This study evaluated the growth of Listeria monocytogenes in cottage cheese with reduced salt content and supplemented with probiotic bacteria. Cottage cheese formulations with varying salt replacements and probiotic additions were manufactured. L. monocytogenes was inoculated and the products were stored under different temperature conditions. The results showed that L. monocytogenes grew in all treatments stored at 4°C but did not grow above detectable levels in formulations with salt replacements stored at 12°C. Treatments with magnesium chloride and potassium chloride promoted the best survival of probiotic bacteria. Thus, reduced salt cottage cheese formulations maintained viability of probiotics and inhibited growth of L. monocytogenes when stored at refrigerated temperatures.

1. Ana Laura T. Jesus, Meg S. Fernandes, Bruna A. Kamimura, Leonardo Prado-Silva,
Ramon Silva, Erick A. Esmerino, Adriano G. Cruz, Anderson S. Sant'Ana
Accepted 28 December 2015
Published 30 December 2015
Presentation created by
Areesha Ahmad
Microbiologist

2. Introduction

3.  Slightly acidic  pH 4.5 to 6.5
 Produced by  coagulation of pasteurized skimmed milk
 Classified as a low fat content fresh cheese  4% fat
 Moisture  80%
 NaCl content  2.3% to 3.5%
Cottage cheese

4. excessive consumption of sodium

5.  The reduction of sodium in cheeses  vehicles for probiotic
microorganisms
 high water activity, pH value, and high lipid conc  maintain the
viability of probiotic microorganisms
 such parameters provide  ideal conditions for the survival and
multiplication of pathogenic microorganisms such as Listeria
monocytogenes.

6.  Listeria are Gram-positive, flagellate bacteria.
 Capable of multiplying in a wide range of temperatures
(0 to 42 °C), pH values (4 to 9) and at high salt
concentrations (10%).
 The genus Listeria consists of 15 species which only
L.monocytogenes is considered to be consistently
pathogenic
Listeria monocytogenes

7. Caption
Method and material

8. Methods and material
1. Preparation of cell suspensions
2. Starter culture and probiotic strains
3. Production, packaging, inoculation and storage of the cottage cheese
4. Viability of the starter cultures and probiotics
5. Enumeration and detection of L. monocytogenes
6. Determination of the growth potential (δ) of L.monocytogenes and the
probiotic microorganisms

9. L. monocytogenes ATCC
7644 was inoculated in
100 mL of tryptic soy broth
containing 0.6% yeast
extract (TSB-YE) at 37 °C
for 24 h.
10 mL was then
transferred to another
flask containing 100 mL of
TSB-YE, which was
incubated at 37 °C for 24
h.
Procedure was repeated
once more, followed by
centrifugation at 2815
×g for 10 min at 4 °C.
The supernatant being discarded.
The pellet was then washed three
times with ultra-pure sterile water
and the cell concentration
adjusted to an optimal density of
0.5 at 630 nm, which corresponds
to 108 CFU/mL.
The concentration of the
suspension was
subsequently confirmed by
a plate count in Oxford
agar (OXA)
1.Preparation of cell suspensions

10. 2. Starter culture and probiotic strains

11. 3. Production, packaging, inoculation and storage of the cottage cheese

12. For each treatment the salts sodium chloride, potassium chloride and magnesium
chloride and potassium sorbate were added during dressing step
Treatment

13. Each cottage cheese formulation was inoculated with a cell suspension of
L.monocytogenes aiming to reach a concentration of between 103 and 104 CFU/g in
the product.
Inoculation
Storage
Twenty-five gram portions of each formulation inoculated with L. monocytogenes
were then filled into sterile polypropylene pots and stored as follows: I: 4 °C for 28
days. II: 30% of the shelf life (8 days) at 4 °C and 70% (20 days) at 12 °C, and III: 12
°C for 28 days.

14. The pH and water activity were determined in triplicate
immediately after manufacture (zero time) and after 28
days of storage under the different conditions (I, II and
III).
pH value  was determined using a Digimed, model DM-
20 pH-meter at 25 °C, directly inserting the electrode into
the samples.
Water activity (aw)  was obtained by direct
reading using an AQUALAB model 4TEV water
activity meter.

15. 4. Viability of the starter cultures and probiotics
The starter cultures were counted by inoculated in M17 agar
(added 5% of a lactose solution 10% ) incubated at 30 °C for 48 h.
The probiotic culture L. acidophilus La-5 was counted in MRS
agar and the plates were incubated at 37 °C for 72 h.
The culture B. animalis subsp. lactis Bb12 was counted using MRS
agar and after the inoculation, the plates were incubated in
anaerobiosis jars at 37°C for 72 h.

16. 5. Detection of L. monocytogenes
L. monocytogenes was counted in three packs of each formulation at zero time (after
inoculation) and, for each storage condition, in three packs of each formulation at the
end of the product shelf life i.e. 28 days. The count was carried out by homogenizing
25 g of sample in 225mL of 0.1% peptone water for all the serial dilutions, and
inoculating into Oxford Agar (OXA).The plates were incubated at 37 °C for 48 h, and
the colonies counted and expressed as log CFU/g.

17. The growth potential (δ) of L. monocytogenes and of the probiotic
microorganisms in each formulation was determined by calculating the
difference between the microbial counts at the end of the shelf life (28 days) and
at the beginning (zero time).
6. Determination of the growth potential (δ) of L.monocytogenes and the
probiotic microorganisms

18. Results and discussion

19.  were found in the range from 10 –10 log CFU/g
immediately after processing.
 were found in the range from 7.8–8.8 log CFU/g in
formulations (F2-F6) after manufactured .
starter cultures
Probiotics culture
7 8

20. pH
A significant increase in pH was
observed for the cottage cheeses
during storage for the different
formulations and different storage
conditions (conditions I, II and III )

21.  The better survival of probiotic bacterium were observed in treatment F5 and F6
containing MgCl2 in which the counts of B. lactis Bb12 remained higher in
comparison to the other formulations at the end of shelf-life
 KCl also promoted the growth of probiotics
 Storage  best growth was observed at 4 °C/28 days
 Available water  The values for aw remained between 0.98 and 0.99
 Thus, cottage cheese with reduced salt content appeared as a food matrix capable of
carrying probiotic bacteria
Treatments

22.  L. monocytogenes growth was observed in all the treatments which was stored at 4
°C (condition I) increased the populations between 0.5 and 0.8 log CFU/g
 Higher growth potentials were observed when the cottage cheeses were stored for
30% at 4 °C followed by 70% of the shelf life at 12 °C (condition II)
 L. monocytogenes was only able to grow in F1, while in F2–F6 the populations of this
bacterium were below the quantification level in Oxford Agar at 12 °C (condition III)
Growth potential (δ) of L. monocytogenes

23. Conclusion
Thus, it is concluded that the reduce salt concentration inhibit the growth of
L.monocytogenes when store cottage cheese at 12 °C for 28 days this importance
of maintaining cottage cheese for microbiological safety.

24. Any Question ???