Content uploaded by Mihaela Dumitru
Author content
All content in this area was uploaded by Mihaela Dumitru on Jan 17, 2019
Content may be subject to copyright.
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 (γ-
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 (γ- 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.
REFERENCES
1. Abdhul, K., Ganesh, M., Shanmughapriya, S.,
Vanithamani, S., Kanagavel, M., Anbarasu, K.,
Natarajaseenivasan, K. (2015). Bacteriocinogenic
potential of a probiotic strain Bacillus coagulans
[BDU3] from Ngari. International Journal of
Biological Macromolecules, 79, 800–806.
2. Aruwa, C.E., Olatope, O.A. (2015). Characte-
rization of Bacillus species from convenience
foods with conventional and API Kit Method: A
comparative analysis. Journal of Applied Life
Sciences International, 3 (1), 42-48.
3. FAO 2016. Probiotics in animal nutrition –
Production, impact and regulation. By Bajagai,
Y.S., Klieve, A.V., Dart, P.J., Bryden, W.L. Ed.
H.P.S. Makkar. FAO Animal Production and
Health Paper, 179, Rome, Italy.
(http://www.fao.org/3/a-i5933e.pdf).
4. Campbell, J.M., Crenshaw, J.D., Polo, J. (2013).
The biological stress of early weaned piglets.
Journal of Animal Science and Biotechnology, 4
(19), 1-4.
5. Cutting, S.M. (2011). Bacillus probiotics. Food
Microbiology, 28, 214-220.
6. Davis, M.E., Parrott, T., Brown, D.C., De Rodas,
B.Z., Johnson, Z.B., Maxwell, C.V., Rehberger, T.
(2008). Effect of a Bacillus-based direct fed
microbial feed supplement on growth perfor-
mance and pen cleaning characteristics of gro-
wing finishing pigs. Journal of Animal Science,
88, 3320-3326.
7. Dumitru, M., Sorescu, I., Jurcoane, S., Câm-
peanu, G., Tabuc, C., Hăbeanu, M. (2017). As-
sessing of morphological, cultural, biochemical
profile and enzymatic activity of a Lactobacillus
paracasei CCM 1837 strain. Academy of Ro-
manian Scientists Annals, Series on Biological
Sciences, 6 (2), 22-31.
8. Elshaghabee, F.M.F., Rokana, N., Gulhane, R.D.,
Sharma, C., Panwar, H. (2017). Bacillus as po-
tential probiotics: Status, concerns, and future
perspectives. Frontiers in Microbiology, 8 (1490),
1-15.
9. FAO/WHO (2001). Health and nutritional
properties of probiotics in food including powder
milk with live lactic acid bacteria. Report of a Joint
FAO/WHO Expert Consultation on Evaluation of
Health and Nutritional Properties of Probiotics in
Food including Powder Milk with Live Lactic Acid
Bacteria. In Probiotics in food-Health and nutri-
tional properties and guidelines for evaluation.
FAO Food and Nutrition Paper, 85, 2006, WHO,
FAO. (http://www.fao.org/3/a-a0512e.pdf).
10. Guo, X., Li, D., Lu W., Piao, X., Chen, X. (2006).
Screening of Bacillus strains as potential pro-
biotics and subsequent confirmation of the in vivo
effectiveness of Bacillus subtilis MA139 in pigs.
Antonie van Leeuwenhoek, 90 (2), 139–146.
11. Habeanu, M., Lefter, N., Gheorghe, A., Tabuc,
C., Dumitru, M., Ciurescu, G., Palade, M. (2017).
Effects of dietary peas mixed with linseed (3:1)
on the growth performance, enteritis and certain
serum parameter in weaned piglets. Food and
Feed Research, 44 (2), 173-180.
12. Hosoi, T., Ametani, A., Kiuchi, K., Kaminogawa,
S. (2000). Improved growth and viability of lacto-
bacilli in the presence of Bacillus subtilis (natto),
catalase or subtilisin. Canadian Journal of Micro-
biology, 46, 892-897.
13. Jeon, H.L., Yang, S.J., Son, S.H., Kim, W.S.,
Lee, N.K., Paik, H.D. (2018). Evaluation of pro-
biotic Bacillus subtilis P229 isolated from
cheonggukjang and its application in soybean
fermentation. Food Science and Technology, 97,
94-99.
14. Jung, J.H., Chang, H.C. (2012). Evaluation of the
probiotic potential of Bacillus polyfermenticus CJ6
isolated from meju, a Korean soybean fermen-
tation starter. Journal of Microbiology and Bio-
technology, 22, 1510-1517.
15. Kaewtapee, C., Burbach, K., Tomforde, G., Har-
tinger, T., Silva, A.C., Heinritz, S., Seifert, J., Wil-
tafsky, M., Mosenthin, R.F., Rosenfelder-Kuon, P.
(2017). Effect of Bacillus subtilis and Bacillus
licheniformis supplementation in diets with low-
and high-protein content on ileal crude protein
and amino acid digestibility and intestinal micro-
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
biota composition of growing pigs. Journal of
Animal Science and Biotechnology, 8 (37), 1-15.
16. Lee, J., Park, I., Choi, Y., Cho, J. (2012). Bacillus
strains as feed additives: In vitro evaluation of its
potential probiotic properties. Revista Colom-
biana de Ciencias Pecuarias, 25, 577-585.
17. Lei, Y., Kim, I.H. (2014). Effect of Phaffia rho-
dozyma on performance, nutrient digestibility,
blood characteristics, and meat quality in finishing
pigs. Journal of Animal Science, 92, 171–176.
18. Lese, T.D., Knarreborg, A., Worm, J. (2007). Ger-
mination and outgrowth of Bacillus subtilis and
Bacillus licheniformis spores in the gastroin-
testinal tract of pigs. Journal of Applied Micro-
biology, 104, 1025-1033.
19. Link, R., Kovac, G. (2006). The efect of probiotic
Bioplus 2B on feed efficiency and metabolic
parameters in swine. Biologia, Bratislava, Section
Cellular and Molecular Biology, 61 (6), 783-787.
20. Maneewan, C., Yamauchi, K., Thirabunyanon,
M., Siri, S., Mekbungwan, A., Thongwittaya, N.
(2011). Development of Bacillus subtilis MP and
effective utilization on productivity and micro-
organisms in feces of suckling piglets. The Inter-
national Journal of Applied Research in Veteri-
nary Medicine, 9 (4), 382-387.
21. Markowiak, P., Śliżewska, K. (2018). The role of
probiotics, prebiotics and synbiotics in animal
nutrition. Gut Pathogens, 20 (21), 1-20.
22. Meng, Q.W., Yan, L., Ao, X., Zhou, T.X., Wang,
J.P., Lee, J.H., Kim, H. (2010). Influence of
probiotics in different energy and nutrient density
diets on growth performance, nutrient digestibility,
meat quality, and blood characteristics in
growing-finishing pigs. Journal of Animal Science,
88, 3320–3326.
23. Merchant, H.A., McConnell, E.L., Liu, F., Ramas-
wamy, C., Kulkarni, R.P., Basit, A.W., Murdan, S.
(2011). Assessment of gastrointestinal pH, fluid
and lymphoid tissue in the Guinea pig, rabbit and
pig and implications for their use in drug
development. European Journal of Pharmaceuti-
cal Science, 42, 3-10.
24. Mishra, S., Behera, N. (2018). Amylase activity of
a starch degrading bacteria isolated from soil
receiving kitchen wastes. African Journal of Bio-
technology, 7 (18), 3326-3331.
25. Ortiz, A.C.S., González, A.L., Campa-Córdova,
A.I., Montes, R.E., María del Carmen, F.M.,
Mazón-Suástegui, J.M.M. (2015). Isolation and
characterization of potential probiotic bacteria
from pustulose ark (Anadara tuberculosa)
suitable for shrimp farming. Latin American
Journal of Aquatic Research, 43 (1), 123-136.
26. Breed, R.S., Murray, E.G.D., Smith, N.R. (1957).
Bergey's Manual of Systematic Bacteriology,
Seventh Edition, Williams and Wilkins, Baltimore,
Md, USA.
27. Ritter, A.C., Folmer Correa, A.P., Veras, F.F.,
Brandelli, A., (2018). Characterization of Bacillus
subtilis available as probiotics. Journal of Micro-
biology Research, 8 (2), 23-32.
28. Seker E. (2010). Identification of Candida species
isolated from bovine mastitic milk and their in
vitro hemolytic activity in Western Turkey. Myco-
pathologia, 169, 303 - 308.
29. Siddalingeshwara, K.G., Huchesh, C.H., Put-
taraju, H.P., Karthic, J., Sudipta, K.M., Pramod,
T., Vishwanatha, T. (2010). Screening and cha-
racterization of protease from Bacillus sp. Inter-
national Journal of Applied Biology and Pharma-
ceutical Technology, 1 (2), 575-581.
30. Singh, V., Sharma, R., Sharma, P. (2015). Iso-
lation, screening and optimization of amylase pro-
ducing Bacillus sp. from soil. Asian Pacific
Journal of Health Science, 2 (3), 86-93.
31. Sorokulova, I.B., Pinchuk, I.V., Denayrolles, M.,
Osipova, I.G., Huang, J.M., Cutting, S.M., Urdaci,
M.C. (2008). The safety of two Bacillus probiotic
strains for human use. Digestive Diseases and
Science, 53 (4), 954–963.
32. Stoica, C., Sorescu I. (2017). ABIS online -
Advanced Bacterial Identification Software, an
original tool for phenotypic bacterial identification.
Regnum Prokaryotae (www.tgw1916.net).
33. Nagaraju, E.V., Divakar, G. (2012). Screening
and characterization of protease producing Ba-
cillus spp. from spoiled vegetables and fruits.
International Journal of Advances in Pharmacy,
Biology and Chemistry, 1 (4), 485-488.
34. Vishwanatha, T., Spoorthi, N.J., Reena, V., Di-
vyashree, B.C., Siddalingeshwara, K.G., Karthic,
J., Sudipta, K.M. (2010). Screening of substrates
for protease production from Bacillus lichenifor-
mis. International Journal of Engineering Science
and Technology, 2 (11), 6550-6554.
35. Yang, F., Hou, C., Zeng, X., Wqiao, S. (2015).
The use of lactic acid bacteria as a probiotic in
swine diets. Pathogens, 4, 34-45.
36. Yirga, H. (2015). The use of probiotics in animal
nutrition. Journal of Probiotic Health, 3, 1-10.
37. Zaid, A.A. (2018). Study the effect of probiotic
bacteria isolated from foods on pathogens.
Biomedical Research, 29 (12), 2509-2515.
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
