J. Dairy Sci.

102:124–134
https://doi.org/10.3168/jds.2018-14983
© American Dairy Science Association®, 2019.

Probiotic potential and biochemical and technological properties

of Lactococcus lactis ssp. lactis strains isolated
from raw milk and kefir grains
Oktay Yerlikaya*
Ege University, Faculty of Agriculture, Department of Dairy Technology, 35100, Bornova, İzmir, Turkey

ABSTRACT Key words: Lactococcus lactis ssp. lactis, dairy starter,

probiotics, kefir grain, raw milk
Lactococcus lactis ssp. lactis is one of the most im-
portant starter bacteria used in dairy technology and
INTRODUCTION
it is of great economic importance because of its use
in the production of dairy products, including cheese, Lactic acid bacteria (LAB) are used in the produc-
butter, cream, and fermented milks. Numerous studies tion of many fermented food products. They are gram-
have evaluated the biochemical and probiotic proper- positive, facultative anaerobic, catalase-negative, and
ties of lactococci; however, limited studies on the probi- motile. The LAB do not constitute cytochrome and do
otic characteristics of lactococci were conducted using not form spores. They belong to the order Eubacteriales
strains originating from raw milk and dairy products. under Streptococcaceae and Lactobacillaceae families.
Characterizing the probiotic properties of strains iso- The genera that comprise LAB are Lactobacillus, Lac-
lated from raw milk and fermented milk products is tococcus, Bifidobacterium, Tetragonococcus, Vagococ-
important in terms of selecting starter culture strains cus, Weissella, Streptococcus, Leuconostoc, Aerococcus,
for the production of functional dairy products. In this Oenococcus, Carnobacterium, Enterococcus, Sporolac-
study, biochemical properties (including antibiotic sen- tobacillus, and Pediococcus. The LAB play important
sitivity, lipolytic activity, amino acid decarboxylation, roles in ensuring the protection and safety of dairy
antioxidant activity) and probiotic properties (includ- products through production of antimicrobial agents
ing antimicrobial activity, growth in the presence of including lactic acid, diacetyl, hydrogen peroxide,
bile salts, bile salts deconjugation, and hydrophobicity) and bacteriocins (Salminen et al., 1993; Axelsson and
of 14 Lactococcus lactis strains isolated from raw milk Ahrné, 2000). Bacteriocins, which have the potential
and kefir grains were investigated. Strains originating to serve as food preservatives, have been identified in
from kefir grains had better characteristics in terms dairy starter cultures (Kojic et al., 2006; Balciunas et
of antimicrobial activity and bile salt deconjugation, al., 2013).
whereas strains from raw milk had better hydrophobic- Strains of the genus Lactococcus have cocci mor-
ity and antioxidant activity characteristics. None of the phologies and form single cocci pairs or short chain
strains were able to grow in the presence of bile salt and formations (0.5–1.5 μm) in their growth medium. Their
did not show amino acid decarboxylation or lipolytic optimum growth temperature is 30°C and they can
activities. Biochemical and probiotic properties of L. grow between 10°C and 45°C. Their ability to grow
lactis strains varied depending on the strain and some above 40°C, to grow at higher salt concentrations (>4%
of these strains could be used as functional cultures sodium chloride), and to produce acid from different
depending on their properties. However, these strains sugars (arabinose, lactose, mannitol, and raffinose) vary
did not possess all of the properties required to meet depending on the species (Schlegel, 1997; Furet et al.,
the definition of a probiotic. 2004; Khemariya et al., 2017). They cannot grow in the
presence of 6.5% NaCl or at pH 9.6 (Buyukyoruk, 2007;
Üstün et al., 2009). Their ability to ferment lactose is
critically important, particularly in their use as starter
cultures in the dairy industry. In addition to strains of
lactococci of human origin, it is important to character-
Received April 27, 2018.
Accepted September 13, 2018. ize the probiotic properties of strains isolated from raw
*Corresponding author: oktay.yerlikaya@​ege​.edu​.tr milk, vegetables, and fermented milk products in the

124
 PROBIOTIC POTENTIAL AND PROPERTIES OF LACTOCOCCUS LACTIS 125

selection of starter culture strains. The consumption of MATERIALS AND METHODS

food-origin lactococci at high levels, especially in raw
milk and fermented milk products, is the basic criterion Materials
of their adaptation to the intestinal flora (Delgado et
al., 2007; Parracho et al., 2007; Najera-Dominguez et Isolation and screening of LAB were conducted using
al., 2014; Cavanagh et al., 2015). Lactococcus lactis ssp. 15 raw milk samples (7 cow and 8 goat) obtained from
lactis adapts easily to environments including milk or different regions of Izmir, Turkey, and 5 traditional
containing milk or dairy products and hence quickly kefir grain samples obtained from Department of Dairy
and easily metabolizes lactose (Gutiérrez-Méndez et Technology, Faculty of Agriculture, Ege University,
al., 2010). Probiotic bacteria include Lactobacillus and Turkey.
Bifidobacterium spp., and some yeasts, including Sac-
charomyces boulardii, have probiotic properties (Yadav Methods
et al., 2009).
Benefits of probiotic bacteria to human health in- Ten grams of each raw milk or kefir grain sample
clude improving the gut microbiota balance and de- was weighed into a filtered stomacher bag and mixed
fending against pathogens. Other beneficial effects of with 90 mL of sterile 0.1% (wt/vol) peptone water
probiotic microorganisms include stimulation of the (Merck, Darmstadt, Germany). All samples were seri-
immune system, reduction of blood cholesterol lev- ally diluted and 1 mL of each dilution was plated onto
els, synthesis of vitamins (particularly the vitamin B M17-lactose agar (Merck) containing nalidixic acid
group), and anticarcinogenic and antimicrobial activi- (Fluka, Steinheim, Switzerland). The M17 agar plates
ties. Other criteria for a product to be regarded as a were incubated at 30°C under aerobic conditions for
probiotic are consumer acceptance and survival of the 48 h. Following the incubation, appropriate colonies
probiotic microorganisms through the gastrointestinal that have different morphologies (color, appearance,
tract. The recommended microorganism count in sub- and shape) were transferred to Elliker broth (Difco,
jects consuming 100 g/d of Lactobacillus acidophilus, Fluka). Samples were incubated at 30°C for 48 h (Cor-
Bifidobacterium animalis ssp. lactis, Lactobacillus casei, roler et al., 1998; Buyukyoruk and Soyutemiz, 2010).
and other probiotic bacteria is 6 to 7 log cfu/g or log A simple stain (methylene blue) was applied follow-
cfu/mL (Sarao and Arora, 2017). The health benefits ing morphological examination, and all gram-positive
of probiotic lactic acid bacteria vary depending on the and catalase-negative (H2O2, 2% vol/vol) isolates were
strain, and these effects take place by different mecha- purified and observed under a light microscope. The
nisms. strains isolated from raw cow milk, raw goat milk, and
There are various criteria for selecting the appropri- kefir grains were coded as K (K1, K2, K3, K5, K7, K8,
ate bacteria to be used as probiotics (Radulović et al., K9, and K10), Z (Z3, Z6, Z9, and Z10), and KR (KR4
2010; Soccoll et al., 2010; Giraffa, 2012). Lactococcus and KR9), respectively. Isolates were screened for their
lactis can mostly be isolated from dairy products in- ability to ferment 6 carbohydrates critical in L. lactis
cluding raw milk and kefir grains. It is also used in ssp. lactis identification (sorbitol, mannitol, arabinose,
starter cultures used for production of different dairy raffinose, xylose, and rhamnose). Lactococcus lactis ssp.
products including cheese. Based on its history of use lactis NRRL-B-1821 was used as the reference strain.
in food fermentations, L. lactis has GRAS (generally All isolates were coded and stored in M17 broth con-
regarded as safe) status. Lactococcus lactis produces taining equal amounts of 40% sterile glycerol at −20°C.
lactic acid in dairy starter cultures and help to break After phenotypic identification, all bacteria were identi-
down milk proteins during fermentation, contributing fied by PCR.
to the sensory and microbiological properties of the
product (Cavanagh et al., 2015). In vitro and in vivo DNA Isolation and Sequence Analysis
studies on the probiotic properties of L. lactis have sug-
gested its use as a potential probiotic strain, in addi- Cultures were activated twice in Elliker broth and
tion to improving the nutritional value of foods (Yadav inoculated by swiping on M17-lactose agar. Bacterial
et al., 2009; Lee et al., 2015). Raw milk maintains a DNA isolation, PCR, and sequencing analysis of the
suitable growing environment for Lactococcus species. bacteria were carried out at REFGEN Gene Research
Therefore, the aim of this study was to investigate and and Biotechnology Laboratories (METU Technokent,
characterize the probiotic potential and biochemical Ankara, Turkey). Qiagen DNeasy Blood and Tissue
and technological properties of L. lactis strains isolated DNA isolation kit (Qiagen, Hilden, Germany) was used
from raw milk and kefir grains. for DNA isolation. For PCR, 50 µL of 1× Taq buffer,

Journal of Dairy Science Vol. 102 No. 1, 2019

126 YERLIKAYA

1.5 mM MgCl, 0.2 mM deoxynucleotide triphosphates, ism/mL) and a standard opacity was formed. Once the
0.3 pmol/µL of each primer [27F (5′-AGAGTTTGAT- agar had solidified, the dishes were stored for 2 h in a
CATGGCTCAG-3′) and 1492R (5′-GGTTACCTTGT- refrigerator. Four wells were made and filled using 10
TACGACTT-3′)], and 1.25 U of Taq DNA polymerase μL of the cell-free filtrate prepared previously. Petri
(Thermo, Waltham, MA) were used. The PCR analysis dishes were incubated at 37°C for 24 h. The inhibitory
was performed following these steps: pre-denaturation spectrum of the antimicrobial agent produced by the
for 5 min at 94°C; 30 cycles of denaturation for 30 s at isolates of L. lactis strains against different indicator
94°C; annealing for 30 s at suitable temperature for each bacteria was determined by disk diffusion assay using
primer pair; extension for 30 s at 72°C; and final exten- Whatman 6-mm-diameter sterile disks (Rammelsberg
sion for 5 min at 72°C. For DNA sequencing reactions, and Radler, 1980).
Applied Biosystems BigDye v3.1 Cycle Sequencing kit Growth in the Presence of Bile Salt. The ability
(Amersham Pharmacia Biotech Inc., Piscataway, NJ) of L. lactis strains to grow in the presence of bile salt
was used. Following the sequencing reactions, samples was determined according to Vinderola and Reinheimer
were analyzed by using an ABI 3100 Genetic Analyzer (2003), with modifications. Strains were inoculated
(Applied Biosystems, Carlsbad, CA). The results were (1% vol/vol) into Elliker broth (Difco, Fluka) and were
compared with the National Center for Biotechnology incubated twice at 30°C for 24 h, and cells were cen-
Information gene bank (http:​/​/​www​.ncbi​.nlm​.nih​.gov) trifuged and suspended. Then, M17 agar containing 0,
and aligned with ClustalW (https:​/​/​www​.genome​.jp/​ 0.3, 0.5, 1, or 2% (wt/vol) bile salt (Sigma Chemical
tools​-bin/​clustalw); the phylogenetic tree was drawn Co., St. Louis, MO) was inoculated with 10 µL of ac-
using the neighbor-joining method. tive culture. Results were evaluated as “growth” or “no
growth.”
Probiotic Properties of L. lactis ssp. lactis Strains Bile Salt Deconjugation. Bile salt deconjugation
ability of L. lactis strains was determined according
Antimicrobial Activity. The optical densities (OD) to the method of Taranto et al. (1995), with minor
of the active cultures were standardized by adjusting to modifications. Elliker agars were prepared by adding
0.6 ± 0.2 at 600 nm using a spectrophotometer (Ana- 0.5% (wt/vol) sodium salts of taurocholic acid, tauro-
lytik Jena AG, Jena, Germany). To confirm that the deoxycholic acid, glycocholic acid, and glycodeoxycholic
antimicrobial activity was not related to acidity, cell- acid (Sigma Chemical Co.). Elliker agar without bile
free culture supernatants at neutral pH were used, and salts was prepared as the control. The Petri dishes were
the antimicrobial assays were prepared by growing the inoculated with 10 µL of active L. lactis culture and
isolates in M17-lactose broth (Merck) at 30°C for 24 h incubated aerobically at 30°C for 72 h. The presence
and centrifuged at 6,654 × g for 20 min at 4°C. The an- of precipitated bile acid around colonies (opaque halo)
timicrobial activity of the cell-free culture supernatant was considered a positive result.
was determined by disk diffusion assay according to Hydrophobicity. The ability of the L. lactis strains
the method of Tagg and McGiven (1971). This was fol- to adhere to hydrocarbons as a measure of their hydro-
lowed by filtration of the supernatant through a 0.22- phobicity was determined according to the method of
µm pore size filter (Merck) to obtain a cell-free filtrate. Vinderola and Reinheimer (2003), with modifications.
The antimicrobial activity of the isolated LAB (cell-free Cultures of the strains were harvested in stationary
filtrate) against microorganisms such as Salmonella en- phase by centrifugation at 8,000 × g for 10 min at 4°C,
terica ssp. enterica CECT 443, Salmonella choleraesuis washed twice in 50 mM K2HPO4 (pH 6.5) buffer, and
ssp. choleraesuis ATCC 13076, Bacillus cereus CECT resuspended in the same buffer. The cell suspension
131, Listeria monocytogenes CECT 932, Listeria mono- was adjusted to absorbance (at 560 nm, A560) of ap-
cytogenes ATCC 7644, Staphylococcus aureus ATCC proximately 1.0 with buffer; then, 3 mL of the bacterial
12600, Escherichia coli ATCC 25922, Escherichia coli suspension was mixed with 0.6 mL of n-hexadecane and
CECT 4267, and Enterobacter aerogenes ATCC 13048 vortexed for 120 s. The 2 phases were left to separate
was assessed by disk diffusion assay (Warminska-Radyo for 30 min at 37°C. The aqueous phase was carefully
et al., 2002). The pathogenic test bacteria were incu- removed and A560 was measured. The decrease in the
bated in tryptic soy broth (Oxoid, Basingstoke, UK) absorbance of the aqueous phase was taken as a mea-
at the appropriate temperature (30–37°C) for 24 h. sure of the cell surface hydrophobicity (H, %), which
Petri dishes containing 20 mL of tryptic soy agar (Ox- was calculated by using the formula; H (%) = [(A0
oid) were prepared and inoculated with the incubated – A)/A0] × 100, where A0 and A are the absorbance
pathogenic bacteria. The microorganisms tested were values before and after extraction with n-hexadecane,
adjusted according to McFarland 0.5 (108 microorgan- respectively.

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 PROBIOTIC POTENTIAL AND PROPERTIES OF LACTOCOCCUS LACTIS 127

Biochemical and Technological Properties of L. MO) according to Balakrishnan and Agrawal (2012),
lactis ssp. lactis Strains with modifications. Bacteria cultures were subcultured
twice in M17 broth, transferred to reconstituted milk
Screening for Antibiotic Susceptibility. The containing 12% nonfat DM at a 1% ratio, and incu-
disk diffusion method was used to screen the antibi- bated at 30°C for 24 h. The samples were cooled to
otic susceptibility of isolates using 6-mm-diameter 4°C at the end of the incubation, and centrifuged at
disks (Becton, Dickinson and Co., Franklin Lakes, NJ) 6,654 × g for 20 min at 4°C. The separated supernatant
containing 1 or 2 doses of ampicillin (10 and 25 μg), was filtered through a sterile filter (Minisart, Sartorius,
bacitracin (10 μg), cefsulodin (30 μg), chloramphenicol Göttingen, Germany) with 0.22-μm-diameter pore size.
(10 μg), clindamycin (2 and 10 μg), erythromycin (10 One milliliter of 0.1 mM DPPH (prepared in methanol)
and 15 μg), gentamycin (10 and 120 μg), kanamycin was mixed with 1.5 mL of methanol and 0.5 mL of su-
(30 μg), nalidixic acid (30 μg), neomycin (30 μg), novo- pernatant and kept at 30°C for 30 min; absorbance was
biocin (5 μg), oxacillin (1 and 5 μg), penicillin (10 μg), measured at 517 nm. Absorbance of the control sample
polymyxin B (300 μg), streptomycin (50 and 300 μg), was measured by mixing 1.5 mL of DPPH and 1.5 mL
tetracycline (30 μg), and vancomycin (30 μg). Blank of methanol. Antioxidant activity of the bacteria was
disks were used as the negative control. The tests were calculated using the following formula.
performed according to the criteria of the Clinical and
Laboratory Standards Institute (CLSI) using Elliker Antioxidant activity (%) = A – [(B − C) × 100/A],
agar. Inhibition-zone diameters were measured after
aerobic incubation at 30°C for 24 h. To facilitate the where A = absorbance of DPPH solution with no sam-
evaluation and interpretation of the results, the inhibi- ple; B = absorbance of sample + DPPH solution; and
tion zones were evaluated as follows: 6 to 8 mm = −; 9 C = absorbance of blank, which did not contain DPPH.
to 14 mm = +, 15 to 20 mm = ++, and 21 to 30 mm
= +++ (Wikler, 2006; Shazali et al., 2014).
RESULTS AND DISCUSSION
Decarboxylase Activity. The ability to produce
biogenic amines by decarboxylation of AA was tested Based on the phenotypic and sequence analyses, the
on a medium designed by Bover-Cid and Holzapfel 14 strains with the best morphological and growth
(1999), which contained either of the precursor AA, properties were L. lactis ssp. lactis, and the biochemical
Tyr or Lys. To induce decarboxylase activity before the and probiotic properties of those strains were analyzed.
screening test, the L. lactis strains were subcultured Lactococcus lactis ssp. cremoris and L. lactis ssp. lactis
twice in M17 broth (Merck) containing 0.1% of each are mostly found in dairy products, including raw milk,
precursor AA and 0.005% pyridoxal-5-phosphate. The soft and hard cheeses, and sour cream (Ward et al.,
latter compound was previously shown to be impor- 2002; Casalta and Montel, 2008). However, some stud-
tant for inducing decarboxylase activity (Recsei et al., ies have reported that Lactococcus species are found
1983). Ten-microliter volumes of each bacteria culture not only in milk and dairy products but can be isolated
were then spotted onto agar plates with or without from other sources, such as German sauerkraut (Harris
each AA and the plates were incubated aerobically at et al., 1992), fermented sausages (Noonpakdee et al.,
37°C for 2 to 5 d. Plates were observed for a purple 2003), river water (Zendo et al., 2003), and human milk
color surrounding the formed colonies. (Beasley, 2004).
Lipolytic Activity. Lactococcus lactis strains were
grown overnight at 30°C in M17 broth. Then, 10 µL Probiotic Properties of L. lactis ssp. lactis Strains
of fresh culture was placed on tributyrin agar contain-
ing 10 mL/L neutral tributyrin (glycerol tributyrate; Antimicrobial Activity. Inhibitory effects of
Merck; Leuschner et al., 1997). Plates were incubated LAB are due to the natural protective organic acids,
at 30°C for 5 d and observed daily for halo formation hydrogen peroxide, diacetyl, bacteriocins, and specific
around the colonies. The radius of the halo formation substances, such as antiviral peptides or low-molecular-
(in mm) at the end of incubation was measured. For weight peptides (reuterin, reutericyclin; Fan and Song,
better monitoring of zone formation, 5% acetic acid 2013; Viana de Souza and Silva Dias, 2017). The LAB
solution was poured onto the Petri dishes, making the inhibit the development of pathogenic microorganisms
zones on the surface become more visible. and food-degrading microorganisms by competing for
Antioxidant Activity. The antioxidant activity of food with pathogens that shorten the shelf life of food
L. lactis strains was estimated by 1,1-diphenyl-2-pic- products. The most commonly known bacteriocins pro-
rylhydrazyl (DPPH) assay (Sigma-Aldrich, St. Louis, duced by lactococci are nisin, lactococcin A, lactococ-

Journal of Dairy Science Vol. 102 No. 1, 2019

128 YERLIKAYA

cin B, lactococcin MN, lactococcin G, lactococcin 972,

lacticin 481, lacticin 3147, lacticin FS92, lacticin RM,

ATCC 13048
Enterobacter
aerogenes
lacticin NK24, lactococcin R, lactococcin MMT24, and

12
13
11
13
10
11
10
11
8
12
10
11
12

−
lactococcin MMFII, (Davidson and Hoover, 1993; Rat-
tanachaikunsopon and Phumkhachorn, 2010; Šušković
et al., 2010). Table 1 shows that none of the Lacto-
coccus lactis strains studied here showed antimicrobial

CECT 4267
Escherichia
activity against Bacillus cereus CECT131. Lactococcus

coli

10
11
9
10
lactis K5 had an effect only on Enterobacter aerogenes

−
−
−
−
−
−
−
−
−
−
ATCC 13048, whereas K9 had an effect only on Liste-
ria monocytogenes ATCC 7644. However, K9 had no

Table 1. Antimicrobial activity (as measured by average zone diameter, mm) of Lactococcus lactis ssp. lactis strains against different pathogens
effect on Listeria monocytogenes CECT 932. Similarly,

Strains K1 to K10 were derived from raw cow milk; Z3, Z6, Z9, and Z10 were from raw goat milk; and KR4 and KR9 were from kefir grains.
ATCC 25922
Escherichia
a lower level of inhibitory activity was observed against
Escherichia coli CECT 4267, whereas activity against

coli

11
12
12
12
12
9

−
−
−
−
−
−
−
−
Escherichia coli ATCC 25922 was higher. Strains Z9
and Z3 were not effective against Bacillus cereus CECT
131 but had the highest antimicrobial activity against
other pathogens. Additionally, the lowest antimicrobial

Staphylococcus

ATCC 12600
activity was observed against Salmonella choleraesuis

aureus

11
10
14
10
12
12
ssp. choleraesuis ATCC 13076 and Escherichia coli

−
−
−
−
−
−
−
−
CECT 4267 bacteria. Only 4 of the L. lactis strains
(Z3, Z6, Z9, and Z10) were effective against these 2
indicator bacteria. In generally, most of the L. lactis
strains isolated and identified in the study were effec- monocytogenes
tive against Enterobacter aerogenes ATCC 13048; only ATCC 7644
Listeria

14
12
13
14
10
15
14
10
strain K10 had no effect on this pathogen. The best- 14

−
−
−
−
−
defined bacteriocin is nisin, which can be produced by
some strains of L. lactis.
Growth in Bile Salts at Different Concentra-
monocytogenes

tions. The physiological concentration of bile salts in

CECT 932
Listeria

the intestinal tract varies between 0.3 and 0.5% (Begley

14
10
12
13
11
12

−
−
−
−
−
−
−
−

et al., 2005). The resistance to bile salts property of

probiotic microorganisms is associated with activity of
bile salt hydrolase, which reduces the inhibitory effect
of bile by hydrolyzing conjugated bile salts (Oh et al.,
CECT 131

2000; Mourad and Nour-Eddine, 2006). The growth of

Bacillus
cereus

L. lactis strains in the presence of various concentra-

−
−
−
−
−
−
−
−
−
−
−
−
−
−

tions of bile salts is shown in Table 2. None of the

strains were able to grow in the presence of 0.3, 0.50,
1, or 2% bile salt. In contrast to the results obtained
choleraesuis ssp.

in the current study, Vinderola and Reinheimer (2003)

ATCC 13076
choleraesuis
Salmonella

reported that the viability of 9 strains of L. lactis in

10
11
13
11

−
−
−
−
−
−
−
−
−
−

the presence of 0.3% bile salt varied between 21.3 and

Negative (no antimicrobial activity).

76.6%. Vinderola and Reinheimer (2003) found that

probiotic bacteria are more resistant to bile salt than
are LAB; specifically, most Streptococcus thermophilus
species are susceptible to 0.5% bile salt. Additionally,
enterica ssp.

CECT 443
Salmonella

enterica

previous research (Kim et al., 1999; Kimoto-Nira et al.,

16
11
13
14
14
11
13
−2

−
−
−
−
−
−

2009; Kaya Ozdogan et al., 2012) reported that L. lac-

tis was resistant even to 1% bile salt, whereas Ertekin
and Çon (2014) showed that 2 different L. lactis strains
could grow even in the presence of 9% bile salt. Klijn
Strain1

et al. (1995) reported that Lactobacillus delbrueckii ssp.

KR9
KR4
K10

Z10
K9
K8
K7
K5
K3
K1
K2

bulgaricus and Lactococcus spp. were resistant to bile

Z9
Z6
Z3

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 PROBIOTIC POTENTIAL AND PROPERTIES OF LACTOCOCCUS LACTIS 129
Table 2. Probiotic properties of Lactococcus lactis ssp. lactis strains

Growth in the presence of bile salts Deconjugation of bile salts2

Hydrophobicity
Strain1 Control 0.3% 0.5% 1% 2% Control TC TDC GDC GLC (% ± SE)
K1 + − − − − g + + − g 89.76 ± 0.04
K2 + − − − − g g g − g 33.25 ± 0.11
K3 + − − − − g w+ w+ − g 19.09 ± 0.06
K5 + − − − − g g g − g 49.33 ± 0.07
K7 + − − − − g + g − g 37.19 ± 0.06
K8 + − − − − g g g − g 33.19 ± 0.06
K9 + − − − − g g g − g 7.56 ± 0.14
K10 + − − − − g + g − g 85.65 ± 0.05
Z3 + − − − − g + + wg + 51.94 ± 0.06
Z6 + − − − − g w+ w+ − w+ 57.18 ± 0.02
Z9 + − − − − g g g − + 6.30 ± 0.05
Z10 + − − − − g w+ g − w+ 88.20 ± 0.05
KR4 + − − − − g w+ + − + 26.00 ± 0.10
KR9 + − − − − g + + − + 3.20 ± 0.05
1
Strains K1 to K10 were derived from raw cow milk; Z3, Z6, Z9, and Z10 were from raw goat milk; and KR4 and KR9 were from kefir grains.
2
TC = taurocholic acid, TDC = taurodeoxycholic acid, GDC = glycodeoxycholic acid; GLC = glycocholic acid; − = negative, no growth; + =
positive, growth and deconjugation of the bile salts; w+ = weak positive, wg = weak growth, g = growth.

salts. Todorova et al. (2007) reported that L. lactis Four strains showed deconjugation activity in the pres-
strains isolated from human vaginal secretions could ence of glycodeoxycholic bile salt, and weak deconjuga-
grow in the presence of 0.3% bile salt. Lactococcus lactis tion activity detected in 2 strains. Eight strains were
strains isolated from raw milk and kefir grains cannot able to grow in this bile salt. Vinderola and Reinheimer
grow in the presence of bile salt and this is regarded (2003) reported that LAB do not present deconjuga-
as a negative characteristic regarding probiotic proper- tion properties; however, Lactobacillus bulgaricus and
ties. However, the results regarding the ability to grow Streptococcus thermophilus strains were affected by bile
in bile salt were ignored in this study because even salts (taurocholic acid, taurodeoxycholic acid, glyco-
though the lack of growth in bile salts is an unfavor- cholic acid, and glycodeoxycholic acid). Thus, L. lactis
able property of a probiotic, the use of these strains as strains were not able to grow in the presence of bile
functional cultures cannot be overlooked because of the salts at concentrations of 0.3 to 2%, but they were able
positive characteristics of the strains, including bile salt to grow or deconjugate bile salts such as taurocholic
deconjugation. acid, taurodeoxycholic acid, and glycodeoxycholic acid.
Bile Salt Deconjugation Properties. Bile salt Hydrophobicity. Bacterial adhesion was determined
deconjugation has an important role in regulating the to assess the adherence potential of microorganisms to
gut microflora and reducing serum cholesterol levels. surface hydrocarbons, which is a measure of adhesion
Accordingly, the presence of this activity is significant to epithelial cells of the gut (Yadav et al., 2016). Table
when selecting species that will be used as fortification 2 shows the hydrophobicity of L. lactis strains. Hydro-
or adjunct cultures (Corzo and Gilliland, 1999; Vin- phobicity values varied between 3.20 and 89.76%; the
derola and Reinheimer, 2003). Bile salt deconjugation highest hydrophobicity was determined in strain K1.
abilities of L. lactis on 4 bile salts are given in Table 2. Hydrophobic potential is organism- and strain-specific
Five of the L. lactis strains (K1, K7, Z3, Z6, and KR9) and can be affected by the age and surface chemistry of
deconjugated taurocholic acid, 4 strains had weak (K3, strains as well as by the composition of the culture me-
Z6, Z10, and KR4) deconjugation effects, whereas 5 dium (Puniya et al., 2016). Vinderola and Reinheimer
strains (K2, K5, K8, K9, and Z9) had no deconjuga- (2003) found hydrophobicity values of 9 L. lactis strains
tion activity, but showed growth in the presence of bile to range between 14.9 and 31.3%. Hydrophobicity
salts. Four strains fully deconjugated taurodeoxycholic can be advantageous for strains that are in competi-
acid, 2 of the strains had a weak deconjugation activity, tion with other bacteria in the gastrointestinal system
and 8 of the strains were not able to grow in the pres- (Naidu et al., 1999; Todorova et al., 2007). Ouwehand
ence of that bile salt. None of the strains were able to et al. (1999) reported a correlation between surface hy-
deconjugate or grow in the presence of glycodeoxycholic drophobicity and adhesion abilities of bacteria. Kaya-
acid, and only 1 strain presented weak growth activity. Ozdogan (2011) reported that the optical density of a

Journal of Dairy Science Vol. 102 No. 1, 2019

130 YERLIKAYA

cell suspension of L. lactis strain isolated from Turkey

erythromycin (10 and 15 μg); CFS30 = cefsulodin (30 μg); GM10 and GM120 = gentamycin (10 and 120 μg); S50 and S300 = streptomycin (50 and 300 μg); C10 = chloramphenicol
NA30 = nalidixic acid (30 μg); CC2 and CC10 = clindamycin (2 and 10 μg); TE30 = tetracycline (30 μg); NB5 = novobiocin (5 μg); C = negative control, antibiotic-free disc;
B10 = bacitracin (10 μg); N30 = neomycin (30 μg); VA30 = vancomycin (30 μg); AM10 and AM25 = ampicillin (10 and 25 μg); PB300 = polymyxin B (300 μg); E10 and E15 =
++ +++
+++ +++
P10

++
++
++
++
++
++
++
++
++
++
++
was between 0.93 and 1.09 before treatment with n-

+
hexadecane and between 0.15 and 0.18 after treatment,

CFS30 GM10 GM120 S50 S300 C10 K30 OX1 OX5

whereas hydrophobicity was 83.48 ± 1.45%. Although

+
+
+
−
+
+
+
+
+
+
+
+
there is no standard value of hydrophobicity sought in
bacteria, high hydrophobicity is a positive feature in

−
+
+
−
−
−
−
+
−
+
−
−
−
−
terms of probiotic properties.

++

−
−
−
−
−
−
+
−
−
−
−
−
−
Biochemical and Technological Properties

++
++
++
++
++
++
++
++
++
++
++

+
−
−
of L. lactis Strains

++
++
++

+
+
+
+
+
+
+
+
+
+
+
Antibiotic Susceptibility. Antibiotic susceptibility

Strains K1 to K10 were derived from raw cow milk; Z3, Z6, Z9, and Z10 were from raw goat milk; and KR4 and KR9 were from kefir grains.
and resistance of L. lactis strains to various antibiotics

++

+
+
+
+
−
+
+
+
+
+
+
+
+
in different concentrations are given in Table 3. All L.
lactis strains in the study were resistant to 30 µg of

++
++

+
−
+
+
+
+
+
+
+
+
+
+
nalidixic acid and were able to grow in this concentra-
tion. Only L. lactis Z6 showed resistance to clindamycin
at 2 µg, whereas all strains were susceptible to 10 µg

−
−
+
−
−
+
−
−
−
−
−
−
−
−
of clindamycin; 1 strain (Z6) was susceptible to 30 µg
and 4 strains (Z3, Z6, Z10, and KR4) were susceptible

++
++

+
+
+
−
+
+
+
+
+
+
+
+
to 10 µg of tetracycline; 2 strains (Z3 and Z6) were
susceptible to 10 µg of bacitracin; and 2 strains (Z10
Antibiotic and concentration3

++ +++
E15

++
++ ++
++
++
++
++
++ ++
++
++
++
++
++
++ ++
and KR4) were susceptible to 30 µg of neomycin. All
L. lactis strains were susceptible to 30 µg of vanco-

(10 μg); K30 = kanamycin (30 μg); OX1 and OX5 = oxacillin (1 and 5 μg); P10 = penicillin (10 μg).
B10 N30 VA30 AM10 AM25 PB300 E10

mycin, to 10 and 25 µg of ampicillin, and to 10 IU

+
+
+
+
+
+
+
+
+
+
of penicillin, and the diameters of the susceptibility
zones that formed increased as the antibiotic concen-

++
++

−
−
−
−
−
−
−
−
−
−
−
−

trations increased. Three strains (Z3, Z10, and KR9)

Inhibition zones: − = 6–8 mm; + = 9–14 mm; ++ = 15–20 mm; +++ = 21–30 mm.
were susceptible to 300 U of polymyxin B, whereas 1

+++
+++
+++
+++

++
++
++
++
++
++
++
++
++
++

strain (Z6) was resistant to 30 µg of cefsulodin. All

L. lactis strains were susceptible to erythromycin; the

++
++
++
++
++
++
++
++
++
++
++
++
++
++

higher concentration affected the resistance that some

strains have shown. Twelve strains were resistant to
Table 3. Antibiotic sensitivity1 of Lactococcus lactis ssp. lactis strains

+++

10 µg but only 2 strains (Z9 and KR9) were resistant ++

++
++
++
++
++
++
++
++
++

+
+
+

to 120 µg of gentamicin. Similarly, 2 strains (Z6 and

KR9) were resistant to 50 µg of streptomycin and none
−
+
+
−
−
−
−
−
−
−
−
−
−
−

were resistant to 300 µg. It was found that most strains

++
++
++

were susceptible to 10 µg of chloramphenicol and, of

++
++
++
++
++
++
++

+
−
−
+

the 14 strains, only 2 (Z3 and Z6) were resistant. In

+++

++
++
++
++
++
++

contrast, most strains (9 strains) were resistant to 30

+
+
+
+
+
+
+
C

µg of kanamycin. Five strains (K1, K2, K3, Z3, and Z6)

NA30 CC2 CC10 TE30 NB5

were resistant to 1 µg of oxacillin, whereas only 1 strain

++
++
++

+
−
−
+
−
−
+
+
+
+
+

(Z6) was resistant to 5 µg. Kaya-Ozdogan (2011) re-

+++ +++
+++ +++

ported that the nisin A–producing L. lactis LL27 strain

++
++
+++ ++
++
++
++
++
++
++

+
+
−

originating in Turkey was susceptible to penicillin G,

ampicillin, cefazolin, meropenem, imipenem, vanco-
+++
++
++
++
++
++
++
++
++
++
++

mycin, amoxicillin-clavulanic acid, ceftiofur, amikacin,

gentamicin, kanamycin, spectinomycin, tetracycline,
++
++
++
++
++
++
++
++
++
++
++
++
++

chloramphenicol, erythromycin, azithromycin, florfeni-

col, ciprofloxacin, lincomycin, and ofloxacin, but was
−
−
−
−
−
−
−
−
−
−
−
−
−
−

resistant to streptomycin, polymyxin B, and neomycin.

In general, L. lactis strains are susceptible to broad-
Strain2

spectrum antibiotics and β-lactam antibiotics, which

KR9
KR4
K10

Z10
K9
K8
K7
K5
K3
K2
K1

are effective against gram-positive bacteria; however,

Z9
Z6
Z3

Journal of Dairy Science Vol. 102 No. 1, 2019

 PROBIOTIC POTENTIAL AND PROPERTIES OF LACTOCOCCUS LACTIS 131

L. lactis strains are resistant to cefoxitin, amygdalin, characteristics, decarboxylation activity of L. lactis
gentamycin, and kanamycin, antibiotics are effective strains varies depending on the strain.
against gram-negative bacteria. As we observed, the Lipolytic Activity. Lipolysis is the process whereby
susceptibility to tetracycline, cephalothin, nitrofuran- lipids are hydrolyzed to glycerin and fatty acids by the
toin, and cefotetan varies from strain to strain. As in effects of lipolytic enzymes such as lipase. The lipase
lactobacilli, some rare L. lactis strains are resistant to activity of LAB varies depending on genus and species,
chloramphenicol, clindamycin, streptomycin, erythro- and many LAB show limited and weak lipase activity.
mycin, and tetracycline (Flórez et al., 2005; Ammor Although lipolytic activity in products such as milk,
et al., 2007; Khemariya et al., 2017). Sahnouni et al. yogurt, and butter is not highly desired, a certain level
(2012) reported that L. lactis MT and ST2 strains were of lipolysis is desirable in some types of cheese in terms
susceptible to penicillin, chloramphenicol, tetracycline, of formation of aroma and structure. None of the L. lac-
amikacin, and erythromycin, and resistant to vancomy- tis ssp. lactis strains in the study had lipolytic activity
cin and nalidixic acid. (Table 4). Katz et al. (2002), in their study on lipolytic
Decarboxylation Activity. To prevent the forma- activity of LAB, found that L. lactis O233 strain was
tion of biogenic amines in commercial food products, not able to hydrolyze triglycerides including tributyrin.
it is important to fortify these products with starter Sahnouni et al. (2012) reported that L. lactis strains
cultures that do not have AA decarboxylase activity. isolated from the gastrointestinal tract of coastal fish
Biogenic amines are agents that can be formed during had no lipolytic activity. Researchers have noted that
the production, processing, and storage of fermented some bacterial strains had no lipolytic activity in tri-
protein-rich foods, and excessive intake of biogenic butyrin agar containing glycerol tributyrate, although
amines via foods can result in toxic effects (Naila et al., these strains could hydrolyze milk fat and form free
2010; Spano et al., 2010; Linares et al., 2011). Table 4 fatty acids (Martínez-Moreno, 1976; Serio et al., 2010).
shows the decarboxylation activity of L. lactis strains Therefore, strains that had no lipolytic activity in agar
on lysine and tyrosine. None of the strains decarboxyl- did show have lipolytic activity in dairy products.
ated either Lys or Tyr. In contrast, Sahnouni et al. Antioxidant Activity. Several studies have reported
(2012) found that L. lactis MT and ST2 strains pro- antioxidant activities in lactobacilli using milk casein as
duced biogenic amines from lysine, ornithine, histidine, substrate. Several methods can be used to evaluate the
and tyrosine. All strains of L. lactis isolated from raw antioxidant activity of the protein hydrolysate; for ex-
milks and kefir grains tested in the current study failed ample, DPPH, ferrous ion chelating activity, reducing
to decarboxylate lysine and tyrosine, and this is re- power, 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic
garded as a positive characteristic. As with many other acid radical scavenging activity, and linoleic acid and

Table 4. Decarboxylation of AA, lipolytic activity, and antioxidant activity properties of Lactococcus lactis
ssp. lactis strains

Decarboxylation of AA
Lipolytic Antioxidant activity
Strain1 Lysine Tyrosine activity (DPPH % ± SE)2
K1 −3 − − 29.67 ± 0.03
K2 − − − 32.26 ± 0.04
K3 − − − 23.10 ± 0.05
K5 − − − 24.95 ± 0.05
K7 − − − 33.74 ± 0.06
K8 − − − 25.97 ± 0.03
K9 − − − 16.34 ± 0.06
K10 − − − 16.07 ± 0.03
Z3 − − − 32.72 ± 0.03
Z6 − − − 24.39 ± 0.01
Z9 − − − 20.97 ± 0.03
Z10 − − − 22.08 ± 0.02
KR4 − − − 10.35 ± 0.05
KR9 − − − 17.27 ± 0.03
1
Strains K1 to K10 were derived from raw cow milk; Z3, Z6, Z9, and Z10 were from raw goat milk; and KR4
and KR9 were from kefir grains.
2
DPPH = 1,1-diphenyl-2-picrylhydrazyl assay.
3
Where − = negative, measured as the purple color surrounding the formed colonies (presence of Tyr or Lys).

Journal of Dairy Science Vol. 102 No. 1, 2019

132 YERLIKAYA

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