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Received: 11 May 2016 | Revised: 12 June 2016 | Accepted: 14 October 2016

DOI 10.1111/jfpp.13253


Inuence of probiotic (Lactobacillus acidophilus NCFM,

L. paracasei LPC37, and L. rhamnosus HN001) strains
on starter cultures and secondary microora in Swiss- and
Dutch-type cheeses

Marek Aljewicz | Graz_ yna Cichosz

Department of Dairy Science and Quality

Management, Faculty of Food Science,
University of Warmia and Mazury in The aim of this study was to determine the inuence of three probiotic strains on microbial quality
Olsztyn, Olsztyn, 10-719, Poland of industrially produced Swiss- and Dutch-type cheeses. The levels of starter cultures were higher
Correspondence in Swiss-type cheeses containing L. paracasei LPC37 and L. rhamnosus HN001. Mesophilic citrate-
Marek Aljewicz, Department of Dairy
fermenting bacteria were more abundant in cheeses containing L. acidophilus NCFM. The greatest
Science and Quality Management, Faculty
of Food Science, University of Warmia and reduction in Propionibacterium sp. counts was observed in cheeses produced with the addition of
Mazury in Olsztyn, Olsztyn 10-719, Poland. L. paracasei LPC37. All adjunct Lactobacillus sp. cultures stimulated changes in Enterococcus sp.
Email: counts and signicantly inuenced the counts of yeasts and molds in cheeses. Populations of coli-
Funding information form bacteria were lower in experimental cheeses than in control cheeses. The counts of butyric
National Science Centre, Grant Number:
acid bacteria and anaerobic sulte-reducing bacteria were reduced in experimental Swiss-type
312 469640; Polish Ministry of Science
and Higher Education, Grant Number: cheeses. Lactobacillus sp. used in the study are characterized by good viability and have no nega-
52807030802 tive inuence on starter cultures in cheese production.

Practical applications
Eect of three Lactobacillus sp. strains on technologically harmful microora in cheeses. The pro-
tective (antibacterial) eect of bacteria depends on the type of cheese, temperature of ripening
and storage, and the applied strain.

1 | INTRODUCTION fermented milk (De Oliveira, Augusto, Da Cruz, & Cristianini, 2014),
ice-creams (Mohammadi, Mortazavian, Khosrokhavar, & Cruz, 2011),
The metabolic activity of starter cultures is one of the main factors inu- lent, 2015).
and butters (Erkaya, Mustafa, Bayram, & Bu
encing the microbiological safety of dairy products. The positive impact of In the initial stages of production, starter cultures are the predomi-
lactic acid bacteria, including probiotic cultures, on human health has been nant microora, and their counts are regularly reduced during cheese
described in many research papers and review articles (Nagpal et al., ripening. Unlike starter cultures, the counts of nonstarter lactic acid
2012). Their health-promoting properties have been used in the produc- bacteria gradually increase in cheese during ripening and storage
tion of functional foodmainly cheeses and fermented milk drinks. (Cichosz et al., 2014; Ganesan et al., 2014; Ross, Fitzgerald, Collins, &
Most scientic research papers analyze the viability or survival Stanton, 2002).
rates of various bacterial cultures and their inuence on physicochemi- The microbiological quality of raw milk is one of the key determi-
cal (glycolytic, lipolytic, and proteolytic) changes in dierent types of nant of the quality of ripened cheese. The counts of secondary micro-
cheese, such as Minas cheese (Dantas et al., 2016), English-type Ched- ora, including technologically harmful microorganisms (Enterococcus
dar (Ganesan et al., 2014), cheese-like products (Cichosz, Aljewicz, & sp., coliform bacteria, yeasts, molds, anaerobic sulte-reducing bacteria
Nalepa, 2014), white cheese (Yerlikaya & Ozer, 2014), cottage cheese Clostridium perfringens, and butyric acid bacteria), may increase during
(Abada-Garca et al., 2013) as well as other dairy products such as cheese ripening, in particular in cheeses that had been salted in a brine
pez, Palou,
yogurts (Ainaz & Ehsani, 2008; Batista et al., 2015; Mani-Lo bath (Bintsis, 2006). Milk and ingredients of low microbiological quality,
 pez-Malo, 2014), probiotic beverages (Cusato et al., 2013),
& Lo used in the production, can contribute to cheese defects consequence

J Food Process Preserv. 2017;e13253. V

C 2017 Wiley Periodicals, Inc. | 1 of 10

of proliferation of: psychrotrophic bacteria, coliform bacteria, Entero- to AOAC (2005), fat content by the Van Gulik method (ISO, 2008). The
coccus sp., yeasts and molds, and Clostridium sp. (Beresford, Fitzsimons, pH of cheese slurry was measured according to the method proposed
Brennan, & Cogan, 2001; Temelli, Anar, Sen, & Akyuva, 2006). by Cichosz et al. (2014).
The growth of secondary microora in cheese is dependent,
among others, on the rate of milk fermentation. Nonspecic metabo- 2.3 | Microbiological analysis
lites such as lactic acid, acetic acid, and hydrogen peroxide, whose con-
Cheeses suspensions were prepared according to the method pro-
centrations and proportions are determined by the strain composition
posed by Cichosz et al. (2014). The number of L. rhamnosus HN001
of starter cultures, are among the factors limiting bacterial growth
was determined according to (Aljewicz & Cichosz, 2015), and L. aci-
(Dimitrijevi et al., 2009; Tma, Kuerova, & Plockova, 2008). Due to
dophilus NCFM was determined according to ISO (2006). Total L. para-
their synergistic eect, lactic acid and acetic acid are more eective in
casei LPC37 counts were determined on MRS agar modied according
inhibiting the growth of secondary microora when used in combina-
to Aljewicz and Cichosz (2015). The total counts of starter bacteria
tion than alone (Cherrington, Hinton, Mead, & Chopra, 1991; Theron &
were determined on M17 agar (Merck, Darmstadt, Germany). Samples
Rykers, 2011). An important role is also played by specic metabolites,
were incubated aerobically at 30 8C for 48 hr (mesophilic cultures) and
including diacetyl, acetoin, and bacteriocins: nisin, lacticin 481, and lac-
45 8C for 48 hr (thermophilic cultures). Total counts of citrate ferment-
~ez, 2011). The above metabolites are expen-
ticin 3147 (Medina & Nun
ing LAB (only in control and experimental cheeses with L. acidophilus
sive to produce, and they are rarely used on an industrial scale.
NCFM) were determined according to Nickels and Leesment (1964). In
The purpose of the present study was to dene the inuence of
order to identify (Lactobacillus sp. and Lactococcus sp.) strains to the
three Lactobacillus (L. acidophilus NCFM, L. paracasei LPC37, and L.
genus level, randomly selected bacterial colonies were subjected to the
rhamnosus HN001) strains on the levels of starter cultures and second-
Gram stain test (Olympus BX51, Japan). Total counts of Propionibacte-
ary microora (Enterococcus sp., coliform bacteria, yeasts, molds, anaer-
rium sp. were determined according to Drinan and Cogan (1992).
obic sulte-reducing bacteria, and butyric acid bacteria) and,
Total Enterococcus sp. counts were determined on Kanamycin
consequently, on the microbiological safety and storage stability of
esculin azide agar (Merck) according to Merck Manual (Merck, 2010).
industrially produced Swiss- and Dutch-type cheeses. Samples were incubated aerobically at 37 8C for 48 hr. Number of
yeasts and molds were determined according to PN-ISO (2009). Total
coliform counts were determined on MacConkey agar (BTL, o
Poland) Merck Manual (Merck, 2010). Samples were incubated aerobi-
2.1 | Experimental design cally at 32 8C for 18 hr. Total butyric acid bacteria counts were deter-

The experimental ripened cheeses produced in an industrial plant in mined on Bryant Burkey broth with resazurin and lactate (Merck)

Giz_ ycko. The production process was coincident with the industrial Merck Manual (Merck, 2010). Samples were incubated aerobically at

standards for Dutch- and Swiss-type cheese. Each cheese was pro- 37 8C for 168 hr. Total anaerobic sulte-reducing bacteria counts were

duced on the same day from the same material. determined according to PN-ISO (2005).

Cheeses were produced according to the method proposed by

Cichosz et al. (2014). DVS-starters: Dutch-type cheeseChoozit classic 2.4 | Statistical analysis
111 (0.06%) and in addition in Swiss-type cheeseChoozit TA 52 The signicance of dierences between means was analyzed by Dun-
(0.015%) and Choozit Eye (0.01%) were used. In experimental cheeses cans test. The results were prepared with Statistica software (Statsoft;
0.03% vol/vol of L. acidophilus NCFM or L. rhamnosus HN001 or L. par- 2011, Krakow, Poland) at p < 0.05 for n 5 3. All received data (micro-
acasei LPC-37 (DuPont, Poznan, Poland) were added with cheese- biological analysis, biochemical and physicochemical parameters) are
starters directly to batches. In experimental cheeses the Lactobacillus presented as means 6 standard deviation. Each replicate trial was man-
sp. were added in the amount of no less than 8 log10 CFU g21 per g of ufactured on the same day from the same milk.
experimental cheese.
The conditions of cheese ripening were as follows: Dutch-type
cheese6 weeks at 12 8C; Swiss-type cheese2 weeks at 1214 8C, 2
weeks at 1920 8C followed by 2 weeks at 1214 8C. The relative 3.1 | Chemical composition of cheeses
humidity (RH) during ripening of the rooms must be 85%. Trials of
The average water content of all cheeses was 43% (Table 1). The aver-
brined cheeses ripened for 6 weeks and stored for 3 months (RH 85%;
age fat content of control Dutch-type cheeses was determined
4 8C) were collected for analysis. The time of ripening was typical of
at 24.8%, and it was (p < 0.05) lower than in experimental cheeses
cheeses produced for the Polish market.
(Table 1). Swiss-type cheeses had a similar fat content of approximately
27%. Dutch-type cheeses were characterized by a comparable sodium
2.2 | Chemical composition
chloride content of approximately of 1.65%. Control Swiss-type cheese
Samples of cheeses were gathered as indicated by AOAC 955.30 contained 1.7% sodium chloride on average, and it diered signicantly
(AOAC, 2005). Salt and moisture content were determined according (p < 0.05) from experimental cheeses (Table 1).

T A B LE 1 Composition of control and experimental Swiss- and Dutch-type cheeses (%)

Cheese with Cheese with Cheese with

Parameter Control cheese L. acidophilus NCFM L. paracasei LPC37 L. rhamnosus HN001

Swiss-type cheeses

Moisture 43.90 6 0.14b 43.02 6 0.52a 43.20 6 0.64ab 43.37 6 0.17a

Fat 27.50 6 0.50a 27.51 6 0.37a 27.29 6 0.37a 27.04 6 0.51a

FDM 49.02 6 1.01a 48.27 6 0.26a 48.04 6 0.16a 47.74 6 0.76a

MFFS 60.55 6 0.61b 59.34 6 0.44a 59.41 6 0.58ab 59.44 6 0.21a

Salt 1.66 6 0.03b 1.33 6 0.02a 1.34 6 0.04a 1.75 6 0.06b

SDM 2.96 6 0.05b 2.34 6 0.04a 2.35 6 0.07a 3.09 6 0.11b

Dutch-type cheeses

Moisture 42.51 6 0.13a 42.88 6 0.34ab 43.18 6 0.22b 41.82 6 0.28c

Fat 24.84 6 0.77a 27.73 6 0.28c 26.71 6 0.17b 28.30 6 0.27d

FDM 43.2 6 1.36b 48.54 6 0.48a 47.00 6 0.21c 48.65 6 0.58a

MFFS 56.56 6 0.65c 59.33 6 0.45b 58.91 6 0.21ab 58.33 6 0.50a

Salt 1.63 6 0.05a 1.64 6 0.02a 1.71 6 0.03c 1.62 6 0.03a

SDM 2.83 6 0.08a 2.87 6 0.04a 3.01 6 0.05c 2.78 6 0.05a

FDM 5 fat in dry matter; MFFS 5 moisture in the fat free substance; SDM 5 salt in dry matter. Analyses were conducted after brining of Swiss- and
Dutch-type cheeses. Values (%) are means 6 standard deviation; means in rows with the same superscript letters are not signicantly dierent (p < 0.05).

3.2 | Acidity of Swiss- and Dutch-type cheeses increase was higher in cheeses containing L. paracasei LPC 37 (0.43
and 1.07 log CFU g21) than in cheeses containing L. rhamnosus HN001
Average acidity reached a pH of 5.69 in ripened Dutch-type cheeses
(0.32 and 0.95 log CFU g21). Signicant correlations were determined
and it was signicantly lower than in ripened Swiss-type cheeses (pH
between changes in the populations of SLAB 30 versus L. paracasei
5.51, Table 2). During 3 months of storage, acidity decreased signi-
LPC37 (r 5 .61; p < 0.05) and L. rhamnosus HN001 (r 5 .79; p < 0.05)
cantly (p < 0.05) in both Dutch- and Swiss-type cheeses (experimental
(Table 4).
and control) and the noted drop was always higher in Swiss-type
Unlike in Swiss-type cheeses, experimental cultures did not exert a
cheeses (control DpH 0.22; HN001 DpH 0.25; NCFM DpH 0.16 and
signicant inuence on SLAB 30 counts in Dutch-type cheeses during
DpH LPC37 0.20) than in Dutch-type cheeses (control DpH 0.17;
ripening. In experimental Dutch-type cheeses, the abundance of meso-
HN001 DpH 0.20; NCFM DpH 0.07, and DpH LPC37 0.09).
philic starter cultures was reduced during ripening. In cheeses with L.
paracasei LPC37, the populations of mesophilic starter cultures
3.3 | The eect of experimental cultures
increased insignicantly by 0.28 log CFU g21 (Table 3). A very strong
(Lactobacillus sp.) on the microora of cheeses
correlation (r 5 .94; p < 0.05) was observed between changes in the
A statistical analysis revealed that Lactobacillus sp. cultures contributed abundance of SLAB 30 and L. acidophilus NCFM.
to changes in the counts of mesophilic starter cultures (SLAB 30) in The populations of thermophilic starter cultures (SLAB 45) in
Swiss-type and Dutch-type cheeses. In Swiss-type cheeses containing experimental Swiss-type cheeses produced with the addition of L. aci-
L. paracasei LPC37 and L. rhamnosus HN001, the average counts of dophilus NCFM and L. paracasei LPC37 did not change signicantly dur-
mesophilic starter cultures increased signicantly (p < 0.05) during rip- ing ripening and storage. In cheeses containing L. rhamnosus HN001,
ening and storage relative to control cheeses (Table 3), and the noted SLAB 45 increased (p < 0.05) by 1 and 1.66 log CFU g21, respectively.

T A B LE 2Average pH values in cheese after 6 weeks of ripening and 3 months of storage. Values are means 6 standard deviation; means in
rows with the same superscript letters are not signicantly dierent (p < 0.05)

Cheese with Cheese with Cheese with

Period Control cheese L. acidophilus NCFM L. paracasei LPC37 L. rhamnosus HN001

Dutch-type cheese Ripening 5.68 6 0.03a 5.73 6 0.03ab 5.64 6 0.02a 5.72 6 0.03b

Storage 5.84 6 0.02a 5.80 6 0.03ab 5.75 6 0.03b 5.75 6 0.02b

Swiss-type cheese Ripening 5.50 6 0.05a 5.56 6 0.02a 5.54 6 0.06a 5.43 6 0.03b

Storage 5.72 6 0.03a 5.72 6 0.05a 5.74 6 0.05a 5.68 6 0.02a

4 of 10

T A B LE 3 Average count (log CFU g21) of microorganisms during ripening and storage of Dutch- and Swiss-type cheeses

Microorganism Swiss-type cheese Dutch-type cheese

With With With With With With
Control L. acidophilus L. paracasei L. rhamnosus Control L. acidophilus L. paracasei L. rhamnosus
cheese NCFM LPC37 HN001 cheese NCFM LPC37 HN001


Mesophilic Lactococcus sp. 8.50 6 0.37a 8.46 6 0.33a 8.93 6 0.03b 8.82 6 0.36b 9.01 6 0.39ab 8.95 6 0.47ab 8.77 6 0.37a 9.19 6 0.40ab

Thermophilic Lactococcus sp. 7.18 6 1.06a 7.06 6 1.15a 7.71 6 0.94a 8.22 6 1.50a

NSLAB 6.44 6 1.93bc 7.44 6 0.23c 9.37 6 0.20a 9.32 6 0.20a 6.73 6 2.18bc 7.82 6 0.52c 9.56 6 0.37a 9.2 6 0.39a

Citrate-fermenting bacteria 6.05 6 0.64a 6.90 6 0.50a 7.6 6 1.25a 8.14 6 0.93ab

L. rhamnosus HN001 9 6 0.49a 9.32 6 0.17a

L. acidophilus NCFM 7.39 6 0.27a 7.82 6 0.45a

L. paracasei LPC37 9.6 6 0.51a 9.43 6 0.19a

Propionibacterium sp. 6.3 6 0.81a 5.50 6 0.95a 4.91 6 0.73a 5.45 6 0.81ab

Enterococcus sp. 3.14 6 0.36a 3.99 6 1.26a 3.47 6 0.90a 3.16 6 1.16a 2.31 6 0.81a 2.90 6 1.05a 2.86 6 1.14a 2.81 6 0.97a

Coliform bacteria 2.98 6 0.54b 2.00 6 0.79ab 2.44 6 0.93ab 2.85 6 0.65b 3.18 6 0.40b 2.39 6 0.47ab 1.86 6 0.17a 2.82 6 0.41b

Yeast and molds 2.89 6 0.56a 2.25 6 1.41ab 1.70 6 1.40ba 1.56 6 1.23b 3.02 6 0.51a 2.41 6 2.04ab 2.32 6 1.86a 2.33 6 0.37a

Anaerobic sulte-reducing bacteria 1.59 6 0.80ab 1.24 6 1.03ab 1.10 6 0.93ab 0.54 6 0.56ab 0.42 6 0.14a 0.83 6 0.10b 0.23 6 0.16a 0.56 6 0.13b

Butyric acid bacteria 0.89 6 0.71ab 0.26 6 0.47a 1.57 6 0.34b 0.09 6 0.29a nd nd nd nd


Mesophilic Lactococcus sp. 7.66 6 0.31a 7.86 6 0.26a 8.73 6 0.30bc 8.61 6 0.20bc 8.65 6 0.16ab 7.83 6 0.53a 9.06 6 0.24c 8.48 6 0.81abc

Thermophilic Lactococcus sp. 7.06 6 0.52a 7.07 6 0.33a 6.94 6 1.38a 8.72 6 0.22b

NSLAB 7.33 6 0.45a 7.45 6 0.21a 8.89 6 0.34b 8.82 6 0.36b 8.53 6 0.98ab 7.53 6 0.21c 9.07 6 0.25b 8.73 6 0.31ab

Citrate-fermenting bacteria 4.88 6 0.62a 5.22 6 1.02a 7.83 6 0.30b 6.03 6 0.47a

L. rhamnosus HN001 8.42 6 0.24a 8.48 6 0.33a

L. acidophilus NCFM 5.92 6 0.75a 7.08 6 0.43a

L. paracasei LPC37 8.90 6 0.22a 9.12 6 0.25a

Propionibacterium 7.27 6 0.55a 6.35 6 0.89a 7.39 6 1.0a 6.36 6 0.41a


Enterococcus sp.

4.86 6 0.91a 4.63 6 1.39ac 5.11 6 0.51a 4.74 6 0.71a 2.35 6 0.71c 4.05 6 0.57ac 4.92 6 0.47c 2.28 6 0.99c

The counts of mesophilic citrate-fermenting bacteria in experimen-

2.06 6 0.92ab
L. rhamnosus

2.74 6 0.68b

0.62 6 0.04a
tal Swiss-type cheese produced with the addition of L. acidophilus
NCFM increased (p > 0.05) during ripening (0.84 log CFU g21) and


NSLAB 5 nonstarter lactic acid bacteria (lactobacilli). Values are means 6 standard deviation (n 5 three sets of data analyzed in duplicate); means in a row with the same superscript letters are not
storage (0.34 log CFU g21). A similar increase was noted in Dutch-type

cheeses, but only during ripening (Table 3).
Lactobacillus sp. cultures contributed to changes in the size of Pro-

0.20 6 0.18b
1.06 6 0.59a

4.23 6 0.40a
L. paracasei
pionibacterium sp. populations during ripening and storage. After 6
LPC37 weeks of ripening, cheeses containing L. paracasei LPC37 were charac-

terized by the highest decrease (1.39 log CFU21; p > 0.05), and cheeses

containing L. acidophilus NCFM and L. rhamnosus HN001by the low-
est decrease (0.82 log CFU g21) in Propionibacterium sp. counts. The
1.08 6 0.60ab
L. acidophilus

4.18 6 0.40a

0.76 6 0.66a
opposite was observed during storage. A greater average decrease
(0.91 log CFU g21) in Propionibacterium sp. populations was noted in

experimental cheeses containing L. acidophilus NCFM and L. rhamnosus


HN001 than in control cheeses. A signicant correlation was also

Dutch-type cheese

found between the increase in L. acidophilus NCFM (r 5 2.81; p < 0.05)

and L. rhamnosus HN001 (r 5 2.86; p < .05) counts and the decrease in
2.14 6 1.15ab

3.22 6 0.42ab

0.61 6 0.15a

the average size of Propionibacterium sp. populations during ripening


(Table 3).

An analysis of variance revealed that Enterococcus sp. counts were

determined by cheese type and the duration of ripening and storage.
The average abundance of Enterococcus sp. increased during ripening
3.08 6 0.67ab
L. rhamnosus

1.71 6 0.41b

0.70 6 0.59b

0.19 6 0.41a

in all Swiss-type cheeses, where the highest increase was observed in


cheeses produced with the addition of L. acidophilus NCFM (0.84 log


CFU g21), and the lowest increasein products containing L. rhamnosus

HN001 (0.02 log CFU g21). Enterococcus sp. counts did not change sig-
nicantly in experimental cheeses during storage. Similarly to Swiss-
0.66 6 0.71abc
4.14 6 0.45ab

1.38 6 1.31b
1.02 6 0.70a

type cheeses, Dutch-type cheeses were also characterized by a small

L. paracasei

increase in the size of bacterial populations of the genus Enterococcus.


The increase in Enterococcus sp. counts was signicantly correlated

with a decrease in the abundance of Lactobacillus sp. cultures (Table 3).
An analysis of variance revealed correlations between changes in
0.51 6 0.54abc

the counts of coliform bacteria and the size of L. acidophilus NCFM, L.

0.77 6 0.54ab

3.91 6 0.54ab
L. acidophilus

0.76 6 0.89b

paracasei LPC37, and L. rhamnosus HN001 populations. Coliform bacte-


ria were less abundant in experimental Swiss-type and Dutch-type


cheeses during ripening and storage. The greatest reduction in the size
Swiss-type cheese

of coliform colonies was noted during ripening and storage in experi-

mental Swiss-type cheeses containing L. acidophilus NCFM (1 log CFU
3.48 6 0.80ab
2.17 6 0.85b

2.62 6 0.47a

1.31 6 0.27c

g21; p > 0.05), whereas the smallest decrease was noted in cheese

enhanced with L. rhamnosus HN001 (0.1 log CFU g21; p > 0.05). In

signicantly dierent (p < 0.05); nd, not detected.

Dutch-type cheeses, the highest decrease in coliform counts was

observed in cheeses containing L. paracasei LPC37 (1.32 log CFU g21;
p < 0.05), and the lowest decrease, similarly to Swiss-type cheesesin
Anaerobic sulte-reducing bacteria

cheese with L. rhamnosus HN001 (0.36 log CFU g21; p > 0.05). The
decrease in coliform counts was correlated with changes in the size of
Lactobacillus sp. populations (Table 3).

The abundance of yeasts and molds in experimental cheeses was

Butyric acid bacteria
Coliform bacteria

largely determined by the type of the Lactobacillus sp. culture added to

Yeast and molds

the analyzed products. In experimental Swiss-type cheeses containing

L. paracasei LPC37 and L. rhamnosus HN001, the decrease in yeast and
T A B LE 3

mold counts during ripening was similar at 1.17 log CFU g21 (p > 0.05)
and 1.33 log CFU g21 (p < 0.05), respectively. The experimental cheese

T A B LE 4 Correlation between Lactobacillus counts and microora of Swiss- and Dutch-type cheeses

Microorganism Swiss-type cheese with Dutch-type cheese with



Mesophilic Lactococcus sp. 0.98* 0.61* 0.79* 0.94* 20.54 20.64*

Thermophilic Lactococcus sp. 20.73* 20.23

NSLAB 0.93* 0.45 0.46 0.93* 0.80* 20.80*

Citrate-fermenting bacteria 20.73* 0.41

Propionibacterium sp. 20.81* 0.13 20.86*

Enterococcus sp. 20.94* 20.07 20.94* 20.76* 0.21 20.65*

Coliform bacteria 0.96* 20.36 0.82* 0.59* 20.40 20.76*

Yeast and molds 20.95* 0.16 20.90* 20.77* 0.36 20.47

Anaerobic sulte-reducing bacteria 20.92* 0.73* 20.72* 20.39 0.42 0.27

Butyric acid bacteria 20.64* 0.62* 20.53


Propionibacterium sp. 20.31 20.58* 20.25

Enterococcus sp. 0.59* 20.16 20.42 0.79* 20.64* 20.26

Coliform bacteria 0.61* 0.60* 20.74* 0.81* 0.75* 0.08

Yeast and molds 0.07 20.64* 20.13 0.90* 0.70* 0.72*

Anaerobic sulte-reducing bacteria 0.93* 0.30 20.79* 0.60* 0.59* 0.58*

Butyric acid bacteria 0.71* 0.83* 20.62*

NCFM 5 L. acidophilus NCFM; LPC37 5 L. paracasei LPC37; HN001 5 L. rhamnosus HN001; NSLAB 5 nonstarter lactic acid bacteria (lactobacilli). An
asterisk denotes signicant correlation (p < 0.05).

enhanced with L. acidophilus NCFM was characterized by a lower increased by 0.28 and 0.07 log CFU g21, respectively. The decrease in
decrease in yeast and mold counts at 0.64 log CFU g (p > 0.05). Dur- the abundance of anaerobic sulte-reducing bacteria was correlated
ing storage, yeast and mold populations increased in cheeses contain- with changes in the size of L. acidophilus NCFM populations in both
ing L. acidophilus NCFM (0.43 log CFU g21) and L. paracasei LPC37 Swiss-type cheese and Dutch-type cheeses. The counts of butyric acid
(0.66 log CFU g21). Both types of experimental cheeses were charac- bacteria reduced by the addition of the Lactobacillus sp. culture. Experi-
terized by lower abundance of yeasts and molds than control cheeses. mental cheeses containing L. acidophilus NCFM and L. rhamnosus
In Dutch-type cheeses, the drop in yeast and mold counts in all experi- HN001 were characterized by the highest decrease in the populations
mental cheeses during ripening was similar at approximately 0.67 log of butyric acid bacteria during ripening (0.6 and 0.8 log CFU g21, respec-
CFU g . Similarly to Swiss-type cheeses, the size of yeast and mold tively) and storage (0.8 and 1.12 log CFU g21, respectively). A strong
populations increased during storage in cheeses containing L. acidophi- negative correlation was observed between changes in the abundance
lus NCFM (0.96 log CFU g21; p > 0.05) and L. paracasei LPC37 (1.01 of butyric acid bacteria, L. acidophilus NCFM and L. rhamnosus HN001.
log CFU g21; p > 0.05). Yeast and mold counts decreased by 0.48 log Butyric acid bacteria were not detected in Dutch-type cheeses.
CFU g21 in cheese containing L. rhamnosus HN001.
Lactobacillus sp. cultures contributed to changes in the population
size of anaerobic sulte-reducing bacteria during ripening and storage, 4 | DISCUSSION
which are responsible for the late-blowing defect in Swiss-type cheese.
In experimental cheeses, the drop in the counts of anaerobic sulte- Chemical composition of cheeses (experimental and control) were dif-
reducing bacteria was always higher during storage than ripening. Exper- ferent. A slight dierence in chemical composition was noted between
imental cheeses containing L. rhamnosus HN001 were characterized by experimental Dutch-type cheese and control cheese. Our previous
the greatest decrease (1.30 log CFU g21) and cheeses containing L. para- ndings (Cichosz et al., 2014) and the results of other studies (Burns
casei LPC37by the smallest decrease (0.87 log CFU g ) in the counts et al., 2012; Ong & Shah, 2009) validate the above observation. The
of anaerobic sulte-reducing bacteria. In experimental Dutch type inuence of added bacteria (Lactobacillus sp.) on the chemical composi-
cheeses produced with the addition of L. acidophilus NCFM and L. rham- tion of the cheeses is negligible. Produce ripened cheeses with identical
nosus HN001, the size of anaerobic sulte-reducing bacteria populations chemical composition is practically impossible.

In this research, changes in the pH of cheeses during ripening and in English cheese (45 log CFU g21; Gardiner et al., 1998) and French
storage were due to the increase in population of NSLAB, release of cheese (26 log CFU g21; Jamet et al., 2012), but signicantly lower
ammonia by slow degradation of casein, and degradation of lactic acid than in Spanish cheese (68 log CFU g21; Martn-Platero, Valdivia,
to lactate. The much greater decrease in the acidity of Swiss-type Maqueda, & Martnez-Bueno, 2009). A gradual increase in Enterococcus
cheese during ripening and storage resulted from higher peptidolytic sp. counts during ripening was probably induced by a higher content of
activity of Propionibacterium sp. which metabolize lactate to acetate, low molecular-weight products of casein degradation (McSweeney,
propioniate, carbon dioxide, and water (Cichosz et al., 2014; McSwee- 2004) that are essential for bacterial growth and proliferation (Serio,
ney, 2004). pez, Paparella, & Suzzi, 2010). The use of L. acidophilus
The viability and metabolic activity of starter cultures are deter- NCFM, L. paracasei LPC37, and L. rhamnosus HN001 cultures in cheese
mined by the availability of readily available sources of carbon and production reduced coliform counts, in contrast to Enterococcus sp.
nitrogen, including bacterial metabolites (low-molecular weight pep- The inhibitory eect of Lactobacillus sp. on coliform bacteria was
tides and free amino acids). A minor reduction in the number of meso- also noted in other studies. L. acidophilus La-5 and L. acidophilus 4962
philic lactic acid bacteria during ripening and storage of dierent kinds reduced the levels of E. coli O157:H7 populations in white cheese. On
of cheese, even in the absence of appropriate substrates, is a conse- the other hand the L. paracasei and L. plantarum isolated from artisanal
quence their capability to accumulate energy in the form of phosphoe- raw milk cheese reduced the levels of Escherichia coli O26, Escherichia
nolpyruvate. The subsequent inhibited the growth of technologically coli O157:H7, and Escherichia coli ATCC 45922 (Geria, Dambrosio, Nor-
harmful microora by added Lactobacillus sp. was increased the amount manno, Lorusso, & Caridi, 2014). The average counts of yeasts and
of easily available substrates used by culture starters. By contrast, Lac- molds in control Swiss-type and Dutch-type cheeses were determined
tobacillus sp. had no inuence on the viability of starter cultures in at 2.88 and 3.00 log CFU g21, respectively, and they were lower than
English-type cheese (Ong & Shah, 2009). that reported by other authors (Darehabi & Nikmaram, 2011). In rip-
The survival rates of LAB during ripening and storage of the ana- ened Spanish-type cheeses, average yeast and mold counts reached 6
lyzed cheeses were higher than in cheddar because cheese was salted log CFU g21 (Tornadijo, Fresno, Sermiento, & Carballo, 1998), and in
in a brine bath and not by adding salt to the cheese mass. Low (1.52%) Portuguese cheeses, they were determined in a wider range of 36 log
sodium chloride concentrations in the cheese mass did not reduce the CFU g21 (Pereira-Dias, Potes, Marinho, Malfeito-Ferreira, & Loureiro,
level of Propionibacterium sp. populations. Good viability of Propionibac- 2000).
terium sp. counts (67 log CFU g21) in control and experimental Lower counts of butyric acid bacteria, anaerobic sulte-reducing,
cheeses probably resulted from high concentrations of L-lactate in the yeast, and mold in experimental cheeses can be attributed to higher
substrate, which stimulates the production of intracellular pyruvate concentrations of lactic acid, propanoic acid, acetic acid, and diacetyl
(White, Broadbent, Oberg, & McMahon, 2003). (Aljewicz, unpublished data) as well as, most probably, bacteriocins, lac-
In early stages of ripening of Swiss- and Dutch-type cheeses, Lac- tones, cyclic dipeptides and hydrogen peroxide produced by highly
tobacillus sp. competes with Leuconostoc mesenteroides ssp. mesenter- abundant (89 log CFU g21) starter cultures and Lactobacillus sp.
oides and Lactcoccus lactis ssp. lactis var. diacetylactis for access to strains used in the study (Christiansen et al., 2005; Tharmaraj & Shah,
citrates, and in Swiss-type cheeses, also with Propionibacterium sp. for 2009; Tma et al., 2008).
access to L-lactate. Unlike mesophilic and thermophilic starter cultures, Lactic acid bacteria are responsible for producing high content of
the level of Propionibacterium sp. populations was reduced in experi- carboxylic acids, which increase the acidity of the environment and
mental Swiss type cheeses. The above can probably be attributed to reduces the number of other bacteria. The antibacterial eect of
high average counts of L. rhamnosus HN001 (8.71 log CFU g21) and L. organic acids is also dependent on the form in which they appear in
paracasei LPC37 (9.30 log CFU g21) that use citrates as an easily avail- the product. The undissociated forms of weak organic acids (propionic,
able source of carbon during cheese production and ripening. The acetic) can diuse through the bacterial cell membrane. Upon diusion
antagonistic inuence of citrate-fermenting bacteria on Propionibacte- inside the cell wall, the carboxylic acids can dissociate, but the the
rium sp. cells results mainly from the inhibitory eects of acetate and eectiveness of acid dissociated depends on the hydrogen ion concen-
formate synthesized during citrate breakdown. Their inhibitory eects tration in the cytoplasm. The survival of the bacteria in the stationary
could have been additionally stimulated by high (approximately 4 mg phase or eventual death of susceptible bacteria is consequence of the
100 g21 of cheese) concentrations of zinc ions (data not shown) that H1 ions released during the dissociation can acidify the cytoplasm to
form chelate complexes with citrates, thus reducing the metabolic cause damage of the electrochemical proton gradient (Piard & Desma-
activity of Propionibacterium sp. (Frohlich-Wyder, Bachmann, & Casey, zeaud, 1991).
2002). Smaller undissociated fragments of organic acids can diuse faster
High Enterococcus sp. counts in salted cheeses can be attributed to via the cell membrane into the cell to cause more damage. The antibac-
low-temperature pasteurization and the bacterias high thermal resist- terial eect of organic acids depends on the pH of the environmental
ance and low salt concentrations in dry cheese mass (2.33.1%). Imme- and on their undissociated form. The ability of lactic acid bacteria to
diately after salting, average Enterococcus sp. counts were determined reduce pH by producing lactic acid and acetic acid, and to synthesize
at 24 log CFU g . They were similar to those noted by other authors short-chain lipophilic organic acids (in large quantities under favorable

conditions), may be associated with their ability to inhibit technologi- Polish Ministry of Science and Higher Education (Grant No.
cally harmful microora (Shabanova, Gosteva, Klitsunova, & Bondar- 52807030802).
enko, 2009; Zalan, Ne
meth, Barath, & Hal
asz, 2005). In comparison
with most Lactobacillus sp. cultures, L. rhamnosus and L. acidophilus can R EF ER E N CE S
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age of Iranian white cheese and probiotic cheese. Global Veterinaria,
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