Growth-Death Kinetics of Listeria Monocytogenes in Strained Yogurt

Abstract

In this research, viability of Listeria monocytogenes was studied in strained yogurt (Turkish name: Torba yogurt) samples prepared in laboratory conditions and contaminated with various levels of L. monocytogenes before and after incubation. When strained yogurt samples (pre-incubation inoculation, A1 and post-incubation inoculation, B1) were contaminated with a 10⁶ cfu/ml level of L. monocytogenes, their pH value reached to 4.08, and the presence of L. monocytogenes was defined only by using the enrichment method at the 30^th day of the storage at the pH of 3.55, since the resistance of the bacteria had been lost in those yogurts. However, L. monocytogenes was completely inhibited at the 7^th day of the storage for the strained yogurt samples (pre-incubation inoculation, A2 and post-incubation inoculation, B2) contaminated with the level of 10⁴ cfu/ml when the pH value was 3.58. As a result, it can be stated that viability of Listeria monocytogenes may not be possible if the pH of the yogurt is around the ≤3.59 after the 7^th day of the storage.

Keywords:

• Listeria monocytogenes
• Behaviour
• Strained yogurt

INTRODUCTION

One of the oldest known ways of preserving milk is lactic acid fermentation. Yogurt, a fermented milk product, shows positive effects on human nutrition and health.[1] Traditionally produced concentrated yogurt types, for example, kurut (dried), torba (bag) yogurt, tulum (encased in sheep or goat skin) and peskuten, are popular in Turkey.[2] In order to make bag yogurt (strained yogurt), the milk is first mixed with yogurt. This yogurt/milk mixture is then placed in cloth bags and hung from a high place and thus a major part of the water within the yogurt mixture is drained.[3]

Yogurt is a product achieved with the fermentation of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. Bulgaricus, which are thermophilic homofermentative lactic acid bacteria in milk.[4] Metabolic capabilities of lactic acid bacteria have been utilized for a long time in order to preserve various foods and for their production. These bacteria are used in the industrial processing of milk, production of fermented milk products such as buttermilk and cheeses.[5] Lactic acid bacteria have various mechanisms to inhibit some pathogenic bacteria.[6] These mechanisms include organic acids, bacteriocins, hydrogen peroxide, and ethanol.[7]

Lactic acid bacteria are inhibitory agent for the growth and survival of pathogens during the milk fermentations.[8] Yogurt is also known as inhibitory to pathogens and spoilage microorganism like Salmonella spp. and Listeria monocytogenes.[9,33] Although yogurt is inhibitory, several investigators showed that pathogens survived during the fermentation of yogurt and then they were inactivated at various rates during storage of the products such as yogurt and ayran.[8,10] Schaack and Marth[8] reported that when the pH of fermented milk dropped below 4.0, L. monocytogenes could no longer survive. This emphasises the importance of an active starter culture and proper fermentation temperature to achieve high quality, microbiologically safe fermented dairy products.

L. monocytogenes is the second most likely food poison induced infection factor requiring hospitalized treatment and leading to mortality in some cases.[11] Research carried out in 8 developed countries showed that 22% of milk product related food infections are caused by L. monocytogenes.[12] Again some research has revealed that many milk products such as milk, cheese, buttermilk are contaminated with L. monocytogenes.[13,14] Although L. monocytogenes isolation in yogurt has not been reported so far, possibility of contamination may exists.[15] The purpose of this study was to define the growth-death kinetics of L. monocytogenes in strained yogurt contaminated with L. monocytogenes at levels 10⁴–10⁶ cfu/cells before and after fermentation phases.

MATERIAL AND METHODS

L. monocytogenes Strain

L. monocytogenes 1/2 b strain obtained from Prof. M. P. Doyle (Center for Food Safety Quality Enrichment Department of Food Science Technology, The University of Georgia Griffin, Geogia, USA) was used in the experimental inoculations of this study.

Preparation of Inoculations

In this study, after the heating the milk in laboratory conditions at 90^oC for 5 min, it was cooled down to 43^oC, and it was separately contaminated at the levels of 10⁴ and 10⁶ cfu/ml with the reference strain of L. monocytogenes 1/2 b.

Production and Storage of Trial Strained Yogurts

After being contaminated with reference strain L. monocytogenes 1/2 b, the milk was inoculated with 2% starter cultures containing Lb. delbrueckii ssp. bulgaricus and S. thermophilus (Chr. Hansens, YC-180). After inoculation, the milk samples were distributed into four different sterile glass jars (250 ml for each). Firstly, each of two lots (A1:10⁶ cfu/ml inoculation; A2:10⁴ cfu/ml inoculation) was also inoculated with 10⁴ cfu/ml and 10⁶ cfu/ml levels of L. monocytogenes and these lots were incubated at 43^oC for 4 hours for yogurt production. Secondly, each of other two lots (B1:10⁶ cfu/ml inoculation; B2:10⁴ cfu/ml inoculation) was inoculated with 10⁴ cfu/ml and 10⁶ cfu/ml levels of L. monocytogenes after yogurt production.

After the incubation, the yogurts were left for 6 hours for resting, and then they were transferred into the sterilized cloth bags and left to strain for 24 hours. After the straining, the yogurts were distributed into 250 ml covered sterile glass jars. Thus two different trial groups were obtained from the strained yogurts and stored at 4 ^oC for a period of 45 days.

Microbiological Analysis

L. monocytogenes count was carried out in the milk samples after inoculation. After straining and on the 3^rd, 7^th, 15^th, and 30^th days of the storage, the number of L. monocytogenes, total aerobic mesophilic bacteria (TAMB), streptococci and lactobacilli in the trial yogurts were determined.

Sampling and Preparation of Dilutions

In order to analyse TAMB, streptococci and lactobacilli, 10 g samples of yogurt were measured into each sterile stomacher bag after which 90 ml of sterile peptone serum physiologic (0.85% NaCl + 0.1% peptone) was added in each bag (Interscience, UK) and homogenized in the stomacher bags for 2 minutes. After diluting the samples at a scale of 1:10, decimal solutions were prepared up to 10⁷. In order to determine the number of L. monocytogenes in the milk and yogurt samples, sterile samples of 10 ml of milk and 10 g of yogurt were taken and placed in a 500 ml capacity stomacher bag. Later, 90 ml of Tryptone Soya Broth (TSB) was homogenized in the stomacher (Interscience, UK) and again diluted at a scale of 1:10 with TSB resulting in sample decimal homogenised solutions which were prepared up to10⁻⁷.

Plate Enumeration and Counting

The prepared dilutions were enumerated on Plate Count Agar (PCA, Oxoid, CM325) for TAMB, Lactobacillus Agar (MRS; Oxoid, CM 361) for Lb. delbrueckii ssp. bulgaricus and M17 Agar (Oxoid, CM 0785) for S. thermophilus. The PCA incubated aerobes at 30^oC in 2–3 days, the MRS at 42 ^oC incubated anaerobes in 2–3 days and the M17 incubated aerobes at 40^oC within 2–3 days. Microbial counts were reported as the log number of colony forming units (log cfu/g).[16,17]

L. monocytogenes Direct Count Method

The direct count method was used to determine the number of L. monocytogenes in the milk that was experimentally inoculated with L. monocytogenes. The dilutions which were prepared up to 10⁷ cfu/ml were enriched with LSA (Listeria Selective Agar, Oxoid CM856 + Listeria Selective Supplement, Oxoid SR140) and enumerated using the drop plaque method after which they were left to incubate at 35^oC for 48 hours. At the end of the incubation period, typical colonies surrounded with black halos and 1–3 mm in diameter was evaluated as suspicious for L. monocytogenes.[18] After incubation, each of the Listeria suspicious colonies developing in each plate were enriched with Tryptic Soy Agar-Yeast Extract (Difco, 0370) for purification and after incubating at 30°C for 24 hours the colonies were inspected morphologically and for purity by carrying out gram staining. In the tests, colonies which proved gram and catalase positive, oxidase negative, reproducing umbrella style in SIM medium were evaluated as Listeria spp. β-hemolyse in hematite agar, xylose, L-rhamnose, salicine, dulcite, methil red, Voges Preskauer, nitrate reduction and CAMP tests were utilized to identify Listeria isolated from the samples.[19,20] Microbact™ TM 12L Listeria identification system (Oxoid, MB1128) were used according to manufacturer's instructions to certify identified isolates.

Isolation and Identification of L. monocytogenes with the Enrichment Method

The enrichment method was used to isolate and identify L. monocytogenes in yogurt and buttermilk which was experimentally inoculated with L. monocytogenes and which did not reveal L. monocytogenes. 25 g of each analyzed sample was taken and placed in sterile stomacher bags which were enriched with 225 ml of Listeria Enrichment Broth (LEB, Oxoid, CM863 + SR142), after which the contents were homogenized in the stomacher bags (Bagmixer, Interscience) for 2 minutes and incubated in aerobe conditions at 30°C for 24 hours. Following incubation 0.1 ml homogenized from the LEB was transferred into tubes containing 10 ml each Fraiser Broth (Oxoid, CM895 + SR156) and incubated again at 30°C for 24 hours. The 0.1 ml homogenized acquired after this procedure was drawn in to Palcam Agar (Oxoid, CM877 + SR150) and Oxford Agar (Oxoid, CM856 + SR140) and each plate was left to incubate at 30°C for 48 hours. Each of the Listeria suspicious colonies developing on the plate after incubation was treated with Tryptic Soy Agar-Yeast Extract (Difco, 0370) for purification, after incubating at 30°C for 24 hours the colonies were inspected morphologically and for purity by carrying out gram staining. In the tests, colonies which proved gram and catalase positive, oxidase negative, reproducing umbrella style in SIM medium, were evaluated as Listeria spp.β-hemolyse in hematite agar, xylose, L-rhamnose, salicine, dulcite, methil red, Voges Preskauer, nitrate reduction and CAMP tests were utilized to identify Listeria isolated from the samples.[18,19,20,21,22] Microbact™ TM 12L Listeria identification system (Oxoid, MB1128) were used according to manufacturer's instructions to certify identified isolates.

Statistical Analysis

Statistical analysis of the data was performed using SAS® Software.³⁶ When analysis of variance (ANOVA) revealed a significant effect (P < 0.05), the data means were compared by the least significant difference (Tukey's Studentized Range test; HSD) test.

RESULTS AND DISCUSSION

In this study, the growth-death kinetics of L. monocytogenes was studied in the production and storage phases of strained yogurts (A1 and A2) made from milk which was fermented in the presence of L. monocytogenes at levels of 10⁴ or 10⁶ cfu/ml. In A1 strained yogurt which was contaminated with L. monocytogenes at a level 10⁶ cfu/ml at the pH level of 4.08 after straining, the L. monocytogenes level decreased to the level of 5.68 log cfu/g. At the 7^th day of storage when the pH was 3.58, level of the bacteria decreased to 3.41 log cfu/g. At the 15^th day of storage when the pH was 3.57, the L. monocytogenes could not be determined by direct count method, but it was observed by an enrichment method. Finally, they lost their resistance in the yogurt by the 30^th day of storage when the pH was 3.55 (Table 1). In the A2 strained yogurt that was contaminated with L. monocytogenes at the level of 10⁴ cfu/ml, at the pH of 4.08 after straining, the level of L. monocytogenes decreased to 3.78 log cfu/g. At the 7^th day of storage,e when the pH level was 3.55, the L. monocytogenes lost its resistance completely (Table 1

Table 1 Logarithmic counts (log cfu/g) of L. monocytogene s in strained (Torba) yogurt during the storage at 4 °C

Samples	Groups	pH	L. monocytogenes	S. thermophilus	Lb. delbrueckii ssp. bulgaricus	TAMB
First hour	A1	6.32 ± 0.00 ^a	6.75 ± 0.00 ^a	^−	−	−
	A2	6.32 ± 0.01 ^a	4.78 ± 0.02 ^b	^−	−	−
	B1	6.31 ± 0.00 ^a	6.75 ± 0.01 ^a	−	−	−
	B2	6.32 ± 0.02 ^a	4.78 ± 0.07 ^b	−	−	−
1^st day	A1	4.08 ± 0.00 ^a	5.68 ± 0.00 ^a	7.45 ± 0.04 ^b	4.58 ± 0.10 ^a	7.57 ± 0.19 ^a
	A2	4.07 ± 0.00 ^ab	3.78 ± 0.02 ^c	7.69 ± 0.00 ^a	4.50 ± 0.24 ^a	7.32 ± 0.00 ^a
	B1	4.05 ± 0.02 ^b	5.20 ± 0.01 ^b	7.49 ± 0.00 ^b	4.48 ± 0.07 ^a	7.00 ± 0.04 ^b
	B2	4.08 ± 0.01 ^a	1.60 ± 0.00 ^d	7.36 ± 0.03 ^c	4.72 ± 0.00 ^a	7.34 ± 0.00 ^a
3^rd day	A1	3.58 ± 0.00 ^a	4.66 ± 0.54 ^a	7.48 ± 0.00 ^b	4.73 ± 0.08 ^a	7.40 ± 0.08 ^a
	A2	3.59 ± 0.00 ^a	1.70 ± 0.04 ^b	7.53 ± 0.00 ^b	4.90 ± 0.16 ^a	7.43 ± 0.06 ^a
	B1	3.58 ± 0.01 ^a	4.07 ± 0.06 ^a	8.32 ± 0.07 ^a	4.96 ± 0.05 ^a	7.41 ± 0.00 ^a
	B2	3.58 ± 0.01 ^a	1.00 ± 0.00 ^b	7.40 ± 0.08 ^b	4.90 ± 0.04 ^a	7.20 ± 0.00 ^b
7^th day	A1	3.55 ± 0.00 ^b	3.41 ± 0.07 ^a	7.26 ± 0.07 ^ab	4.68 ± 0.16 ^a	7.41 ± 0.25 ^a
	A2	3.55 ± 0.00 ^b	ND ^c	7.08 ± 0.07 ^b	4.56 ± 0.05 ^a	7.08 ± 0.02 ^a
	B1	3.56 ± 0.01 ^ab	1.48 ± 0.00 ^b	7.41 ± 0.09 ^a	4.48 ± 0.02 ^a	7.08 ± 0.07 ^a
	B2	3.57 ± 0.00 ^a	ND ^c	7.10 ± 0.04 ^b	4.58 ± 0.00 ^a	7.04 ± 0.05 ^a
15^th day	A1	3.59 ± 0.00 ^a	≤10	7.44 ± 0.00 ^a	3.92 ± 0.07 ^ab	7.41 ± 0.25 ^a
	A2	3.58 ± 0.01 ^a	ND	7.48 ± 0.26 ^a	4.10 ± 0.08 ^a	7.08 ± 0.02 ^a
	B1	3.57 ± 0.00 ^a	ND	7.26 ± 0.05 ^a	3.78 ± 0.07 ^bc	7.08 ± 0.07 ^a
	B2	3.59 ± 0.03 ^a	ND	7.43 ± 0.06 ^a	3.68 ± 0.02 ^c	7.04 ± 0.05 ^a
30^th day	A1	3.55 ± 0.02 ^a	ND	7.32 ± 0.22 ^a	4.55 ± 0.00 ^a	7.30 ± 0.20 ^a
	A2	3.56 ± 0.00 ^a	ND	7.30 ± 0.24 ^a	4.25 ± 0.20 ^a	7.35 ± 0.29 ^a
	B1	3.55 ± 0.01 ^a	ND	7.26 ± 0.25 ^a	4.32 ± 0.24 ^a	7.45 ± 0.00 ^a
	B2	3.55 ± 0.00 ^a	ND	7.30 ± 0.23 ^a	4.33 ± 0.24 ^a	7.50 ± 0.22 ^a
−: not analyzed; ND: not detected; TAMB: Total aerobic mesophilic bacteria. ^a,b,c: Letters in a column indicate statistically differences among groups at p < 0.05.

). Additionally, B1 and B2 samples were similar to A1 and A2 in terms of survival of L. monocytogenes. At the 7^th day of the storage, when the pH level was 3.57, the L. monocytogenes was completely inhibited (Table 1). Statistically, significant difference (p < 0.05) was obtained for the counts of L. monocytogenes of the groups (A1, A2, B1, B2) during the storage time (Table 1). The other properties of the strained yogurt (pH value and the counts of yoghurt bacteria) had low different or were not different from each other at p > 0.05 significance level (Table 1).

Recently, L. monocytogenes has been identified as one of the major pathogens resulting in infections from milk and milk products.[12] Several researchers studied the behaviour of L. monocytogenes in yogurts during production and storage. After milk contaminated with 10³ and 10⁷ cfu/ml of L. monocytogenes was used in yogurt production by Massa et al.[23] and then stored at 4^oC. After the fermentation, the pH levels in yogurts inoculated with 10³ and 10⁷ cfu/ml of L. monocytogenes decreased to 4.9 and 5.0 cfu/ml, respectively. At the second day of storage, yogurts inoculated with 10³ cfu/ml of L. monocytogenes could be determined by utilizing the an enrichment method while at the 5^th day of storage when the pH was 4.2, the L. monocytogenes completely lost its resistance. At the 7^th day of storage of the yogurts which had been inoculated with 10⁷ cfu/ml of L. monocytogenes, it could not be counted through the regular method but only through the enrichment method. The L. monocytogenes completely disappeared by the 15^th day when the pH level had dropped to 4.2. The milk for yogurt production was contaminated with 4.69 log cfu/ml of L. monocytogenes by Gulmez and Guven[24]. The initial level of L. monocytogenes was around 3.0 log cfu/ml when the pH was 4.2, then decreased to 0.6 log cfu/ml at the 10^th day of storage at pH 4.1. Tipparaju et al. [15] inoculated low fat and fat free yogurts with 10⁴ and 10⁷ cfu/ml of L. monocytogenes, and after the fermentation they stored the yogurts at 8 ^oC. At the 31^st day of the storage period, the pH levels of the low fat and fat free yogurts which had been inoculated with 10⁴ cfu/ml of L. monocytogenes had dropped down to 4.31 and 4.39 respectively, additionally, the number of L. monocytogenes decreased to approximately 1.5 log cfu/ml with 2.5 log reduction. Again, at the 31^st day of storage of the low fat and fat free yogurts which had been inoculated with 10⁷ cfu/ml of L. monocytogenes, the pH levels were reduced to 4.28 and 4.36 respectively while the L. monocytogenes decreased to around 4.0 log cfu/ml level with 3.0-log reduction.

The quality and safety of food products depend on the microorganisms, the food characteristics and the process. Considerable effort has been made to develop nonthermal processes that act by killing of microorganisms in decades.[34] The survival ability of L. monocytogenes at low pH and water activity makes it an important hazard in foods.[35] The high acid levels of yogurt and pasteurization of milk are considered to be the most effective barrier between the product and the contaminated L. monocytogenes.[25] The inhibitor effects of lactic acid bacteria (LAB) on pathogens are well known. LAB has the ability to produce acid and other metabolites.[15] Acid type fermentation and pH drops below ≤ 5.2 reduce the development ratio of L. monocytogenes and have a major effect on its vitality. However, L. monocytogenes generally ceased the growth at the pH of ≤ 4.5.[26,27] This study revealed that the pH levels of A1 and A2 strained yogurts contaminated with L. monocytogenes at the levels of 10⁶ and 10⁴ cfu/ml respectively decreased to 3.55 at the 30^th day, and at the 7^th day of storage with the pH decreased to 3.58, the L. monocytogenes was completely ceased (Table 1). In the experiments carried out with plain yogurt by Massa et al.,[23] it was discovered that the L. monocytogenes eliminated completely when the pH dropped to 4.2. Gulmez and Guven[24], and Tipparaju et al.[15] reported that pH levels of 4.1 and 4.28–4.36 respectively had a reducing effect on L. monocytogenes. In strained yogurts, the reduction of L. monocytogenes is more significant compared to the plain yogurts due to the lower pH level As a matter of fact, Schaack and Marth[8] reported that large additions of casein into yogurt during fermentation and following storage period might protect L. monocytogenes. This study revealed that even with a low pH value, from a public health point of view, strained yogurts contaminated with L. monocytogenes might prove the danger. The viability of L. monocytogenes causing mastitis in cattle may not be possible if the pH of the strained yogurt is less than 3.59. The presence of L. monocytogenes in the faeces of the cattle may cause the contamination of the milk.[28,29,30] If pasteurized milk contaminated with L. monocytogenes is fermented, its products will also be contaminated with L. monocytogenes.[8] L. monocytogenes is naturally resistant to conditions involving temperature, acidity and environmental stress factors including low water activity.[31] Yogurts which are re-contaminated with a sufficient number of L. monocytogenes following the heat treatment are usually produced by small plants, and the houses using insufficient sanitation methods without required hygiene conditions may become an additional potential public health hazard [15] in terms of L. monocytogenes.

CONCLUSION

The life span of L. monocytogenes in yogurts was dependent upon inoculation amount, pH level, and storage temperature. In order to obtain microbiologically safe and high quality fermented milk products, it is important to apply appropriate fermentation temperature and strict sanitation procedures for the milk, and the thermophilic starter culture to be used. In addition, to make the strained yogurt consumption be safe for human, Good Manufacturing Practices (GMP) and Hazard Analysis and Critical Control Points (HACCP) systems must be applied properly to ensure all phases of milk production; from milking to consumer delivery are strictly carried out.