0.05). However, hatch of transfer was significantly reduced by T2 compared to T1, T3, and T4 (P<0.001). T2 had significantly higher percentages of late dead embryos and pips when compared to the other treatments (P=0.002 and P<0.001, respectively). Chicks hatched from T2 were not vigorous and, thus, not used for the grow-out trial. No differences were observed for growth performance characteristics for any of the treatments (P>0.05). For bacterial enumeration, the ileum had equal or fewer bacterial counts for T3 and T4 when compared to T1 on most sampling days, except on d21 where T4 had higher aerobic and coliform counts (P≤ 0.0001). For the ceca, T3 and T4 had equal or fewer bacterial counts than T1 on every sampling day (P≤ 0.0001). These data demonstrate that not all B. subtilis evaluated are detrimental to hatchability, but rather, serotype dependent. In addition, different B. subtilis serotypes can modify the intestinal microflora with potential to reduce pathogenic bacteria present in young broilers, without impacting overall performance.
... The presence of spores as bacterium protection enable Bacillus spp. to withstand environmental conditions such as pH, bile salts, temperature, radiation, pressure, and chemical agents, and factors that can destroy the vegetative form (Bernardeau et al., 2017). Dumitru et al. (2019;2018a) reported that B. subtilis ATCC 6051a tolerated well GIT conditions such as low pH and bile salts, making it a commensal bacterium for animals that ingest it (Vasques, 2016). The Bacillus subtilis ATCC 6051a strain also tolerated 80 °C temperature for two hours which allows its inclusion in animal diets even if it is ground and pelleted . ...
... MD was responsible for conducting and monitoring the experiment, in vitro testing probiotic properties, which were presented in another study (Dumitru et al., 2018a), and preparing bacterial culture based on Bs. MH was involved in the feeding trial and performed statistical analysis. ...
Article
Full-text available
This article evaluated the effects of supplemental probiotic Bacillus subtilis (Bs) ATCC 6051a (1.6x10 9 cfu/mL) in diets for weaned piglets on their performance and on the occurrence of diarrhoea. Sixty piglets, 30 ±3 days old with initial bodyweight of 8.41±0.92 kg, were allotted randomly to six pens of ten piglets. There were two replicates of each treatment, namely a control diet (C), a diet supplemented with 1% Bs (E1), and a diet supplemented with 3% Bs (E2). Feed was provided ad libitum as flour in two meals per day. Feed materials were examined for total numbers of fungi, aerobic mesophilic bacteria (TNG), Coliforms, Escherichia coli and Salmonella spp. The addition of Bs did not influence (P >0.05) bodyweight (BW) or average daily weight gain (ADWG). However, across the experimental period ADWG was greater in E2 and E1 than in C (>1.12 and 1.08 times compared with C). Feed intake (ADFI) by pigs fed C was greater than pigs fed E1 and E2. Feed efficiency was higher in E1 and E2 than the C diet. Addition of 1% Bs decreased (P<0.05) diarrhoea occurrence around 8% compared with C, and 4% compared with 3% Bs. A total of 23.4% of the piglets produced soft faeces. Diarrhoea scores of 2 (mild diarrhoea) and 3 (severe diarrhoea) were observed in 43.75% and 32.81% of the pigs. No differences (P >0.05) were detected between the treatments. The results suggested that E1 could positively affect growth performance and mitigate the occurrence of diarrhoea. _______________________________________________________________________________________
... Blood agar plates [Trypticase soy agar (TSA, Sanimed) containing 5% (w/v) sheep blood], were used to test hemolysis activity [15]. The interpretation was followed after incubation at 37°C, for 24 h. ...
... Our strain had no clear transparent or greenish zone surrounding colonies on TSA agar. The results are similar to Kavitha et al. [24] and Dumitru et al. [15]. ...
Article
Full-text available
The present study was planned to identify the Bacillus probiotic characteristics from a strain knows as Bacillus spp. in with the aim of use as a probiotic candidate in broiler chickens feed. The strain was identified phenotypically and evaluated for their viability through total plate count method, colony morphology, catalase test, hemolysis activity, pH (2 and 3) and bile salts (0.3% w/v) tolerance under simulated gastrointestinal tract (GIT) conditions, rate of survivability (%), spores resistance at high temperature and antibiotics susceptibility tests. Registered under the code IBNA 66, the strain was identified by API 50CHB (ID 99.9%) and ABIS online (91.8% similarity) as Bacillus megaterium. The strain presented a viable count of 3 x 10 11 CFU/mL after 22±2h of incubation at 37°C, 120 rpm with positive catalase, and non-hemolytic activity results. Also, Bacillus megaterium showed a significant resistance and survivability at pH 2 (P <0.0001, 62.09%) and pH 3 (P <0.0001, 77.53%), bile salts (P <0.0001, 84.27%), with a high ability to produce spores (after 120 min. at 80°C show 9.47 Log CFU/mL, P <0.0001). The antibiotic susceptibility test showed 100.00% resistance of strain to oxacillin (1 μg/mL), with multiple antibiotic resistance indices above 0.5. In conclusion, Bacillus megaterium can be an ideal probiotic candidate that can potentially be formulated and applied in the poultry feed for improving performance and modulated GIT microflora.
... Some of Bacillus spp. that are used as probiotics can produce toxins (Gaggia et al., 2010); however, in case of Bacillus subtilis and Bacillus licheniformis has been listed as non-toxigenic bacteria without hemolysis pathogenicity (Dumitru et al., 2018;Dumitru et al., 2019), involving that biological supplements to livestock diets (EFSA, 2010). Also, probiotics have been reported to decrease blood cholesterol (Ooi and Liong, 2010), reducing metabolic reactions that produce toxic substances, secret vitamins and antimicrobial compounds such as bacteriocins (Hemaiswarya et al., 2013). ...
... Bacterial strain, culture medium and growth conditions Bacillus subtilis ATCC 6051a (BS) and Bacillus licheniformis ATCC 21424 (BL) as bacterial strains used as DFM was acquired from the American Tissue Culture Collection (ATCC) in the form of freeze-dried. The probiotic properties of BS and BL strain were analyzed in vitro and presented in previous studies (Dumitru et al., 2018;Dumitru et al., 2019;. The cultures growth was done following parameters described by . ...
Article
Full-text available
In this study, an experiment was conducted to investigate the effects of two levels of Bacillus subtilis ATCC 6051a (BS), respectively Bacillus licheniformis ATCC 21424 (BL) administered as direct-fed microbial DFM probiotic bacteria on plasma biochemical profile of weaning piglets fed diets. A total of 100 piglets, 30±3 day-old, with initial average body weight (BW) of 8.53±0.17 kg were randomly distributed to 5 homogeneous groups (C, E1-BS 1%, E2-BS 3%, E3-BL 1%, E4-BL 3%,), 2 replicates/group with 10 piglets/pens, for 16 days of biological trial. The doses used were: 1% low dose in a concentration of 1.6 x 109 CFU spores g-1 feed, and 3% high dose in a concentration of 4.8 x 109 CFU spores g-1 feed. Results showed that BS supplementation affected plasma concentration of GGT (P<0.017), TG (P<0.048) and Mg (P<0.0001). No effect (P>0.05) as diet*day interaction on plasma metabolic profile was observed. In conclusion, administration of Bacillus spp. maintains the concentration of blood plasma parameters within the physiological ranges, without affecting the piglet’s health status.
... Microbial flora was assessed for Lactobacillus spp. [LABs on MRS agar (Man, Rogosa and Sharpe)], Escherichia coli biotype β-haemolytic [Trypticase soy agar (TSA, Sanimed) + 5% sheep blood (w/v), Dumitru et al., 2018], Salmonella spp. (Slanetz-Bartley agar, Oxoid). ...
... Our pH values are in concordance with the results reported by Heo et al. (2012), which confirms that the pH of different areas of the gastrointestinal tract of piglets in the weaning crisis is, for example, in range of 6.0 to 7.4 in ileum segment, respectively 5.4 to 6.7 in caecum content. Dumitru et al. (2018; presents some results of the probiotic properties of Bacillus spp. including pH and bile salts resistance, these being significant criteria for selecting a probiotic product for use in animal nutrition. ...
Article
Full-text available
Direct-fed microbial (DFM) supplementation in piglet's nutrition may offer high benefits to the young animals by diminishing the bacteria pathogens from the gastrointestinal tract and environment. In this study, we evaluated the efficiency of Bacillus subtilis ATCC 6051a (BS, 1.6 x 10 9 CFU/mL) on the piglet's microbiota in the weaning crisis. A total of 60 piglets, 30 days ± 3 days of age, were allotted in 3 homogeneous groups (C, E1 and E2, 10 piglets/6 pens, 2 replicates/group) supplemented with BS 1% (E1), respectively 3% (E2)/kg feed. At the end of the trial (46 ± 3 d-old), 6 piglets were slaughtered (2 piglets/group) and intestinal content (ileum and cecum) were collected for evaluating the microbiota and intestinal pH values. The piglet's faces were collected for microbial analysis on 1-d, 8d, and 16 d. The numbers of lactic acid bacteria (LAB), Coliforms bacteria, Enterococcus spp., Clostridium spp. and Bacillus spp. from intestinal content were modified at the addition of BS, whereas, from faeces samples, the microbiota was insignificant (P ≥ 0.05). The addition of BS 1% and 3% decreased the numbers of Escherichia coli biotype β-hemolytic from piglet's intestinal content (P ≤ 0.05) and faces vs. C group. Salmonella spp. was not present. The intestinal pH from the ileum and cecum segment was observed to be lower in E1 + BS 1%, while in E2 + BS 3% the pH was higher vs. C group. In conclusion, 1% supplementation of Bacillus subtilis ATCC 6051a in piglets feed had a positive effect during the post-weaning period on endogenous microbiota, fecal microbial count and intestinal pH evolution.
... It is well known that Bacillus genus is very famous for alpha-amylase production [6]. Some Bacillus strains as B. subtilis, B. licheniformis, B. cereus, B. stearothermophilus, and B. amyloliquefaciens are isolated for amylase production and their utilization are involved in raw starch degradations [7][8][9][10][11]. In addition, solid-state fermentation (SSF) can be used as a carbon and energy source for cultivation of microorganisms on moist solid supports [12]. ...
Article
In the present investigation, the production of amylase, cellulase, and protease activity by Bacillus subtilis ATCC 6051a was evaluated on different raw materials (soybean meal, peas, sorghum flour, corn) and combined feed (CF). The effect of various fermentation conditions (24-72 h) on enzymatic production through shake-flask culture (Erlenmeyer 100 mL) in optimum conditions (150 rpm, pH 7.0±0.2, 37•C) was investigated.
... Blood agar plates [Trypticase soy agar (TSA, Sanimed) containing 5% (w/v) sheep blood] were used to test hemolysis activity. Interpretation was followed after incubation at 37 • C, for 24 h[29]. ...
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