- Email: [email protected]
Effectiveness of different methodologies for the selective enumeration of Lactobacillus acidophilus La5 from yoghurt and Prato cheese
Accepted Manuscript Effectiveness of different methodologies for the selective enumeration of Lactobacillus acidophilus La5 from yoghurt and Prato cheese Clarice Gebara, Maria Cecília E. Ribeiro, Karina S. Chaves, Ana Lourdes N. Gandara, Mirna L. Gigante PII:
S0023-6438(15)00343-6
DOI:
10.1016/j.lwt.2015.04.061
Reference:
YFSTL 4650
To appear in:
LWT - Food Science and Technology
Received Date: 14 May 2014 Revised Date:
15 April 2015
Accepted Date: 24 April 2015
Please cite this article as: Gebara, C., Ribeiro, M.C.E., Chaves, K.S., Gandara, A.L.N., Gigante, M.L., Effectiveness of different methodologies for the selective enumeration of Lactobacillus acidophilus La5 from yoghurt and Prato cheese, LWT - Food Science and Technology (2015), doi: 10.1016/ j.lwt.2015.04.061. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Effectiveness of different methodologies for the selective enumeration of
2
Lactobacillus acidophilus La5 from yoghurt and Prato cheese
3
Clarice Gebara1*, Maria Cecília E. Ribeiro1, Karina S. Chaves1, Ana Lourdes N.
4
Gandara2, Mirna L. Gigante1
5
1
6
Campinas, 13082-862, Campinas, S.P., Brazil
7
2
8
*Corresponding author Tel.: +55 19 35213993
9
*E-mail address: [email protected] (C. Gebara)
RI PT
Department of Food Technology, Faculty of Food Engineering, University of
SC
Technical High School of Campinas, 13020-060, Campinas, S.P., Brazil.
M AN U
10 ABSTRACT
12
Prior to the selective enumeration of probiotic microorganisms in fermented dairy
13
products, a preliminary evaluation of different culture media and incubation conditions
14
is required to choose a suitable culture medium. The objective of this study was to
15
evaluate the effectiveness of different methodologies for the selective enumeration of L.
16
acidophilus in probiotic yoghurt and Prato cheese. For selective enumeration of L.
17
acidophilus, four methods were investigated under anaerobic conditions, as follows:
18
MRS agar containing bile solution; MRS agar containing clindamycin solution; and
19
MRS agar containing sorbitol solution, each one incubated at 37 °C for 72 hours, and
20
MRS agar containing sorbitol incubated at 45 °C for 72 hours. These methodologies
21
were used for enumeration of both the pure cultures and the microorganisms from
22
probiotic yoghurt and Prato cheese. The results showed that the best selective medium
23
for enumeration of Lactobacillus acidophilus from yoghurt was MRS agar containing
24
bile incubated at 37 ° C for 72 hours under anaerobic conditions. For enumeration of
AC C
EP
TE D
11
1
ACCEPTED MANUSCRIPT 25
this probiotics from Prato cheese, MRS agar containing sorbitol incubated at 45 º C for
26
72 hours under anaerobic conditions was proven to be the best selective medium.
27 Keywords: Lactobacillus acidophilus, selective enumeration, probiotic yoghurt,
29
probiotic cheese
RI PT
28
30 31
1.
Introduction
The probiotic microorganisms play an important role in the functional foods
33
industry, contributing to a market involving billions of dollars (O’Flaherty &
34
Klaenhammer, 2010). Although they can be added in different food products, dairy
35
products are considered ideal systems for delivery of these microorganisms,
36
contributing to the maintenance of viability during passage through the gastrointestinal
37
tract (Ross et al., 2002). To provide health benefits, the probiotic microorganism must
38
be in products in minimal viable numbers, usually 106-107 CFU / g. Therefore, the
39
adequate evaluation of the viability of these microorganisms in the product should be
40
achieved by efficient methodologies for enumeration (Lahtinen et al., 2011; Shah, 2000;
41
Vasiljevic & Shah, 2008).
EP
TE D
M AN U
SC
32
Several methods for enumeration of L. acidophilus and Bifidobacterium spp.
43
have been proposed; however, most of them are based on enumeration of pure cultures,
44
but not in complex matrices such as foods (Lahtinen et al., 2011; Shah, 2000; Vasiljevic
45
& Shah, 2008). The choice of a methodology suitable for the selective enumeration of
46
probiotic strains in combination with lactic acid starters depends on both the
47
microorganisms and the matrix in question, since a culture medium alone is not suitable
48
for a variety of applications (Karimi et al., 2012; Tharmaraj & Shah, 2003; Van De
49
Casteele et al. 2006).
AC C
42
2
ACCEPTED MANUSCRIPT Traditionally, the enumeration of probiotic microorganisms in food has been
51
performed by plate count technique, and the result is expressed as colony forming units
52
(CFU) (Lahtinen et al., 2011). This enumeration can be obtained using differential or
53
selective media. The selective enumeration is achieved through the addition of
54
inhibitory compounds (such as antibiotics), pH changes, or incubation conditions
55
(temperature and oxygen levels). The selective media allow direct enumeration of
56
colony on the plates without the need for additional analysis, being easy to perform
57
(Lahtinen et al., 2011; Oberg et al., 2011). Many different media were developed for the
58
selective enumeration of probiotics in dairy products, but few have been validated in
59
comparative studies with adequate quantification, recovery, and differentiation from
60
other microorganisms in the product (Tharmaraj & Shah, 2003).
M AN U
SC
RI PT
50
A summary of the studies found in the literature with the selective
62
methodologies used for enumerating Lactobacillus acidophilus in probiotic yoghurt
63
(Table 1) and cheese (Table 2) is presented. Among the selective methodologies, the
64
MRS agar containing bile and incubated at 37 °C for 72 hours under anaerobic
65
conditions was described by International Dairy Federation - IDF (1995) for selective
66
enumeration of Lactobacillus acidophilus in the presence of yoghurt cultures
67
(Streptococcus thermophilus and Lactobacillus bulgaricus) and bifidobacteria. The
68
efficiency of this medium in the selective enumeration of Lactobacillus acidophilus in
69
the presence of yoghurt cultures was confirmed by Mortazavian et al. (2007) and
70
Vinderola & Reinheimer (1999). Bile MRS Agar was also used for the selective
71
enumeration of L. acidophilus in Argentinian fresh cheese produced with Lactococcus
72
lactis and B. bifidum (Vinderola et al., 2000).
AC C
EP
TE D
61
73
The Lactobacillus acidophilus La5 is one of the most studied probiotic strains,
74
with application in different dairy products. For selective enumeration of this
3
ACCEPTED MANUSCRIPT microorganism in the presence of mesophilic cultures, the manufacturer recommends
76
MRS agar containing clindamycin incubated at 37 °C for 72 h under anaerobic
77
conditions (Christian Hansen, 2007). As reported by Van De Casteele et al. (2006), this
78
culture medium was effective for enumeration of L. acidophilus in the presence of
79
commercial lactic acid starters added during the manufacturing processes of yoghurt
80
and cheese.
RI PT
75
On the other hand, the MRS agar containing sorbitol incubated at 37 °C for 72
82
hours under anaerobic conditions was selective for the enumeration of Lactobacillus
83
acidophilus in the presence of Streptococcus thermophilus, Lactobacillus bulgaricus
84
and bifidobacteria in a study on pure cultures (Dave & Shah, 1996). Subsequent studies
85
have used this medium for selective enumeration of L. acidophilus in Cheddar cheese
86
manufactured using Lactococcus lactis (Ong et al, 2006; Ong & Shah, 2009), and
87
turkish white-brined cheese also manufactured using Lactococcus lactis (Özer et al.,
88
2008). In a recent study, MRS agar containing sorbitol incubated at 37 °C under
89
anaerobic conditions was considered non-selective for enumeration of Lactobacillus
90
acidophilus in Cheddar cheese manufactured using probiotic microorganisms as adjunct
91
cultures (L. acidophilus, L. casei, L. paracasei, Bifidobacterium lactis) and Lactococcus
92
sp. (Oberg et al., 2011). In contrast, in the same study, the MRS agar with sorbitol as the
93
only carbohydrate source incubated at 45 °C under anaerobic conditions was effective
94
for the selective enumeration of Lactobacillus acidophilus in Cheddar cheese.
M AN U
TE D
EP
AC C
95
SC
81
Selective enumeration of starter cultures of lactic acid bacteria can be achieved
96
in the presence of probiotic microorganisms. The literature describes the M17 agar
97
containing lactose incubated at 30 °C for enumeration of lactococci (Terzaghi &
98
Sandine, 1975). This culture medium has been the most used for selective enumeration
99
of Lactococcus lactis in the presence of Lactobacillus acidophilus (Darukaradhya et al.,
4
ACCEPTED MANUSCRIPT 2006; Kasimoglu et al., 2004; Oberg et al., 2011; Ong et al., 2006; Ong & Shah, 2008,
101
2009; Van De Casteele et al., 2006). The M17 Agar is also used for enumeration of
102
Streptococcus thermophilus (Shankar & Davies, 1977; Van De Casteele et al., 2006).
103
Besides the M17 agar, ST agar (Streptococcus thermophilus agar) was effective for
104
enumeration of Streptococcus thermophilus in the presence of Lactobacillus delbrueckii
105
subsp. bulgaricus and Lactobacillus acidophilus (Dave & Shah, 1996). Zacarchenco &
106
Massaguer-Roig (2004) also reported the selectivity of ST agar for enumeration of
107
Streptococcus thermophilus in the presence of Lactobacillus acidophilus and
108
Bifidobacterium longum. Both the MRS Agar pH 5.2 and the Reinforced Clostridial
109
Agar (RCA) pH 5.3 were effective for enumeration of Lactobacillus delbrueckii subsp.
110
bulgaricus in the presence of Streptococcus thermophilus, Lactobacillus acidophilus
111
and Bifidobacterium sp. (Dave & Shah, 1996).
M AN U
SC
RI PT
100
Although literature suggests different conditions for selective enumeration of
113
probiotic microorganisms, the efficiency depends on several factors including the strain,
114
presence of other cultures in the product, and type of product to which the probiotics
115
will be added. Thus, to select the methodology for selective enumeration of probiotics
116
in dairy products, it is important that different methods are tested. Given the variety of
117
culture media described in literature, the aim of this study was to evaluate the
118
effectiveness of different methodologies for selective enumeration of L. acidophilus
119
from probiotic yoghurt and Prato cheese.
AC C
EP
TE D
112
120 121
2.
Material and methods
122
2.1. Cultures
123
Four commercial cultures were used: probiotic culture Lactobacillus acidophilus
124
(La5 - Christian Hansen Ind. e Com, Valinhos, Brazil); two yoghurt starter cultures -
5
ACCEPTED MANUSCRIPT 125
Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp.
126
thermophilus (LB 340 and TA 40 - Danisco A / S Brabrand, Denmark); and type "O"
127
starter culture for cheese manufacture (Lactococcus lactis subsp lactis and Lactococcus
128
lactis subsp cremoris - R704, Christian Hansen Ind. e Com, Valinhos, Brazil).
130
RI PT
129 2.2. Preparation of the inoculum
Lyophilized culture of Lactobacillus acidophilus La5 was rehydrated (at 15 °C
132
for 20 minutes) in sterile reconstituted skimmed milk containing 20% (v / v) glycerol
133
(cryoprotectant), and transferred to micro tubes and kept in a freezer (-18 °C). For
134
processing, this culture was reactivated in MRS broth (2% v / v) for two consecutive
135
times, with incubation at 37 ° C for 15 hours. The reactivated culture was placed in
136
sterile polypropylene tubes and centrifuged at 8,000 g for 10 minutes at 4 °C (Sorvall
137
Instruments RC5C - DuPont). The cell concentrate was resuspended in peptone water
138
(0.1% v / v) and centrifuged again under the same conditions, followed by resuspension
139
in peptone water to obtain a concentration of cells with 9-10 log10 CFU / mL (Gebara et
140
al., 2013). The cell concentrate viability was assessed by pour plating on MRS agar
141
incubated at 37 ° C for 72 hours in anaerobic jars using GasPak anaerobic generator
142
(Becton, Dickinson and Company, Franklin Lakes, USA) (De Man et al., 1960). A cell
143
concentrate of Lactobacillus acidophilus was prepared for each run.
M AN U
TE D
EP
AC C
144
SC
131
The cultures L. delbrueckii subsp. bulgaricus, S. thermophilus, and the starter
145
culture L. lactis subsp. lactis and L. lactis subsp. cremoris were previously activated in
146
sterile reconstituted (11%) skimmed milk and incubated at 45 °C / 6 h; 45 °C / 4 h; and
147
30 °C / 16h, respectively. Then, they were used in the analysis of pure cultures and in
148
the manufacture process, being prepared for each processing.
149
6
ACCEPTED MANUSCRIPT 150
2.3. Media Preparation The culture media described below were selected from literature for selective
152
enumeration of Lactobacillus acidophilus in yoghurt and cheese. The methodologies
153
were strictly followed as described in the references:
154
Bile Agar MRS (MRSB): Commercial MRS Agar (Difco - Becton, Dickinson and
155
Company, Franklin Lakes, USA) was prepared according to manufacturer’s
156
instructions. The rehydrated medium was autoclaved at 121 °C for 15 minutes. For bile
157
solution, 10 g bile (B3883 - Sigma-Aldrich Co., St. Louis, USA) was dissolved in 100
158
mL distilled water, and the solution was autoclaved at 121 °C for 15 minutes.
159
Immediately before use, 1.5 mL bile solution was added to 100 mL MRS agar (IDF,
160
1995).
161
Clindamycin MRS Agar (MRSC): Commercial MRS agar (Difco - Becton, Dickinson
162
and Company, Franklin Lakes, USA) was prepared according to manufacturer's
163
instructions. The rehydrated medium was autoclaved at 121 °C for 15 minutes. For the
164
clindamycin solution, 2 mg Clindamycin hydrochloride were dissolved in 10 mL
165
distilled water, and sterilized by filtration through 0.22 µm membrane filter (Millipore,
166
São Paulo, Brazil). Immediately before use, 0.5 mL clindamycin solution was added to
167
1000 mL MRS agar (Christian Hansen, 2007).
168
MRS Agar + Sorbitol (MRS + S): MRS agar was prepared without glucose (10 g
169
peptone; 8 g meat extract; 4 g yeast extract; 2 g K2HPO4; 5 g sodium acetate; 2 g
170
ammonium citrate; 0.2 g magnesium sulfate; 0.05 g manganese sulfate; 1 mL Tween 80;
171
and 10 g agar dissolved in 1L distilled water) and autoclaved at 121 °C for 15 minutes.
172
For the sorbitol solution, 10 g D-sorbitol were dissolved in 100 mL distilled water, and
173
the solution was sterilized by filtration through 0.22 µm membrane filter (Millipore, São
AC C
EP
TE D
M AN U
SC
RI PT
151
7
ACCEPTED MANUSCRIPT Paulo, Brazil). Immediately before use, 10 mL D-sorbitol solution was added for each
175
90 mL MRS agar (1% final concentration) (Dave & Shah, 1996).
176
MRS-Sorbitol Agar (MRSS): MRS agar was prepared without glucose (10 g peptone;
177
8 g meat extract; 4 g yeast extract; 2 g K2HPO4; 5 g sodium acetate; 2 g ammonium
178
citrate; 0.2 g magnesium sulfate; 0.05 g manganese sulfate; 1 mL Tween 80; and 10g
179
agar dissolved in 1L distilled water) with addition of 10g / L D-sorbitol. The medium
180
was autoclaved at 121 °C for 15 minutes (Oberg et al., 2011).
181
MRS Agar pH 5.2: MRS agar (Difco - Becton, Dickinson and Company, Franklin
182
Lakes, USA) was prepared according to the manufacturer's recommendations, and the
183
pH was adjusted to 5.2 with HCl 1.0 M. The medium was autoclaved at 121 °C for 15
184
minutes (Dave & Shah, 1996).
185
RCA pH 5.3: Reinforced Clostridial Agar (Difco - Becton, Dickinson and Company,
186
Franklin Lakes, USA) was prepared according to the manufacturer's instructions, and
187
the pH was adjusted to 5.3 with HCl 1.0 M. The medium was autoclaved at 121 °C for
188
15 minutes (Dave & Shah, 1996).
189
ST Agar: 10 g tryptone, 10 g sucrose, 5 g yeast extract, 2 g K2HPO4 were dissolved in
190
1L distilled water. The pH was adjusted to 6.8 ± 0.1 and 6 mL bromocresol purple (0.5
191
g / 100mL) and 12 g agar were added to the medium. The medium was autoclaved at
192
121 °C for 15 min (Dave & Shah, 1996).
193
M17 Agar: 5 g tryptone; 5 g soytone; 5 g beef extract; 2.5 g yeast extract; 0.5 g
194
ascorbic acid; 0.25 g magnesium sulfate; 19 g sodium glycerophosphate; and 11 g agar
195
were dissolved in 950 mL distilled water. The medium was autoclaved at 121 °C for 15
196
minutes. For the lactose solution, 10 g lactose were dissolved in 100 mL distilled water,
197
and the solution was sterilized by filtration through 0.22 µm membrane filter (Millipore,
AC C
EP
TE D
M AN U
SC
RI PT
174
8
ACCEPTED MANUSCRIPT 198
Sao Paulo, Brazil). Immediately before use, 5 mL lactose solution was added to each 95
199
mL M17 agar (Terzaghi & Sandine, 1975).
200 For enumeration of Lactobacillus acidophilus, the plates containing MRSB agar,
202
MRSC agar, and MRS + S agar were incubated at 37 °C for 72 hours in anaerobic jars
203
using GasPak anaerobic generator (Becton, Dickinson and Company, Franklin Lakes,
204
USA). The plates containing MRSS agar were incubated at 45 °C for 72 hours under
205
anaerobic conditions. For enumeration of L. delbrueckii subsp. bulgaricus, the plates
206
containing MRS pH 5.2 and RCA pH 5.3 were incubated at 45 °C for 72 hours under
207
anaerobic conditions. S. thermophilus was enumerated using ST agar at 30 °C for 24-48
208
hours, and M17 agar supplemented with lactose incubated at 45 °C for 48-72 hours,
209
both under aerobic conditions. The starter culture L. lactis subsp. lactis and L. lactis
210
subsp. cremoris was enumerated using M17 supplemented with lactose and incubated at
211
30 or 37 °C for 48-72 hours under aerobic conditions.
213
SC
M AN U
TE D
212
RI PT
201
2.4. Yoghurt manufacture
To produce the stirred probiotic yoghurt, UHT milk was standardized (14%
215
solids) by addition of skimmed milk powder, heat-treated (95 °C / 5 minutes), cooled
216
(45 ° C), added of 1.25% culture of S. thermophilus, 1.25% culture of L. bulgaricus, and
217
1% culture of L. acidophilus (prepared as described in Section 2.2.). At the end of
218
fermentation (pH 4.8 ± 0.05), the yoghurt was cooled (10 °C). After 24 hours of cold
219
storage, the product was stirred and subjected to microbiological analysis.
AC C
EP
214
220
9
ACCEPTED MANUSCRIPT 221
2.5. Prato cheese manufacture For the manufacture of Prato cheese, 20 L milk (Atilatte - Fazenda Atibainha,
223
Atibaia, SP) were pasteurized (63 °C/30 min) and cooled to 35 °C. Then, 1% type "O"
224
starter culture was added (Lactococcus lactis subsp. lactis and Lactococcus lactis
225
subsp. cremoris), followed by calcium chloride (250 ppm), annatto dye (80 ppm) and
226
coagulant (Ha La 1175, Christian Hansen) in amount sufficient to coagulate the milk in
227
35 minutes. After 35 minutes, the gel was cut into 1 cm cubes, and slow stirring was
228
applied for 15 minutes. After a 5 minutes-rest, the whey was partially drained (30%
229
initial volume of milk), and hot water (80 °C) was added for the cooking stage up to 42
230
°C at a rate of 1 °C / 3 min. Then, the whey was partially drained, and 1% cell
231
concentrate of L. acidophilus was added. The remaining whey was drained, the curd
232
was distributed into 0.5 kg rectangular plastic molds and pressed (30 psi / 30 min; 50 psi
233
/ 90 min) at room temperature using a vertical pneumatic press. After pressing was
234
completed, the cheeses remained for a period of 5 hours at room temperature for
235
fermentation. Then, the cheeses were placed in brine (20% salt) for 10 hours at 5 ºC.
236
The cheeses were dried for 48 hours at 12 °C and vacuum packed. Then, they were
237
stored at 12 °C and subjected to microbiological evaluation after 1 day of manufacture.
239 240
SC
M AN U
TE D
EP
2.6. Enumeration of microorganisms
AC C
238
RI PT
222
Both the probiotic products and the pure cultures activated in skimmed milk
241
were evaluated using the culture media described in Section 2.3. One gram of each pure
242
culture was transferred to tubes containing 9 mL peptone water (0.1%), followed by
243
successive dilutions and pour plating.
244
To assess the viability of the microorganisms from both yoghurt and Prato
245
cheese after 1 day of manufacture, 25 g sample were transferred to stomacher bags
10
ACCEPTED MANUSCRIPT (Stomacher® 400 Classic Standard Bag - Seward Limited, Worthing, UK) with 225 mL
247
sodium citrate (2%) pH 7.0 for homogenization (5 min at medium speedy). Then, serial
248
dilutions were made in peptone water (0.1%), and the aliquots were pour-plated in the
249
culture media described in Section 2.3. The complete experiment was repeated three
250
times.
RI PT
246
251 252
3. Results and discussion
The results of the selective enumeration of Lactobacillus acidophilus from
254
yoghurt using different culture media are shown in Table 3. It was observed that both
255
MRSB and MRSC were selective for enumeration of L. acidophilus, inhibiting the
256
growth of L. bulgaricus and S. thermophilus. For enumeration of L. bulgaricus, the
257
medium RCA pH 5.3 incubated at 45 °C under anaerobic conditions was considered
258
effective, since the other cultures were inhibited under these conditions. With respect to
259
the enumeration of S. thermophilus, ST agar incubated at 30 °C under anaerobic
260
conditions was selective, since no growth of lactobacilli strains was observed.
TE D
M AN U
SC
253
For the selective enumeration of L. acidophilus from Prato cheese, the MRSS
262
incubated at 45 ° C for 72 hours under anaerobic conditions was effectively selective,
263
once it was observed development of the pure culture of Lactobacillus acidophilus and
264
inhibition of Lactococcus lactis (Table 4). For enumeration of Lactococcus lactis, M17
265
supplemented with lactose and incubated at 30 °C under anaerobic conditions was
266
selective because only Lactococcus lactis strains were able to develop in high counts
267
under these conditions.
AC C
EP
261
268
As shown in Table 4, Lactococcus lactis strains were able to grow in MRSB and
269
MRS+S media in high counts. This can lead to the statement that the MRSB and
270
MRS+S media were not selective for L. acidophilus La5 in the presence of L. lactis in
11
ACCEPTED MANUSCRIPT 271
Prato cheese. The addition of 1.5% bile to the culture medium was not able to inhibit L.
272
lactis. Although the sensitivity of lactococci strains to bile salts is reported in literature,
273
this characteristic is strain dependent (Begley et al., 2005), which may explain the
274
variability in results using this culture medium for different microorganisms.
275
contrast, the addition of bile in MRS agar was able to inhibit the yoghurt starter culture,
276
thus being considered selective for enumeration of L. acidophilus from these products
277
(Table 3). Based on experimental results, we conclude that the high counts in the Prato
278
cheese (> 9.0 log CFU / mL) when the culture media MRSB and MRS + S were used,
279
have probably included Lactococcus lactis strains, and may have also included other
280
microorganisms such as NSLAB. This conclusion was supported by colony morphology
281
and microscopic observations (data not shown). Oberg et al. (2011) also observed the
282
non-selectivity of MRS-S for enumeration of L. acidophilus from cheese.
M AN U
SC
RI PT
In
The replacement of glucose by sorbitol in MRS agar should inhibit the
284
development of the lactic acid bacteria, since these microorganisms cannot ferment
285
sorbitol, such as Streptococcus thermophilus strains (Hardie & Whiley, 2006).
286
However, this behavior was not verified in this study, since pure cultures of
287
Lactococcus lactis, Lactobacillus bulgaricus and Streptococcus thermophilus have
288
developed in the culture medium containing sorbitol at 37 °C (L. lactis) or 45 °C
289
(yoghurt cultures). Possibly the unusual growth of these strains in the presence of
290
sorbitol could be explained by culture rotation, which is a technique performed by
291
several industrial manufacturers of cultures in order to control bacteriophages. The
292
inhibition of Lactococcus lactis in MRSS agar incubated at 45 °C suggests that the
293
incubation temperature was responsible for the lack of development in this medium as
294
Lactococcus lactis does not grow at 45 °C (Nomura et al., 2006; Teuber & Geis, 2006).
295
The incubation temperature was also responsible for the inhibition of L. acidophilus in
AC C
EP
TE D
283
12
ACCEPTED MANUSCRIPT M17 agar at 30 °C from cheeses (Table 4), since this microorganism has the optimum
297
growth temperature between 35 and 40 º C (Hammes & Hertel, 2006; IDF, 1995). The
298
selectivity of the M17 agar is also related to the inhibitory effect of sodium
299
glycerophosphate on different lactobacilli strains (Shankar & Davies, 1977). This
300
inhibitory effect was confirmed by the lower counts of pure cultures of L. acidophilus in
301
M17 incubated at 37 °C (Table 4) or 45 °C (Table 3), and L. bulgaricus in M17
302
incubated at 45 °C (Table 3).
RI PT
296
The addition of clindamycin to the MRS agar did not inhibit the Lactococcus
304
lactis strains in the probiotic cheese. Ammor et al. (2007) reported that despite this
305
genus exhibits susceptibility to different groups of antibiotics, some strains have shown
306
resistant to clindamycin, as well as bacteria belonging to the genus Lactobacillus (Table
307
4).
M AN U
SC
303
Comparing the results using the culture medium RCA pH 5.3 and MRS at pH
309
5.2 (Table 4), we concluded that the components of RCA are capable of inhibiting the
310
growth of L. acidophilus and S. thermophilus. In contrast, the decrease in pH of MRS
311
agar to 5.2 was not sufficient to inhibit these microorganisms. Moreover, the
312
temperature and the conditions of incubation were the same for both culture media,
313
evidencing that the MRS agar at pH 5.2 was not selective for the enumeration of L.
314
bulgaricus from the probiotic yoghurt under the conditions studied, since other strains
315
that may be present in yoghurt are able to grow in this culture medium.
EP
AC C
316
TE D
308
Importantly, although literature reports different selective methodologies for
317
evaluation of microorganisms in probiotic products, there is a need to test the culture
318
media, once the selectivity is directly related to the presence of other strains. A careful
319
evaluation of the efficiency of a selective medium should be performed to avoid
320
overestimated results during the development of the study, since some culture media do
13
ACCEPTED MANUSCRIPT not effectively select probiotic cultures, as observed by several authors (Oberg et al.,
322
2011; Van De Casteele et al., 2006). It is necessary that the culture media are previously
323
evaluated in the matrix in question, since dairy products such as cheese, for example,
324
are complex matrices, and selective media are generally evaluated in model systems
325
using pure cultures, which may compromise the results.
RI PT
321
Further research need to be carried out in order to evaluate the effectiveness of
327
selective media, like MRS with clindamycin or bile with higher incubation temperature
328
as 45ºC for selective counting of Lactobacillus acidophilus in Prato cheese.
SC
326
329 4. Conclusions
M AN U
330
Under the conditions of this study, it was found that the MRS containing bile was
332
selective for enumeration of Lactobacillus acidophilus La5 from yoghurt, while RCA
333
agar pH 5.3 and ST agar were effective for enumeration of Lactobacillus bulgaricus and
334
Streptococcus thermophilus, respectively. For the probiotic Prato cheese, the effective
335
culture medium for enumeration of L. acidophilus was the MRSS incubated at 45 °C for
336
72 hours under anaerobic conditions, while M17 agar containing lactose and incubated
337
at 30 °C under aerobic conditions was effective for enumeration of Lactococcus lactis.
EP
TE D
331
For obtaining reliable results in the selective enumeration of probiotic
339
microorganisms in fermented dairy products is necessary to evaluate different methods,
340
both for the product under study and pure cultures, since the presence of different
341
strains may compromise the results, and therefore lead to misleading conclusions about
342
the presence and number of probiotics in dairy products.
AC C
338
343 344
Acknowledgements
14
ACCEPTED MANUSCRIPT 345
The authors thank the Foundation for Research Support of the State of São Paulo
346
(FAPESP) for financial support to the project 2009/54268-9, the National Council for
347
Scientific and Technological Development (CNPq) and the Coordination of
348
Improvement of Higher Education Personnel (CAPES) for scholarships.
RI PT
349 350
5. References
351
Ammor, M.S., Flórez, A.B., & Mayo, B. (2007). Antibiotic resistence in non-
enterococcal lactic acid bacteria and bifidobacteria. Food Microbiology, 24, 559-
353
570.
Antunes, A. E. C., Cazetto, T. F., & Bolini, H. M. A. (2005). Viability of probiotic
M AN U
354
SC
352
355
micro-organisms during storage, postacidification and sensory analysis of fat-free
356
yogurts with added whey protein concentrate. International Journal of Dairy
357
Technology, 58, 169-173.
359
Begley, M.; Gahan, C. G. M., & Hill, C. (2005). The interaction between bacteria and
TE D
358
bile. FEMS Microbiology Reviews 29, 625–651. Christian Hansen (2007). Enumeration of L. acidophilus, in fermented milk products –
361
Guidelines. Technical bulletin P-10 - L.acidophilus enumeration, nov. 2007.
EP
360
Darukaradhya, J., Philips, M., & Kailasapathy, K. (2006). Selective enumeration of
363
Lactobacillus acidophilus, Bifidobacterium spp., starter lactic acid bacteria and
364 365 366
AC C
362
non-starter lactic acid bacteria from Cheddar cheese. International Dairy Journal, 16, 439–445.
Dave, R. I., & Shah, N. P. (1996). Evaluation of media for selective enumeration of
367
Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus,
368
Lactobacillus acidophilus, and Bifidobacteria. Journal of Dairy Science, 79, 1524-
369
1536.
15
ACCEPTED MANUSCRIPT 370 371 372
De Man, J. C., Rogosa, M., & Sharpe, M. E., (1960). A medium for the cultivation of lactobacilli. Journal of Applied Microbiology, 23, 130-135. Donkor, O. N., Henriksson, A., Vasiljevic, T., & Shah, N. P. (2006). Effect of acidification on the activity of probiotics in yoghurt during cold storage.
374
International Dairy Journal, 16, 1181-1189.
RI PT
373
Espírito Santo, A. P., Perego, P., Converti, A., & Oliveira, M. N. (2012). Influence of
376
milk type and addition of passion fruit peel powder on fermentation kinetics,
377
texture profile and bacterial viability in probiotic yoghurts. LWT - Food Science
378
and Technology, 47, 393-399.
SC
375
Gebara, C., Chaves, K. S., Ribeiro, M. C. E., Souza, F. N., Grosso, C. R. F., & Gigante,
380
M. L. (2013).Viability of Lactobacillus acidophilus La5 in pectin–whey protein
381
microparticles during exposure to simulated gastrointestinal conditions. Food
382
Research International, 51, 872–878.
Hammes, W. P., & Hertel, C. (2006). The Genera Lactobacillus and Carnobacterium.
TE D
383
M AN U
379
In: M. Dworkin, S. Falkow, E. Rosenberg, K. Schleifer, & E. Stackebrandt (Eds.).
385
The Prokaryotes - A Handbook on the Biology of Bacteria. - Volume 4: Bacteria:
386
Firmicutes, Cyanobacter. 3ª ed. (pp. 320-403). Singapore: Springer Science
387
Business Media, LLC.
389 390
Hardie, J. M., & Whiley, R. A. (2006). The Genus Streptococcus – Oral. In: M.
AC C
388
EP
384
Dworkin, S. Falkow, E. Rosenberg, K. Schleifer, & E. Stackebrandt (Eds.). The
Prokaryotes - A Handbook on the Biology of Bacteria. - Volume 4: Bacteria:
391
Firmicutes, Cyanobacter. 3ª ed. (pp. 76-107). Singapore: Springer Science Business
392
Media, LLC.
16
ACCEPTED MANUSCRIPT 393
IDF. International Dairy Federation. (1995). Fermented and non-fermented milk
394
products - Detection and enumeration of Lactobacillus acidophilus - culture media.
395
Bulletin of the IDF, 306, 23-33.
397
Karimi, R., Mortazavian, A. M., & Amiri-Rigi, A. (2012). Selective enumeration of probiotic microorganisms in cheese. Food Microbiology, 29, 1-9.
RI PT
396
398
Kasimoglu, A., Göncüoglu, M., & Akgün, S. (2004). Probiotic white cheese with
399
Lactobacillus acidophilus. International Dairy Journal, 14, 1067-1073.
Lahtinen, S. J., Sanchez, B., Forssten, S., Ouwehand, A. C., & Gueimonde, M. (2011).
SC
400
Enumeration and viability assessment of probiotic bacteria. In: N.P. Shah, A. G.
402
Cruz, & J. A. F. Faria (Eds.). Probiotic and Prebiotic Foods: Technology, Stability
403
and Benefits to Human Health. (pp. 49-76). New York: Nova Science Publishers,
404
Inc.
405
M AN U
401
Mortazavian, A. M., Ehsani, M. R., Sohrabvandi, S., & Reinheimer, J. A. (2007). MRSbile agar: its suitability for the enumeration of mixed probiotic cultures in cultured
407
dairy products. Milchwissenschaft, 62, 270-272.
408
TE D
406
Nomura, M., Kobayashi, M., Narita, T., Kimoto-Nira, H., & Okamoto, T. (2006). Phenotypic and molecular characterization of Lactococcus lactis from milk and
410
plants. Journal of Applied Microbiology, 101, 396–405.
412 413 414
Oberg, C. J., Moyes, L. V., Domek, M. J., Brothersen, C., & Mcmahon, D. J. (2011).
AC C
411
EP
409
Survival of probiotic adjunct cultures in cheese and challenges in their enumeration
using selective media. Journal of Dairy Science, 94, 2220-2230.
O’Flaherty, S., & Klaenhammer, T. R. (2010). The role and potential of probiotic
415
bacteria in the gut, and the communication between gut microflora and gut/host.
416
International Dairy Journal, 20, 262–268.
17
ACCEPTED MANUSCRIPT 417
Ong, L., Henriksson, A., & Shah, N. P. (2006). Development of probiotic Cheddar
418
cheese containing Lactobacillus acidophilus, Lb. casei, Lb. paracasei and
419
Bifidobacterium spp. and the influence of these bacteria on proteolytic patterns and
420
production of organic acid. International Dairy Journal, 16, 446-456. Ong, L., & Shah, N, P. (2008). Influence of probiotic Lactobacillus acidophilus and L.
422
helveticus on proteolysis, organic acid profiles, and ACE-inhibitory activity of
423
cheddar cheeses ripened at 4, 8, and 12ºC. Journal of Food Science, 73, M111-
424
M120.
SC
425
RI PT
421
Ong, L., & Shah, N. P. (2009). Probiotic Cheddar cheese: influence of ripening temperatures on survival of probiotic microorganisms, cheese composition and
427
organic acid profiles. LWT – Food Science and Technology, 42, 1260-1268.
428
Özer, B., Uzun, Y. S., & Kirmaci, H. A. (2008). Effect of microencapsulation on
M AN U
426
viability of Lactobacillus acidophilus LA-5 and Bifidobacterium bifidum BB-12
430
during Kasar cheese ripening. International Journal of Dairy Technology, 61, 237-
431
244.
TE D
429
Philips, M., Kailasapathy, K., & Tran, L. (2006). Viability of commercial probiotic
433
cultures (L. acidophilus, Bifidobacterium sp., L. casei, L. paracasei and L.
434
rhamnosus) in Cheddar cheese. International Journal of Food Microbiology, 108,
435
276-280.
437
AC C
436
EP
432
Ribeiro, M. C. E., Chaves, K. S., Gebara, C., Infante, F. N. S., Grosso, C. R. F., & Gigante, M. L. (2014). Effect of microencapsulation of Lactobacillus acidophilus
438
LA-5 on physicochemical, sensory and microbiological characteristics of stirred
439
probiotic yoghurt. Food Research International, 66, 424–431.
18
ACCEPTED MANUSCRIPT 440
Ross, R. P., Fitzgerald, G., Collins, K., & Stanton, C. (2002). Cheese delivering
441
biocultures – probiotic cheese. The Australian Journal of Dairy Technology, 57,
442
71-78.
443
Saccaro, D. M., Hirota, C. Y., Tamime, A. Y., & Oliveira, M. N. (2012). Evaluation of different selective media for enumeration of probiotic micro-organisms in
445
combination with yogurt starter cultures in fermented milk. African Journal of
446
Microbiology Research, 6, 2239-2245.
449
foods. Journal of Dairy Science, 83, 894-907.
SC
448
Shah, N. P. (2000). Probiotic bacteria: selective enumeration and survival in dairy
Shankar, P. A., & Davies, F. L. (1977). A note on the suppression of Lactobacillus
M AN U
447
RI PT
444
450
bulgaricus in media containing β-glycerophosphate and application of such media
451
to selective isolation of Streptococcus thermophiles from yoghurt. Journal of the
452
Society of Dairy Technology, 30, 28-30.
454
Terzaghi, B.E., & Sandine, W.E. (1975). Improved medium for lactic streptococci and
TE D
453
their bacteriophages. Applied Microbiology, 29, 807-813. Teuber, M., & Geis, A. (2006). The Genus Lactococcus. In: M. Dworkin, S. Falkow, E.
456
Rosenberg, K. Schleifer, & E. Stackebrandt (Eds.). The Prokaryotes - A Handbook
457
on the Biology of Bacteria. - Volume 4: Bacteria: Firmicutes, Cyanobacter. 3ª ed.
458
(pp. 205-228). Singapore: Springer Science Business Media, LLC.
460
AC C
459
EP
455
Tharmaraj, N., & Shah, N. P. (2003). Selective enumeration of Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus,
461
bifidobacteria, Lactobacillus casei, Lactobacillus rhamnosus, and propionibacteria.
462
Journal of Dairy Science, 86, 2288-2296.
463 464
Van De Casteele, S., Vanheuverzwijn, T., Ruyssen, T., Van Assche, P., Swings, J., & Huys, G. (2006). Evaluation of culture media for selective enumeration of probiotic
19
ACCEPTED MANUSCRIPT 465
strains of lactobacilli and bifidobacteria in combination with yoghurt or cheese
466
starters. International Dairy Journal, 16, 1470-1476.
468 469
Vasiljevic, T., & Shah, N. P. (2008). Probiotics – From Metchnikoff to bioactives. International Dairy Journal, 18, 714-728. Vinderola, C. G., & Reinheimer, J. A. (1999). Culture media for the enumeration of
RI PT
467
470
Bifidobacterium bifidum and Lactobacillus acidophilus in the presence of yoghurt
471
bacteria. International Dairy Journal, 9, 497-505.
Vinderola, C. G., Prosello, W., Ghiberto, D., & Reinheimer, J. A. (2000). Viability of
SC
472
probiotic (Bifidobacterium, Lactobacillus acidophilus and Lactobacillus casei) and
474
nonprobiotic microflora in argentinian fresco cheese. Journal of Dairy Science, 83,
475
1905-1911.
476
M AN U
473
Zacarchenco, P. B., & Massaguer-Roig, S. (2004). Enumeration of Streptococcus thermophilus in the presence of Bifidobacterium longum and Lactobacillus
478
acidophilus – effect of incubation temperature and atmospheric conditions.
479
Milchwissenschaft, 59, 370-372.
AC C
EP
TE D
477
20
ACCEPTED MANUSCRIPT 1
Table 1 – Selective culture media used for enumerating Lactobacillus acidophilus in
2
commercial probiotic yoghurt, or in combination with Streptococcus thermophilus and
3
Lactobacillus bulgaricus.
Agar
based on
Incubation conditions
References
37ºC, 72 hours,
IDF (1995); Vinderola & Reinheimer (1999);
anaerobic incubation
Mortazavian et al. (2007); Ribeiro et al. (2014)
37ºC, 72 hours,
Antunes et al. (2005)
RI PT
MRS
Selectivity
Bile aerobic incubation
Agar
Dave & Shah (1996); Tharmaraj & Shah
anaerobic incubation
(2003)
Sorbitol
M AN U
MRS
37ºC, 72 hours,
SC
Base
37ºC, 48 hours,
Donkor et al. (2006)
anaerobic incubation MRS Agar
Clindamycin
37ºC, 72 hours,
Van de Casteele et al. (2006); Espírito Santo et
anaerobic incubation
al. (2012); Saccaro et al. (2012)
AC C
EP
TE D
4
ACCEPTED MANUSCRIPT 1
Table 2 – Selective culture media used for enumerating Lactobacillus acidophilus in
2
probiotic cheese, made with addition of Lactococcus lactis.
MRS Agar
Selectivity based
Incubation
on
conditions
Bile
Cheese type
37ºC, 72 hours,
Argentinian
Vinderola et al.
aerobic incubation
fresh cheese
(2000)
MRS
Clindamycin and
37ºC, 48 hours,
Cheddar
Agar
bromocresol green
anaerobic incubation
cheese Cheddar
Agar
37ºC, 72 hours,
Sorbitol
anaerobic incubation
cheese Kasar cheese
Agar
45ºC, 72 hours,
Sorbitol
Cheddar
M AN U
MRS
anaerobic incubation
AC C
EP
TE D
3
Phillips et al. (2006) Ong et al. (2006);
Ong & Shah (2009)
SC
MRS
References
RI PT
Base
cheese
Özer et al. (2008) Oberg et al. (2011)
ACCEPTED MANUSCRIPT Table 3. Microbiological analysis of yoghurts containing Lactobacillus acidophilus and
2
pure cultures of Lactobacillus acidophilus La5, Lactobacillus bulgaricus and
3
Streptococcus thermophilus, using different culture media (n = 3). Values expressed as
4
log10 CFU / g.
5 Enumeration in pure cultures
Enumeration
L. bulgaricus
S. thermophilus
in yoghurt
MRSB1
9.13 ± 0.33
<2
<2
8.25 ± 1.03
MRSC2
9.01 ± 0.69
<2
<2
7.79 ± 0.75
MRS + S3
9.36 ± 0.08
8.81 ± 0.10
5.91 ± 5.12
8.24 ± 0.55
MRS pH 5,24
8.41 ± 0.87
8.52 ± 0.44
7.40 ± 3.11
8.60 ± 0.08
8.74 ± 0.07
<2
8.96 ± 0.78
2.92 ± 2.62
8.93 ± 0.29
8.98 ± 0.23
<2
9.04 ± 0.21
8.82 ± 0.22
RCA5
<2
M17 456
<2
ST7
<2
TE D
6
M AN U
L. acidophilus
SC
Culture media
RI PT
1
7
1
8
conditions; 2MRS agar containing clindamycin (0.5 ppm) incubated at 37°C for 72 h under anaerobic
9
conditions; 3MRS agar containing 1% sorbitol (solution) incubated at 37°C for 72 h under anaerobic 4
EP
MRS agar containing 1.5% bile solution (10% w/v) incubated at 37°C for 72 h under anaerobic
10
conditions;
11
5
Reinforced Clostridium agar at pH adjusted to 5.3 incubated at 45°C for 72 h under anaerobic conditions;
12
6
M17 agar containing 5% lactose solution (10% w/v) incubated at 45°C for 72 h under aerobic
13
conditions; 7ST agar incubated at 30°C for 72 h under aerobic conditions.
AC C
14
MRS agar at pH adjusted to 5.2 incubated at 45°C for 72 h under anaerobic conditions;
ACCEPTED MANUSCRIPT Table 4. Microbiological analysis of Prato cheese containing Lactobacillus acidophilus
2
and pure cultures of Lactobacillus acidophilus La5 and Lactococcus lactis (L. lactis
3
subsp. lactis and L. lactis subsp. cremoris) using different culture media (n = 3). Values
4
expressed as log10 CFU / g.
5 Enumeration in pure cultures Culture media
RI PT
1
Enumeration in cheese
L. acidophilus
L. lactis
MRSB 1
9.75 ± 0.03
9.13 ± 0.44
MRSC2
9.38 ± 0.56
6.13 ± 1.12
6.23 ± 1.24
MRS + S3
9.71 ± 0.10
9.50 ± 0.29
9.59 ± 0.10
MRSS 4
9.71 ± 0.07
<2
6.31 ±0.18
M17 375
2.42 ± 0.89
9.54 ± 0.26
9.59 ± 0.12
M17 306
<2
9.54 ± 0.24
9.55 ± 0.09
SC
M AN U
6
9.28 ±0.22
7
1
8
conditions; 2MRS agar containing clindamycin (0.5 ppm) incubated at 37°C for 72 h under anaerobic
9
conditions; 3MRS agar containing 1% sorbitol (solution) as the only carbohydrate source, incubated at
10
37°C for 72 h under anaerobic conditions; 4MRS agar containing 1% sorbitol as the only carbohydrate
11
source incubated at 45°C for 72 h under anaerobic conditions; 5M17 agar containing 5% lactose solution
12
(10% w/v) incubated at 37°C for 72 h under aerobic conditions;
13
solution (10% w/v) incubated at 30°C for 72 h under aerobic conditions.
TE D
EP
AC C
14
MRS agar containing 1.5% bile solution (10% w/v) incubated at 37°C for 72 h under anaerobic
6
M17 agar containing 5% lactose
ACCEPTED MANUSCRIPT Highlights
2
Methodologies for selective enumeration of probiotics in dairy products.
3
Effectiveness of the methodologies in yoghurt and Prato cheese.
4
Selective enumeration of Lactobacillus acidophilus from yoghurt using MRS bile agar.
5
MRS sorbitol agar at 45 °C is selective for L. acidophilus from Prato cheese.
AC C
EP
TE D
M AN U
SC
RI PT
1
S0023-6438(15)00343-6
DOI:
10.1016/j.lwt.2015.04.061
Reference:
YFSTL 4650
To appear in:
LWT - Food Science and Technology
Received Date: 14 May 2014 Revised Date:
15 April 2015
Accepted Date: 24 April 2015
Please cite this article as: Gebara, C., Ribeiro, M.C.E., Chaves, K.S., Gandara, A.L.N., Gigante, M.L., Effectiveness of different methodologies for the selective enumeration of Lactobacillus acidophilus La5 from yoghurt and Prato cheese, LWT - Food Science and Technology (2015), doi: 10.1016/ j.lwt.2015.04.061. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Effectiveness of different methodologies for the selective enumeration of
2
Lactobacillus acidophilus La5 from yoghurt and Prato cheese
3
Clarice Gebara1*, Maria Cecília E. Ribeiro1, Karina S. Chaves1, Ana Lourdes N.
4
Gandara2, Mirna L. Gigante1
5
1
6
Campinas, 13082-862, Campinas, S.P., Brazil
7
2
8
*Corresponding author Tel.: +55 19 35213993
9
*E-mail address: [email protected] (C. Gebara)
RI PT
Department of Food Technology, Faculty of Food Engineering, University of
SC
Technical High School of Campinas, 13020-060, Campinas, S.P., Brazil.
M AN U
10 ABSTRACT
12
Prior to the selective enumeration of probiotic microorganisms in fermented dairy
13
products, a preliminary evaluation of different culture media and incubation conditions
14
is required to choose a suitable culture medium. The objective of this study was to
15
evaluate the effectiveness of different methodologies for the selective enumeration of L.
16
acidophilus in probiotic yoghurt and Prato cheese. For selective enumeration of L.
17
acidophilus, four methods were investigated under anaerobic conditions, as follows:
18
MRS agar containing bile solution; MRS agar containing clindamycin solution; and
19
MRS agar containing sorbitol solution, each one incubated at 37 °C for 72 hours, and
20
MRS agar containing sorbitol incubated at 45 °C for 72 hours. These methodologies
21
were used for enumeration of both the pure cultures and the microorganisms from
22
probiotic yoghurt and Prato cheese. The results showed that the best selective medium
23
for enumeration of Lactobacillus acidophilus from yoghurt was MRS agar containing
24
bile incubated at 37 ° C for 72 hours under anaerobic conditions. For enumeration of
AC C
EP
TE D
11
1
ACCEPTED MANUSCRIPT 25
this probiotics from Prato cheese, MRS agar containing sorbitol incubated at 45 º C for
26
72 hours under anaerobic conditions was proven to be the best selective medium.
27 Keywords: Lactobacillus acidophilus, selective enumeration, probiotic yoghurt,
29
probiotic cheese
RI PT
28
30 31
1.
Introduction
The probiotic microorganisms play an important role in the functional foods
33
industry, contributing to a market involving billions of dollars (O’Flaherty &
34
Klaenhammer, 2010). Although they can be added in different food products, dairy
35
products are considered ideal systems for delivery of these microorganisms,
36
contributing to the maintenance of viability during passage through the gastrointestinal
37
tract (Ross et al., 2002). To provide health benefits, the probiotic microorganism must
38
be in products in minimal viable numbers, usually 106-107 CFU / g. Therefore, the
39
adequate evaluation of the viability of these microorganisms in the product should be
40
achieved by efficient methodologies for enumeration (Lahtinen et al., 2011; Shah, 2000;
41
Vasiljevic & Shah, 2008).
EP
TE D
M AN U
SC
32
Several methods for enumeration of L. acidophilus and Bifidobacterium spp.
43
have been proposed; however, most of them are based on enumeration of pure cultures,
44
but not in complex matrices such as foods (Lahtinen et al., 2011; Shah, 2000; Vasiljevic
45
& Shah, 2008). The choice of a methodology suitable for the selective enumeration of
46
probiotic strains in combination with lactic acid starters depends on both the
47
microorganisms and the matrix in question, since a culture medium alone is not suitable
48
for a variety of applications (Karimi et al., 2012; Tharmaraj & Shah, 2003; Van De
49
Casteele et al. 2006).
AC C
42
2
ACCEPTED MANUSCRIPT Traditionally, the enumeration of probiotic microorganisms in food has been
51
performed by plate count technique, and the result is expressed as colony forming units
52
(CFU) (Lahtinen et al., 2011). This enumeration can be obtained using differential or
53
selective media. The selective enumeration is achieved through the addition of
54
inhibitory compounds (such as antibiotics), pH changes, or incubation conditions
55
(temperature and oxygen levels). The selective media allow direct enumeration of
56
colony on the plates without the need for additional analysis, being easy to perform
57
(Lahtinen et al., 2011; Oberg et al., 2011). Many different media were developed for the
58
selective enumeration of probiotics in dairy products, but few have been validated in
59
comparative studies with adequate quantification, recovery, and differentiation from
60
other microorganisms in the product (Tharmaraj & Shah, 2003).
M AN U
SC
RI PT
50
A summary of the studies found in the literature with the selective
62
methodologies used for enumerating Lactobacillus acidophilus in probiotic yoghurt
63
(Table 1) and cheese (Table 2) is presented. Among the selective methodologies, the
64
MRS agar containing bile and incubated at 37 °C for 72 hours under anaerobic
65
conditions was described by International Dairy Federation - IDF (1995) for selective
66
enumeration of Lactobacillus acidophilus in the presence of yoghurt cultures
67
(Streptococcus thermophilus and Lactobacillus bulgaricus) and bifidobacteria. The
68
efficiency of this medium in the selective enumeration of Lactobacillus acidophilus in
69
the presence of yoghurt cultures was confirmed by Mortazavian et al. (2007) and
70
Vinderola & Reinheimer (1999). Bile MRS Agar was also used for the selective
71
enumeration of L. acidophilus in Argentinian fresh cheese produced with Lactococcus
72
lactis and B. bifidum (Vinderola et al., 2000).
AC C
EP
TE D
61
73
The Lactobacillus acidophilus La5 is one of the most studied probiotic strains,
74
with application in different dairy products. For selective enumeration of this
3
ACCEPTED MANUSCRIPT microorganism in the presence of mesophilic cultures, the manufacturer recommends
76
MRS agar containing clindamycin incubated at 37 °C for 72 h under anaerobic
77
conditions (Christian Hansen, 2007). As reported by Van De Casteele et al. (2006), this
78
culture medium was effective for enumeration of L. acidophilus in the presence of
79
commercial lactic acid starters added during the manufacturing processes of yoghurt
80
and cheese.
RI PT
75
On the other hand, the MRS agar containing sorbitol incubated at 37 °C for 72
82
hours under anaerobic conditions was selective for the enumeration of Lactobacillus
83
acidophilus in the presence of Streptococcus thermophilus, Lactobacillus bulgaricus
84
and bifidobacteria in a study on pure cultures (Dave & Shah, 1996). Subsequent studies
85
have used this medium for selective enumeration of L. acidophilus in Cheddar cheese
86
manufactured using Lactococcus lactis (Ong et al, 2006; Ong & Shah, 2009), and
87
turkish white-brined cheese also manufactured using Lactococcus lactis (Özer et al.,
88
2008). In a recent study, MRS agar containing sorbitol incubated at 37 °C under
89
anaerobic conditions was considered non-selective for enumeration of Lactobacillus
90
acidophilus in Cheddar cheese manufactured using probiotic microorganisms as adjunct
91
cultures (L. acidophilus, L. casei, L. paracasei, Bifidobacterium lactis) and Lactococcus
92
sp. (Oberg et al., 2011). In contrast, in the same study, the MRS agar with sorbitol as the
93
only carbohydrate source incubated at 45 °C under anaerobic conditions was effective
94
for the selective enumeration of Lactobacillus acidophilus in Cheddar cheese.
M AN U
TE D
EP
AC C
95
SC
81
Selective enumeration of starter cultures of lactic acid bacteria can be achieved
96
in the presence of probiotic microorganisms. The literature describes the M17 agar
97
containing lactose incubated at 30 °C for enumeration of lactococci (Terzaghi &
98
Sandine, 1975). This culture medium has been the most used for selective enumeration
99
of Lactococcus lactis in the presence of Lactobacillus acidophilus (Darukaradhya et al.,
4
ACCEPTED MANUSCRIPT 2006; Kasimoglu et al., 2004; Oberg et al., 2011; Ong et al., 2006; Ong & Shah, 2008,
101
2009; Van De Casteele et al., 2006). The M17 Agar is also used for enumeration of
102
Streptococcus thermophilus (Shankar & Davies, 1977; Van De Casteele et al., 2006).
103
Besides the M17 agar, ST agar (Streptococcus thermophilus agar) was effective for
104
enumeration of Streptococcus thermophilus in the presence of Lactobacillus delbrueckii
105
subsp. bulgaricus and Lactobacillus acidophilus (Dave & Shah, 1996). Zacarchenco &
106
Massaguer-Roig (2004) also reported the selectivity of ST agar for enumeration of
107
Streptococcus thermophilus in the presence of Lactobacillus acidophilus and
108
Bifidobacterium longum. Both the MRS Agar pH 5.2 and the Reinforced Clostridial
109
Agar (RCA) pH 5.3 were effective for enumeration of Lactobacillus delbrueckii subsp.
110
bulgaricus in the presence of Streptococcus thermophilus, Lactobacillus acidophilus
111
and Bifidobacterium sp. (Dave & Shah, 1996).
M AN U
SC
RI PT
100
Although literature suggests different conditions for selective enumeration of
113
probiotic microorganisms, the efficiency depends on several factors including the strain,
114
presence of other cultures in the product, and type of product to which the probiotics
115
will be added. Thus, to select the methodology for selective enumeration of probiotics
116
in dairy products, it is important that different methods are tested. Given the variety of
117
culture media described in literature, the aim of this study was to evaluate the
118
effectiveness of different methodologies for selective enumeration of L. acidophilus
119
from probiotic yoghurt and Prato cheese.
AC C
EP
TE D
112
120 121
2.
Material and methods
122
2.1. Cultures
123
Four commercial cultures were used: probiotic culture Lactobacillus acidophilus
124
(La5 - Christian Hansen Ind. e Com, Valinhos, Brazil); two yoghurt starter cultures -
5
ACCEPTED MANUSCRIPT 125
Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp.
126
thermophilus (LB 340 and TA 40 - Danisco A / S Brabrand, Denmark); and type "O"
127
starter culture for cheese manufacture (Lactococcus lactis subsp lactis and Lactococcus
128
lactis subsp cremoris - R704, Christian Hansen Ind. e Com, Valinhos, Brazil).
130
RI PT
129 2.2. Preparation of the inoculum
Lyophilized culture of Lactobacillus acidophilus La5 was rehydrated (at 15 °C
132
for 20 minutes) in sterile reconstituted skimmed milk containing 20% (v / v) glycerol
133
(cryoprotectant), and transferred to micro tubes and kept in a freezer (-18 °C). For
134
processing, this culture was reactivated in MRS broth (2% v / v) for two consecutive
135
times, with incubation at 37 ° C for 15 hours. The reactivated culture was placed in
136
sterile polypropylene tubes and centrifuged at 8,000 g for 10 minutes at 4 °C (Sorvall
137
Instruments RC5C - DuPont). The cell concentrate was resuspended in peptone water
138
(0.1% v / v) and centrifuged again under the same conditions, followed by resuspension
139
in peptone water to obtain a concentration of cells with 9-10 log10 CFU / mL (Gebara et
140
al., 2013). The cell concentrate viability was assessed by pour plating on MRS agar
141
incubated at 37 ° C for 72 hours in anaerobic jars using GasPak anaerobic generator
142
(Becton, Dickinson and Company, Franklin Lakes, USA) (De Man et al., 1960). A cell
143
concentrate of Lactobacillus acidophilus was prepared for each run.
M AN U
TE D
EP
AC C
144
SC
131
The cultures L. delbrueckii subsp. bulgaricus, S. thermophilus, and the starter
145
culture L. lactis subsp. lactis and L. lactis subsp. cremoris were previously activated in
146
sterile reconstituted (11%) skimmed milk and incubated at 45 °C / 6 h; 45 °C / 4 h; and
147
30 °C / 16h, respectively. Then, they were used in the analysis of pure cultures and in
148
the manufacture process, being prepared for each processing.
149
6
ACCEPTED MANUSCRIPT 150
2.3. Media Preparation The culture media described below were selected from literature for selective
152
enumeration of Lactobacillus acidophilus in yoghurt and cheese. The methodologies
153
were strictly followed as described in the references:
154
Bile Agar MRS (MRSB): Commercial MRS Agar (Difco - Becton, Dickinson and
155
Company, Franklin Lakes, USA) was prepared according to manufacturer’s
156
instructions. The rehydrated medium was autoclaved at 121 °C for 15 minutes. For bile
157
solution, 10 g bile (B3883 - Sigma-Aldrich Co., St. Louis, USA) was dissolved in 100
158
mL distilled water, and the solution was autoclaved at 121 °C for 15 minutes.
159
Immediately before use, 1.5 mL bile solution was added to 100 mL MRS agar (IDF,
160
1995).
161
Clindamycin MRS Agar (MRSC): Commercial MRS agar (Difco - Becton, Dickinson
162
and Company, Franklin Lakes, USA) was prepared according to manufacturer's
163
instructions. The rehydrated medium was autoclaved at 121 °C for 15 minutes. For the
164
clindamycin solution, 2 mg Clindamycin hydrochloride were dissolved in 10 mL
165
distilled water, and sterilized by filtration through 0.22 µm membrane filter (Millipore,
166
São Paulo, Brazil). Immediately before use, 0.5 mL clindamycin solution was added to
167
1000 mL MRS agar (Christian Hansen, 2007).
168
MRS Agar + Sorbitol (MRS + S): MRS agar was prepared without glucose (10 g
169
peptone; 8 g meat extract; 4 g yeast extract; 2 g K2HPO4; 5 g sodium acetate; 2 g
170
ammonium citrate; 0.2 g magnesium sulfate; 0.05 g manganese sulfate; 1 mL Tween 80;
171
and 10 g agar dissolved in 1L distilled water) and autoclaved at 121 °C for 15 minutes.
172
For the sorbitol solution, 10 g D-sorbitol were dissolved in 100 mL distilled water, and
173
the solution was sterilized by filtration through 0.22 µm membrane filter (Millipore, São
AC C
EP
TE D
M AN U
SC
RI PT
151
7
ACCEPTED MANUSCRIPT Paulo, Brazil). Immediately before use, 10 mL D-sorbitol solution was added for each
175
90 mL MRS agar (1% final concentration) (Dave & Shah, 1996).
176
MRS-Sorbitol Agar (MRSS): MRS agar was prepared without glucose (10 g peptone;
177
8 g meat extract; 4 g yeast extract; 2 g K2HPO4; 5 g sodium acetate; 2 g ammonium
178
citrate; 0.2 g magnesium sulfate; 0.05 g manganese sulfate; 1 mL Tween 80; and 10g
179
agar dissolved in 1L distilled water) with addition of 10g / L D-sorbitol. The medium
180
was autoclaved at 121 °C for 15 minutes (Oberg et al., 2011).
181
MRS Agar pH 5.2: MRS agar (Difco - Becton, Dickinson and Company, Franklin
182
Lakes, USA) was prepared according to the manufacturer's recommendations, and the
183
pH was adjusted to 5.2 with HCl 1.0 M. The medium was autoclaved at 121 °C for 15
184
minutes (Dave & Shah, 1996).
185
RCA pH 5.3: Reinforced Clostridial Agar (Difco - Becton, Dickinson and Company,
186
Franklin Lakes, USA) was prepared according to the manufacturer's instructions, and
187
the pH was adjusted to 5.3 with HCl 1.0 M. The medium was autoclaved at 121 °C for
188
15 minutes (Dave & Shah, 1996).
189
ST Agar: 10 g tryptone, 10 g sucrose, 5 g yeast extract, 2 g K2HPO4 were dissolved in
190
1L distilled water. The pH was adjusted to 6.8 ± 0.1 and 6 mL bromocresol purple (0.5
191
g / 100mL) and 12 g agar were added to the medium. The medium was autoclaved at
192
121 °C for 15 min (Dave & Shah, 1996).
193
M17 Agar: 5 g tryptone; 5 g soytone; 5 g beef extract; 2.5 g yeast extract; 0.5 g
194
ascorbic acid; 0.25 g magnesium sulfate; 19 g sodium glycerophosphate; and 11 g agar
195
were dissolved in 950 mL distilled water. The medium was autoclaved at 121 °C for 15
196
minutes. For the lactose solution, 10 g lactose were dissolved in 100 mL distilled water,
197
and the solution was sterilized by filtration through 0.22 µm membrane filter (Millipore,
AC C
EP
TE D
M AN U
SC
RI PT
174
8
ACCEPTED MANUSCRIPT 198
Sao Paulo, Brazil). Immediately before use, 5 mL lactose solution was added to each 95
199
mL M17 agar (Terzaghi & Sandine, 1975).
200 For enumeration of Lactobacillus acidophilus, the plates containing MRSB agar,
202
MRSC agar, and MRS + S agar were incubated at 37 °C for 72 hours in anaerobic jars
203
using GasPak anaerobic generator (Becton, Dickinson and Company, Franklin Lakes,
204
USA). The plates containing MRSS agar were incubated at 45 °C for 72 hours under
205
anaerobic conditions. For enumeration of L. delbrueckii subsp. bulgaricus, the plates
206
containing MRS pH 5.2 and RCA pH 5.3 were incubated at 45 °C for 72 hours under
207
anaerobic conditions. S. thermophilus was enumerated using ST agar at 30 °C for 24-48
208
hours, and M17 agar supplemented with lactose incubated at 45 °C for 48-72 hours,
209
both under aerobic conditions. The starter culture L. lactis subsp. lactis and L. lactis
210
subsp. cremoris was enumerated using M17 supplemented with lactose and incubated at
211
30 or 37 °C for 48-72 hours under aerobic conditions.
213
SC
M AN U
TE D
212
RI PT
201
2.4. Yoghurt manufacture
To produce the stirred probiotic yoghurt, UHT milk was standardized (14%
215
solids) by addition of skimmed milk powder, heat-treated (95 °C / 5 minutes), cooled
216
(45 ° C), added of 1.25% culture of S. thermophilus, 1.25% culture of L. bulgaricus, and
217
1% culture of L. acidophilus (prepared as described in Section 2.2.). At the end of
218
fermentation (pH 4.8 ± 0.05), the yoghurt was cooled (10 °C). After 24 hours of cold
219
storage, the product was stirred and subjected to microbiological analysis.
AC C
EP
214
220
9
ACCEPTED MANUSCRIPT 221
2.5. Prato cheese manufacture For the manufacture of Prato cheese, 20 L milk (Atilatte - Fazenda Atibainha,
223
Atibaia, SP) were pasteurized (63 °C/30 min) and cooled to 35 °C. Then, 1% type "O"
224
starter culture was added (Lactococcus lactis subsp. lactis and Lactococcus lactis
225
subsp. cremoris), followed by calcium chloride (250 ppm), annatto dye (80 ppm) and
226
coagulant (Ha La 1175, Christian Hansen) in amount sufficient to coagulate the milk in
227
35 minutes. After 35 minutes, the gel was cut into 1 cm cubes, and slow stirring was
228
applied for 15 minutes. After a 5 minutes-rest, the whey was partially drained (30%
229
initial volume of milk), and hot water (80 °C) was added for the cooking stage up to 42
230
°C at a rate of 1 °C / 3 min. Then, the whey was partially drained, and 1% cell
231
concentrate of L. acidophilus was added. The remaining whey was drained, the curd
232
was distributed into 0.5 kg rectangular plastic molds and pressed (30 psi / 30 min; 50 psi
233
/ 90 min) at room temperature using a vertical pneumatic press. After pressing was
234
completed, the cheeses remained for a period of 5 hours at room temperature for
235
fermentation. Then, the cheeses were placed in brine (20% salt) for 10 hours at 5 ºC.
236
The cheeses were dried for 48 hours at 12 °C and vacuum packed. Then, they were
237
stored at 12 °C and subjected to microbiological evaluation after 1 day of manufacture.
239 240
SC
M AN U
TE D
EP
2.6. Enumeration of microorganisms
AC C
238
RI PT
222
Both the probiotic products and the pure cultures activated in skimmed milk
241
were evaluated using the culture media described in Section 2.3. One gram of each pure
242
culture was transferred to tubes containing 9 mL peptone water (0.1%), followed by
243
successive dilutions and pour plating.
244
To assess the viability of the microorganisms from both yoghurt and Prato
245
cheese after 1 day of manufacture, 25 g sample were transferred to stomacher bags
10
ACCEPTED MANUSCRIPT (Stomacher® 400 Classic Standard Bag - Seward Limited, Worthing, UK) with 225 mL
247
sodium citrate (2%) pH 7.0 for homogenization (5 min at medium speedy). Then, serial
248
dilutions were made in peptone water (0.1%), and the aliquots were pour-plated in the
249
culture media described in Section 2.3. The complete experiment was repeated three
250
times.
RI PT
246
251 252
3. Results and discussion
The results of the selective enumeration of Lactobacillus acidophilus from
254
yoghurt using different culture media are shown in Table 3. It was observed that both
255
MRSB and MRSC were selective for enumeration of L. acidophilus, inhibiting the
256
growth of L. bulgaricus and S. thermophilus. For enumeration of L. bulgaricus, the
257
medium RCA pH 5.3 incubated at 45 °C under anaerobic conditions was considered
258
effective, since the other cultures were inhibited under these conditions. With respect to
259
the enumeration of S. thermophilus, ST agar incubated at 30 °C under anaerobic
260
conditions was selective, since no growth of lactobacilli strains was observed.
TE D
M AN U
SC
253
For the selective enumeration of L. acidophilus from Prato cheese, the MRSS
262
incubated at 45 ° C for 72 hours under anaerobic conditions was effectively selective,
263
once it was observed development of the pure culture of Lactobacillus acidophilus and
264
inhibition of Lactococcus lactis (Table 4). For enumeration of Lactococcus lactis, M17
265
supplemented with lactose and incubated at 30 °C under anaerobic conditions was
266
selective because only Lactococcus lactis strains were able to develop in high counts
267
under these conditions.
AC C
EP
261
268
As shown in Table 4, Lactococcus lactis strains were able to grow in MRSB and
269
MRS+S media in high counts. This can lead to the statement that the MRSB and
270
MRS+S media were not selective for L. acidophilus La5 in the presence of L. lactis in
11
ACCEPTED MANUSCRIPT 271
Prato cheese. The addition of 1.5% bile to the culture medium was not able to inhibit L.
272
lactis. Although the sensitivity of lactococci strains to bile salts is reported in literature,
273
this characteristic is strain dependent (Begley et al., 2005), which may explain the
274
variability in results using this culture medium for different microorganisms.
275
contrast, the addition of bile in MRS agar was able to inhibit the yoghurt starter culture,
276
thus being considered selective for enumeration of L. acidophilus from these products
277
(Table 3). Based on experimental results, we conclude that the high counts in the Prato
278
cheese (> 9.0 log CFU / mL) when the culture media MRSB and MRS + S were used,
279
have probably included Lactococcus lactis strains, and may have also included other
280
microorganisms such as NSLAB. This conclusion was supported by colony morphology
281
and microscopic observations (data not shown). Oberg et al. (2011) also observed the
282
non-selectivity of MRS-S for enumeration of L. acidophilus from cheese.
M AN U
SC
RI PT
In
The replacement of glucose by sorbitol in MRS agar should inhibit the
284
development of the lactic acid bacteria, since these microorganisms cannot ferment
285
sorbitol, such as Streptococcus thermophilus strains (Hardie & Whiley, 2006).
286
However, this behavior was not verified in this study, since pure cultures of
287
Lactococcus lactis, Lactobacillus bulgaricus and Streptococcus thermophilus have
288
developed in the culture medium containing sorbitol at 37 °C (L. lactis) or 45 °C
289
(yoghurt cultures). Possibly the unusual growth of these strains in the presence of
290
sorbitol could be explained by culture rotation, which is a technique performed by
291
several industrial manufacturers of cultures in order to control bacteriophages. The
292
inhibition of Lactococcus lactis in MRSS agar incubated at 45 °C suggests that the
293
incubation temperature was responsible for the lack of development in this medium as
294
Lactococcus lactis does not grow at 45 °C (Nomura et al., 2006; Teuber & Geis, 2006).
295
The incubation temperature was also responsible for the inhibition of L. acidophilus in
AC C
EP
TE D
283
12
ACCEPTED MANUSCRIPT M17 agar at 30 °C from cheeses (Table 4), since this microorganism has the optimum
297
growth temperature between 35 and 40 º C (Hammes & Hertel, 2006; IDF, 1995). The
298
selectivity of the M17 agar is also related to the inhibitory effect of sodium
299
glycerophosphate on different lactobacilli strains (Shankar & Davies, 1977). This
300
inhibitory effect was confirmed by the lower counts of pure cultures of L. acidophilus in
301
M17 incubated at 37 °C (Table 4) or 45 °C (Table 3), and L. bulgaricus in M17
302
incubated at 45 °C (Table 3).
RI PT
296
The addition of clindamycin to the MRS agar did not inhibit the Lactococcus
304
lactis strains in the probiotic cheese. Ammor et al. (2007) reported that despite this
305
genus exhibits susceptibility to different groups of antibiotics, some strains have shown
306
resistant to clindamycin, as well as bacteria belonging to the genus Lactobacillus (Table
307
4).
M AN U
SC
303
Comparing the results using the culture medium RCA pH 5.3 and MRS at pH
309
5.2 (Table 4), we concluded that the components of RCA are capable of inhibiting the
310
growth of L. acidophilus and S. thermophilus. In contrast, the decrease in pH of MRS
311
agar to 5.2 was not sufficient to inhibit these microorganisms. Moreover, the
312
temperature and the conditions of incubation were the same for both culture media,
313
evidencing that the MRS agar at pH 5.2 was not selective for the enumeration of L.
314
bulgaricus from the probiotic yoghurt under the conditions studied, since other strains
315
that may be present in yoghurt are able to grow in this culture medium.
EP
AC C
316
TE D
308
Importantly, although literature reports different selective methodologies for
317
evaluation of microorganisms in probiotic products, there is a need to test the culture
318
media, once the selectivity is directly related to the presence of other strains. A careful
319
evaluation of the efficiency of a selective medium should be performed to avoid
320
overestimated results during the development of the study, since some culture media do
13
ACCEPTED MANUSCRIPT not effectively select probiotic cultures, as observed by several authors (Oberg et al.,
322
2011; Van De Casteele et al., 2006). It is necessary that the culture media are previously
323
evaluated in the matrix in question, since dairy products such as cheese, for example,
324
are complex matrices, and selective media are generally evaluated in model systems
325
using pure cultures, which may compromise the results.
RI PT
321
Further research need to be carried out in order to evaluate the effectiveness of
327
selective media, like MRS with clindamycin or bile with higher incubation temperature
328
as 45ºC for selective counting of Lactobacillus acidophilus in Prato cheese.
SC
326
329 4. Conclusions
M AN U
330
Under the conditions of this study, it was found that the MRS containing bile was
332
selective for enumeration of Lactobacillus acidophilus La5 from yoghurt, while RCA
333
agar pH 5.3 and ST agar were effective for enumeration of Lactobacillus bulgaricus and
334
Streptococcus thermophilus, respectively. For the probiotic Prato cheese, the effective
335
culture medium for enumeration of L. acidophilus was the MRSS incubated at 45 °C for
336
72 hours under anaerobic conditions, while M17 agar containing lactose and incubated
337
at 30 °C under aerobic conditions was effective for enumeration of Lactococcus lactis.
EP
TE D
331
For obtaining reliable results in the selective enumeration of probiotic
339
microorganisms in fermented dairy products is necessary to evaluate different methods,
340
both for the product under study and pure cultures, since the presence of different
341
strains may compromise the results, and therefore lead to misleading conclusions about
342
the presence and number of probiotics in dairy products.
AC C
338
343 344
Acknowledgements
14
ACCEPTED MANUSCRIPT 345
The authors thank the Foundation for Research Support of the State of São Paulo
346
(FAPESP) for financial support to the project 2009/54268-9, the National Council for
347
Scientific and Technological Development (CNPq) and the Coordination of
348
Improvement of Higher Education Personnel (CAPES) for scholarships.
RI PT
349 350
5. References
351
Ammor, M.S., Flórez, A.B., & Mayo, B. (2007). Antibiotic resistence in non-
enterococcal lactic acid bacteria and bifidobacteria. Food Microbiology, 24, 559-
353
570.
Antunes, A. E. C., Cazetto, T. F., & Bolini, H. M. A. (2005). Viability of probiotic
M AN U
354
SC
352
355
micro-organisms during storage, postacidification and sensory analysis of fat-free
356
yogurts with added whey protein concentrate. International Journal of Dairy
357
Technology, 58, 169-173.
359
Begley, M.; Gahan, C. G. M., & Hill, C. (2005). The interaction between bacteria and
TE D
358
bile. FEMS Microbiology Reviews 29, 625–651. Christian Hansen (2007). Enumeration of L. acidophilus, in fermented milk products –
361
Guidelines. Technical bulletin P-10 - L.acidophilus enumeration, nov. 2007.
EP
360
Darukaradhya, J., Philips, M., & Kailasapathy, K. (2006). Selective enumeration of
363
Lactobacillus acidophilus, Bifidobacterium spp., starter lactic acid bacteria and
364 365 366
AC C
362
non-starter lactic acid bacteria from Cheddar cheese. International Dairy Journal, 16, 439–445.
Dave, R. I., & Shah, N. P. (1996). Evaluation of media for selective enumeration of
367
Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus,
368
Lactobacillus acidophilus, and Bifidobacteria. Journal of Dairy Science, 79, 1524-
369
1536.
15
ACCEPTED MANUSCRIPT 370 371 372
De Man, J. C., Rogosa, M., & Sharpe, M. E., (1960). A medium for the cultivation of lactobacilli. Journal of Applied Microbiology, 23, 130-135. Donkor, O. N., Henriksson, A., Vasiljevic, T., & Shah, N. P. (2006). Effect of acidification on the activity of probiotics in yoghurt during cold storage.
374
International Dairy Journal, 16, 1181-1189.
RI PT
373
Espírito Santo, A. P., Perego, P., Converti, A., & Oliveira, M. N. (2012). Influence of
376
milk type and addition of passion fruit peel powder on fermentation kinetics,
377
texture profile and bacterial viability in probiotic yoghurts. LWT - Food Science
378
and Technology, 47, 393-399.
SC
375
Gebara, C., Chaves, K. S., Ribeiro, M. C. E., Souza, F. N., Grosso, C. R. F., & Gigante,
380
M. L. (2013).Viability of Lactobacillus acidophilus La5 in pectin–whey protein
381
microparticles during exposure to simulated gastrointestinal conditions. Food
382
Research International, 51, 872–878.
Hammes, W. P., & Hertel, C. (2006). The Genera Lactobacillus and Carnobacterium.
TE D
383
M AN U
379
In: M. Dworkin, S. Falkow, E. Rosenberg, K. Schleifer, & E. Stackebrandt (Eds.).
385
The Prokaryotes - A Handbook on the Biology of Bacteria. - Volume 4: Bacteria:
386
Firmicutes, Cyanobacter. 3ª ed. (pp. 320-403). Singapore: Springer Science
387
Business Media, LLC.
389 390
Hardie, J. M., & Whiley, R. A. (2006). The Genus Streptococcus – Oral. In: M.
AC C
388
EP
384
Dworkin, S. Falkow, E. Rosenberg, K. Schleifer, & E. Stackebrandt (Eds.). The
Prokaryotes - A Handbook on the Biology of Bacteria. - Volume 4: Bacteria:
391
Firmicutes, Cyanobacter. 3ª ed. (pp. 76-107). Singapore: Springer Science Business
392
Media, LLC.
16
ACCEPTED MANUSCRIPT 393
IDF. International Dairy Federation. (1995). Fermented and non-fermented milk
394
products - Detection and enumeration of Lactobacillus acidophilus - culture media.
395
Bulletin of the IDF, 306, 23-33.
397
Karimi, R., Mortazavian, A. M., & Amiri-Rigi, A. (2012). Selective enumeration of probiotic microorganisms in cheese. Food Microbiology, 29, 1-9.
RI PT
396
398
Kasimoglu, A., Göncüoglu, M., & Akgün, S. (2004). Probiotic white cheese with
399
Lactobacillus acidophilus. International Dairy Journal, 14, 1067-1073.
Lahtinen, S. J., Sanchez, B., Forssten, S., Ouwehand, A. C., & Gueimonde, M. (2011).
SC
400
Enumeration and viability assessment of probiotic bacteria. In: N.P. Shah, A. G.
402
Cruz, & J. A. F. Faria (Eds.). Probiotic and Prebiotic Foods: Technology, Stability
403
and Benefits to Human Health. (pp. 49-76). New York: Nova Science Publishers,
404
Inc.
405
M AN U
401
Mortazavian, A. M., Ehsani, M. R., Sohrabvandi, S., & Reinheimer, J. A. (2007). MRSbile agar: its suitability for the enumeration of mixed probiotic cultures in cultured
407
dairy products. Milchwissenschaft, 62, 270-272.
408
TE D
406
Nomura, M., Kobayashi, M., Narita, T., Kimoto-Nira, H., & Okamoto, T. (2006). Phenotypic and molecular characterization of Lactococcus lactis from milk and
410
plants. Journal of Applied Microbiology, 101, 396–405.
412 413 414
Oberg, C. J., Moyes, L. V., Domek, M. J., Brothersen, C., & Mcmahon, D. J. (2011).
AC C
411
EP
409
Survival of probiotic adjunct cultures in cheese and challenges in their enumeration
using selective media. Journal of Dairy Science, 94, 2220-2230.
O’Flaherty, S., & Klaenhammer, T. R. (2010). The role and potential of probiotic
415
bacteria in the gut, and the communication between gut microflora and gut/host.
416
International Dairy Journal, 20, 262–268.
17
ACCEPTED MANUSCRIPT 417
Ong, L., Henriksson, A., & Shah, N. P. (2006). Development of probiotic Cheddar
418
cheese containing Lactobacillus acidophilus, Lb. casei, Lb. paracasei and
419
Bifidobacterium spp. and the influence of these bacteria on proteolytic patterns and
420
production of organic acid. International Dairy Journal, 16, 446-456. Ong, L., & Shah, N, P. (2008). Influence of probiotic Lactobacillus acidophilus and L.
422
helveticus on proteolysis, organic acid profiles, and ACE-inhibitory activity of
423
cheddar cheeses ripened at 4, 8, and 12ºC. Journal of Food Science, 73, M111-
424
M120.
SC
425
RI PT
421
Ong, L., & Shah, N. P. (2009). Probiotic Cheddar cheese: influence of ripening temperatures on survival of probiotic microorganisms, cheese composition and
427
organic acid profiles. LWT – Food Science and Technology, 42, 1260-1268.
428
Özer, B., Uzun, Y. S., & Kirmaci, H. A. (2008). Effect of microencapsulation on
M AN U
426
viability of Lactobacillus acidophilus LA-5 and Bifidobacterium bifidum BB-12
430
during Kasar cheese ripening. International Journal of Dairy Technology, 61, 237-
431
244.
TE D
429
Philips, M., Kailasapathy, K., & Tran, L. (2006). Viability of commercial probiotic
433
cultures (L. acidophilus, Bifidobacterium sp., L. casei, L. paracasei and L.
434
rhamnosus) in Cheddar cheese. International Journal of Food Microbiology, 108,
435
276-280.
437
AC C
436
EP
432
Ribeiro, M. C. E., Chaves, K. S., Gebara, C., Infante, F. N. S., Grosso, C. R. F., & Gigante, M. L. (2014). Effect of microencapsulation of Lactobacillus acidophilus
438
LA-5 on physicochemical, sensory and microbiological characteristics of stirred
439
probiotic yoghurt. Food Research International, 66, 424–431.
18
ACCEPTED MANUSCRIPT 440
Ross, R. P., Fitzgerald, G., Collins, K., & Stanton, C. (2002). Cheese delivering
441
biocultures – probiotic cheese. The Australian Journal of Dairy Technology, 57,
442
71-78.
443
Saccaro, D. M., Hirota, C. Y., Tamime, A. Y., & Oliveira, M. N. (2012). Evaluation of different selective media for enumeration of probiotic micro-organisms in
445
combination with yogurt starter cultures in fermented milk. African Journal of
446
Microbiology Research, 6, 2239-2245.
449
foods. Journal of Dairy Science, 83, 894-907.
SC
448
Shah, N. P. (2000). Probiotic bacteria: selective enumeration and survival in dairy
Shankar, P. A., & Davies, F. L. (1977). A note on the suppression of Lactobacillus
M AN U
447
RI PT
444
450
bulgaricus in media containing β-glycerophosphate and application of such media
451
to selective isolation of Streptococcus thermophiles from yoghurt. Journal of the
452
Society of Dairy Technology, 30, 28-30.
454
Terzaghi, B.E., & Sandine, W.E. (1975). Improved medium for lactic streptococci and
TE D
453
their bacteriophages. Applied Microbiology, 29, 807-813. Teuber, M., & Geis, A. (2006). The Genus Lactococcus. In: M. Dworkin, S. Falkow, E.
456
Rosenberg, K. Schleifer, & E. Stackebrandt (Eds.). The Prokaryotes - A Handbook
457
on the Biology of Bacteria. - Volume 4: Bacteria: Firmicutes, Cyanobacter. 3ª ed.
458
(pp. 205-228). Singapore: Springer Science Business Media, LLC.
460
AC C
459
EP
455
Tharmaraj, N., & Shah, N. P. (2003). Selective enumeration of Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus,
461
bifidobacteria, Lactobacillus casei, Lactobacillus rhamnosus, and propionibacteria.
462
Journal of Dairy Science, 86, 2288-2296.
463 464
Van De Casteele, S., Vanheuverzwijn, T., Ruyssen, T., Van Assche, P., Swings, J., & Huys, G. (2006). Evaluation of culture media for selective enumeration of probiotic
19
ACCEPTED MANUSCRIPT 465
strains of lactobacilli and bifidobacteria in combination with yoghurt or cheese
466
starters. International Dairy Journal, 16, 1470-1476.
468 469
Vasiljevic, T., & Shah, N. P. (2008). Probiotics – From Metchnikoff to bioactives. International Dairy Journal, 18, 714-728. Vinderola, C. G., & Reinheimer, J. A. (1999). Culture media for the enumeration of
RI PT
467
470
Bifidobacterium bifidum and Lactobacillus acidophilus in the presence of yoghurt
471
bacteria. International Dairy Journal, 9, 497-505.
Vinderola, C. G., Prosello, W., Ghiberto, D., & Reinheimer, J. A. (2000). Viability of
SC
472
probiotic (Bifidobacterium, Lactobacillus acidophilus and Lactobacillus casei) and
474
nonprobiotic microflora in argentinian fresco cheese. Journal of Dairy Science, 83,
475
1905-1911.
476
M AN U
473
Zacarchenco, P. B., & Massaguer-Roig, S. (2004). Enumeration of Streptococcus thermophilus in the presence of Bifidobacterium longum and Lactobacillus
478
acidophilus – effect of incubation temperature and atmospheric conditions.
479
Milchwissenschaft, 59, 370-372.
AC C
EP
TE D
477
20
ACCEPTED MANUSCRIPT 1
Table 1 – Selective culture media used for enumerating Lactobacillus acidophilus in
2
commercial probiotic yoghurt, or in combination with Streptococcus thermophilus and
3
Lactobacillus bulgaricus.
Agar
based on
Incubation conditions
References
37ºC, 72 hours,
IDF (1995); Vinderola & Reinheimer (1999);
anaerobic incubation
Mortazavian et al. (2007); Ribeiro et al. (2014)
37ºC, 72 hours,
Antunes et al. (2005)
RI PT
MRS
Selectivity
Bile aerobic incubation
Agar
Dave & Shah (1996); Tharmaraj & Shah
anaerobic incubation
(2003)
Sorbitol
M AN U
MRS
37ºC, 72 hours,
SC
Base
37ºC, 48 hours,
Donkor et al. (2006)
anaerobic incubation MRS Agar
Clindamycin
37ºC, 72 hours,
Van de Casteele et al. (2006); Espírito Santo et
anaerobic incubation
al. (2012); Saccaro et al. (2012)
AC C
EP
TE D
4
ACCEPTED MANUSCRIPT 1
Table 2 – Selective culture media used for enumerating Lactobacillus acidophilus in
2
probiotic cheese, made with addition of Lactococcus lactis.
MRS Agar
Selectivity based
Incubation
on
conditions
Bile
Cheese type
37ºC, 72 hours,
Argentinian
Vinderola et al.
aerobic incubation
fresh cheese
(2000)
MRS
Clindamycin and
37ºC, 48 hours,
Cheddar
Agar
bromocresol green
anaerobic incubation
cheese Cheddar
Agar
37ºC, 72 hours,
Sorbitol
anaerobic incubation
cheese Kasar cheese
Agar
45ºC, 72 hours,
Sorbitol
Cheddar
M AN U
MRS
anaerobic incubation
AC C
EP
TE D
3
Phillips et al. (2006) Ong et al. (2006);
Ong & Shah (2009)
SC
MRS
References
RI PT
Base
cheese
Özer et al. (2008) Oberg et al. (2011)
ACCEPTED MANUSCRIPT Table 3. Microbiological analysis of yoghurts containing Lactobacillus acidophilus and
2
pure cultures of Lactobacillus acidophilus La5, Lactobacillus bulgaricus and
3
Streptococcus thermophilus, using different culture media (n = 3). Values expressed as
4
log10 CFU / g.
5 Enumeration in pure cultures
Enumeration
L. bulgaricus
S. thermophilus
in yoghurt
MRSB1
9.13 ± 0.33
<2
<2
8.25 ± 1.03
MRSC2
9.01 ± 0.69
<2
<2
7.79 ± 0.75
MRS + S3
9.36 ± 0.08
8.81 ± 0.10
5.91 ± 5.12
8.24 ± 0.55
MRS pH 5,24
8.41 ± 0.87
8.52 ± 0.44
7.40 ± 3.11
8.60 ± 0.08
8.74 ± 0.07
<2
8.96 ± 0.78
2.92 ± 2.62
8.93 ± 0.29
8.98 ± 0.23
<2
9.04 ± 0.21
8.82 ± 0.22
RCA5
<2
M17 456
<2
ST7
<2
TE D
6
M AN U
L. acidophilus
SC
Culture media
RI PT
1
7
1
8
conditions; 2MRS agar containing clindamycin (0.5 ppm) incubated at 37°C for 72 h under anaerobic
9
conditions; 3MRS agar containing 1% sorbitol (solution) incubated at 37°C for 72 h under anaerobic 4
EP
MRS agar containing 1.5% bile solution (10% w/v) incubated at 37°C for 72 h under anaerobic
10
conditions;
11
5
Reinforced Clostridium agar at pH adjusted to 5.3 incubated at 45°C for 72 h under anaerobic conditions;
12
6
M17 agar containing 5% lactose solution (10% w/v) incubated at 45°C for 72 h under aerobic
13
conditions; 7ST agar incubated at 30°C for 72 h under aerobic conditions.
AC C
14
MRS agar at pH adjusted to 5.2 incubated at 45°C for 72 h under anaerobic conditions;
ACCEPTED MANUSCRIPT Table 4. Microbiological analysis of Prato cheese containing Lactobacillus acidophilus
2
and pure cultures of Lactobacillus acidophilus La5 and Lactococcus lactis (L. lactis
3
subsp. lactis and L. lactis subsp. cremoris) using different culture media (n = 3). Values
4
expressed as log10 CFU / g.
5 Enumeration in pure cultures Culture media
RI PT
1
Enumeration in cheese
L. acidophilus
L. lactis
MRSB 1
9.75 ± 0.03
9.13 ± 0.44
MRSC2
9.38 ± 0.56
6.13 ± 1.12
6.23 ± 1.24
MRS + S3
9.71 ± 0.10
9.50 ± 0.29
9.59 ± 0.10
MRSS 4
9.71 ± 0.07
<2
6.31 ±0.18
M17 375
2.42 ± 0.89
9.54 ± 0.26
9.59 ± 0.12
M17 306
<2
9.54 ± 0.24
9.55 ± 0.09
SC
M AN U
6
9.28 ±0.22
7
1
8
conditions; 2MRS agar containing clindamycin (0.5 ppm) incubated at 37°C for 72 h under anaerobic
9
conditions; 3MRS agar containing 1% sorbitol (solution) as the only carbohydrate source, incubated at
10
37°C for 72 h under anaerobic conditions; 4MRS agar containing 1% sorbitol as the only carbohydrate
11
source incubated at 45°C for 72 h under anaerobic conditions; 5M17 agar containing 5% lactose solution
12
(10% w/v) incubated at 37°C for 72 h under aerobic conditions;
13
solution (10% w/v) incubated at 30°C for 72 h under aerobic conditions.
TE D
EP
AC C
14
MRS agar containing 1.5% bile solution (10% w/v) incubated at 37°C for 72 h under anaerobic
6
M17 agar containing 5% lactose
ACCEPTED MANUSCRIPT Highlights
2
Methodologies for selective enumeration of probiotics in dairy products.
3
Effectiveness of the methodologies in yoghurt and Prato cheese.
4
Selective enumeration of Lactobacillus acidophilus from yoghurt using MRS bile agar.
5
MRS sorbitol agar at 45 °C is selective for L. acidophilus from Prato cheese.
AC C
EP
TE D
M AN U
SC
RI PT
1