Volume 9, Issue 4, April – 2024 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165 https://doi.org/10.38124/ijisrt/IJISRT24APR399

Isolation of Amylolytic Lactic Acid Bacteria from

Cow, Goat and Sheep Milk in Yola Town,
Adamawa State, Nigeria
*Tyokusa, A. G. Danke, I. C.
Microbiology Department Microbiology Department
Joseph Sarwuan Tarka University Modibbo Adama University
(Formerly Federal University of Agriculture), (Formerly Federal University of Technology),
Makurdi, Nigeria. Yola, Nigeria

Corresponding Author: *Tyokusa, A. G.

Abstract:- In this study, lactic acid bacteria (LAB) were Amylase producing lactic acid bacteria known as
isolated from fresh milk samples from cow, sheep, and amylolytic lactic acid bacteria (ALAB) secretes amylases,
goat using MRS agar as a growth medium. which make it easier to hydrolyze and ferment starch into
Lactobacillusplantarum, Lactobacillusacidophilus, lactic acid in a single step (Reddy etal., 2008).The
Lactobacillusfermentum, Enterococcusgallinarum, and manufacture of food additives like organic acids (like lactic
Enterococcusfaecium were the species of LAB that were acid) and enzymes (like alpha amylase) is done in the food
isolated and identified using the Vitek 2 compact system. processing industries using ALAB enzymes (Panda and
While Lactobacillus fermentum, Enterococcus faecium, Ray, 2016). The ALAB produce amylases, which are starch-
and Lactobacillus plantarum were isolated from goat modifying enzymes with a wider range of uses than
milk, Lactobacillus acidophilus, Lactobacillus gallinarum, chemical starch hydrolysis in the food processing sectors
and Lactobacillus plantarum were isolated from cow (Smerilli et al. 2015).Among these essential enzymes in the
milk.However, from sheep milk, only Lactobacillus realm of biotechnology is amylase.From a fungal base,
plantarum and Lactobacillus acidophilus were isolated. amylase appears to have been the first enzyme manufactured
All the isolates were subjected to amylase production test industrially in 1894.In the treatment of digestive diseases, it
using modified MRS media and the results showed that was utilized as a medicinal acid(Shanmugasundaram etal.,
Lactobacillus plantarum, Lactobacillusacidophilus and 2015). According to Sachdev et al (2016), amylase is one of
Lactobacillusfermentum produced amylase while the leading enzymes used in industry from decade and that
Enterococcus gallinarum and Enterococcus faecium did microorganisms such as Bacillus species are the major
not produce amylase. source of production of amylase because of the ease of
availability, manipulation and operation.In contrast to
Keywords:- Milk, LAB, Vitek 2,Amylase and Modified MRS. chemical starch hydrolysis in the starch processing sector, a
wide range of commercially available microbial amylases
I. INRODUCTION now hold potential applications.Among other bacteria that
produce amylase, the most recently found ALAB strain
One of the most important types of bacteria in the food Lactobacillusparacasei B41 was the first amylolytic
industry are the lactic acid bacteria (LAB).People all over representative of the L. casei group(Bhanwar and Gangul,
the world have long consumed them in dairy products, and 2014).
the majority of them are categorized as "generally
recognized as safe" (GRAS) microorganisms because they Milk is theprimary source of nutrition for young
are non-pathogenic, appropriate for industrial and mammals (including breastfed human infants) before they
technological processes, tolerant of bile and acid, and are able to digest solid food (Van Winckel etal., 2011). The
capable of producing antimicrobial substances (Shehata et primary players in milk fermentation are lactic acid bacteria,
al., 2016).Pediococcus, Enterococcus, Lactobacillus, which work with amylase and other produced enzymes to
Streptococcus, Leuconostoc, and other bacteria are among convert lactose to lactic acid. This increased acidity makes
the cocci and bacilli in the LAB group (Mazzoli et al., the growth conditions of microorganisms other than LAB
2014).Application areas for lactic acid bacteria (LAB) increasingly unfavourable (Fernandez et al., 2015).
include food, agriculture, and medicine (Bintsis,
2018).Furthermore, lactic acid bacteria produce a variety of Considering cattle, sheep and goats rearing as major
enzymes, including lipases, ureases, peptidases, amylases, occupationinAdamawa state of Nigeria, fresh milk is always
and proteases. in abundant supply. Therefore, isolating amylase producing
lactic acid bacteria from cow, sheep and goat milk in the
state will create an avenue for exploring and harnessing the
available resources within the state. Consequently, the aim

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Volume 9, Issue 4, April – 2024 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165 https://doi.org/10.38124/ijisrt/IJISRT24APR399

of this research work was to isolate and identify the amylase Manganese sulphate-0.05 all g/L/, pH-6.5 at 250C) (TITAN
producing lactic acid bacteria from cow, goat and sheep BIOTECH LTD India) for 24 to 48 hours at room
milk from Yola, Adamawa state of Nigeria. temperature (30-370C). Pure isolates were obtained by sub-
culturing a representative sample of distinct colonies spotted
II. METHODOLOGY on MRS agar.The bacteria isolates were identified using the
Vitek 2 compact system (BIOMERIEUX, France) in
A. Sample Collection, Isolation and Identification of addition to the catalase test and microscopic identification.
Bacteria Isolates
Five samples of fresh cow, goat, and sheep milk each B. Detection of Amylase Activity
were collected in sterile containers, transported to the In order to detect amylase activity, the isolated lactic
laboratory, and left for a 24-hour fermentation period. In acid bacteria were grown on modified MRS media
total, fifteen samples were obtained.Serial dilutions were containing 0.5% peptone, 0.7% yeast extract, 0.2% NaCl,
made for all the samples and appropriate dilutions (10-5 to 2% starch, and 1.5% agar. Following a 24-48-hour
10-9) inoculated on MRS agar (Dextrose-20, Agar 12., incubation period at 37°C, Gram's iodine solution (iodine:
Proteose peptone-10, Beef extract-10, Yeast extract-5, 0.2%, KI: 0.4%, distilled water: 100 mL) was added and the
Sodium acetate-5, Ammonium citrate-2, Dipotassium results were monitored (Madhavet al., 2011).
phosphate-2, Tween 80-1, Magnesium sulphate-0.10 and

III. RESULTS

 Results of Bacteria Isolates from Cow, Goat and Sheep Milk

Table 1: Catalase Test and Microscopic Identification of the Isolates

S/N ISOLATE CODE CATALASE TEST GRAM REACTION SHAPE
1 C1 _ + Rods
2 C2 _ + Rods
3 C3 _ + Cocci
4 G1 _ + Rods
5 G2 _ + Rods
6 G3 _ + Cocci
7 S1 _ + Rods
8 S2 _ + Rods
KEY: C=Cow Milk Isolate, S= Sheep Milk Isolate, G= Goat Milk Isolate + = Positive, - = Negative

Table 2: Lactic Acid Bacteria Identified using the Vitek 2 Compact System from the Milk Samples
S/No Source Lactic Acid Bacteria
1 C1 Lactobacillus fermentum
2 C2 Lactobacillus plantarum
3 C3 Enterococcus gallinarum
4 G1 Lactobacillus plantarum
5 G2 Lactobacillus fermentum
6 G3 Enterococcus faecium
7 S1 Lactobacillus acidophilus
8 S2 Lactobacillus plantarum
KEY: C = Cow Milk, G = Goat Milk and S = Sheep Milk

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Volume 9, Issue 4, April – 2024 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165 https://doi.org/10.38124/ijisrt/IJISRT24APR399

 Results of Lactic Acid Bacteria That Produce Amylase

Plate 1: Detection of Lactic Acid Bacteria's Amylase Activity on a Modified MRS Agar Plate

Table 3: Amylase Producing Lactic Acid Bacteria

S/N ISOLATE CODE ISOLATE NAME AMYLASE PRODUCTION
1 C1 Lactobacillus fermentum +
2 C2 Lactobacillus pantarum +
3 C3 Enterococcus gallinarum -
4 G1 Lactobacillus plantarum+
5 G2 Lactobacillus fermentum+
6 G3 Enterococcus faecium-
7 S1 Lactobacillus acidophilus+
8 S2 Lactobacillus plantarum+
KEY: C = isolate from cow milk, G = Isolate from goat milk. S = Isolate from sheep milk,
(+) = produce amylase and (-) = do not produce amylase

IV. DISCUSSION goat milk that are Gram positive. Raw cow milk wasused to
isolate and identify LAB genera, including Lactobacillus,
Based on this finding, the bacteria isolated were Gram Lactococcus, Leuconostoc, Pediococcus, Streptococcus, and
positive and catalase negative.Altogether,a total of six rods Bifidobacterium species(Fesseha etal., 2021). Similarly, the
and two cocci bacteria were isolated (Table 1).The Vitek 2 raw cow milk was also used to isolate the LAB
compact system was used to identify the bacteria isolates namely;Enterococcus, Lactobacillus, Leuconostoc,
(Table 2).The isolates identified were Streptococcus, Lactococcus, and Pediococcus species
Lactobacillusfermentum, Lactobacillusplantarum, (Wassie and Wassie, 2016).Guessas and Kihal (2004), also
Lactobacillusacidophilus, Enterococcusgallinarum and isolated LAB namely Lactococcus sp,
Enterococcusfaecium.L. plantarum was found in all the Streptococcusthermophilus, Leuconostoc sp,
three milk samples while L. fermentum were found in goat Lactobacilluscurvatus, L. helviticus, L. plantarum,L. reuteri,
and sheep milk. L. acidophilus was however, found only in L. casei, L. brevis, L. bulgaricus, L. paracasei and L.
cow milk sample.Similarly, Enterococcusgallinarum was caidophilus from goat milk.
present in cow milk only while Enterococcusfaecium was
isolated only from goat milk.All the isolates are members of The Lactobacilli genera; Lactobacilli plantarum, L.
the lactic acid bacteria (LAB). This agrees with Agagunduz pentosus, L. delbrueckii, L. helveticus, and L. paracasei-
etal (2021), who reported that, a variety of live lactic acid associated with a fermented goat milk product from
bacteria species can be found in fermented dairy Tajikistan were isolated byCho et al, (2018).In contrast,
products.Tyokusa and Owuama (2023), also isolated lactic Lactobacillus plantarum YN.1.3, a lacticacidbacterium, was
acid bacteria(Enterococcusgallinarum and isolated by Yelnetty et al (2020), from goat milk.According
Enterococcusfaecium) and additional bacteria from cow and to Chen etal(2020), Hu sheep milk was used to isolate

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Volume 9, Issue 4, April – 2024 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165 https://doi.org/10.38124/ijisrt/IJISRT24APR399

Lactococcuslactis, Leuconostoc lactis, and a 97-amino acid starch-binding domain (SDB) (Wang et al.,
Sphingomonas.Lactobacillusplantarum, Lactobacillus 2006). A 954 amino acid protein with an open reading frame
rhamnosus, Lactobacillus acidophilus, and of 2862 bp was found to be encoded by the whole nucleotide
Bacillusshackletonii are the four species of lactic acid sequence of the LactobacillusamylovorusamyA gene(Giraud
bacteria (LAB) that were isolated from sheep milk and and Cuny, 1997).
identified using physiological, biochemical, and 16S rRNA
sequencing study (Patil et al., 2019). V. CONCLUSION

All the Lactobacillus species isolated produced Fresh milk from cows, sheep, and goats obtained from
amylase while Enterococcus species did not produce Adamawa State, Nigeria, like any other place,is a good
amylase (Table 3).Amylolytic Lactobacillus species have source of amylase producing lactic acid bacteria such as
been isolated from milk and other sources. Lee et al (2006), Lactobacillusfermentum, Lactobacillusplantarum, and
isolated L. acidophilus and L. fermentum with high amylase Lactobacillusacidophilus. The importance of this enzyme in
activity from intestinal contents of pigs. Also, South African food and other industries cannot be over emphasized. It is
barley was used to isolate strains of Lactobacillus therefore important to harness the readily available milk in
plantarum that producedα-amylase (Hattingh et al., Adamawa state for the commercial production of the
2015).Likewise, from traditional fermented Dadih, Elida amylase enzyme.
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