IMPACT OF PROBIOTICS ON STAPHYLOCOCCUS AUREUS GROWTH IN SOME DAIRY PRODUCTS

Assiut University web-site: www.aun.edu.eg

IMPACT OF PROBIOTICS ON STAPHYLOCOCCUS AUREUS GROWTH

IN SOME DAIRY PRODUCTS

EL-KHOLY, A.M. ¹; HASSAN, G.M. ²; SAMIA I. AFIFI ³ and MOHAMED, E.A.S ⁴.

^1,2 Food Hygiene Department, Faculty of Veterinary Medicine, Beni-Suef University, Egypt.

^3,4 Animal Health Research Institute, Beni-Suef Branch, Dokii, Egypt.

Received: 26 August 2018;     Accepted: 24 September 2018

INTRODUCTION

Milk and dairy products play an essential role in human nutrition therefore, it is preferred that 25% of the daily nutrition intake contains dairy products (Hoven, 1987).

Fermented milk precognitive as a dairy product obtained by the fermentation of milk through the action of Lactic Acid Bacteria "LAB" which resulted in lowering of pH with or without coagulation. These bacteria must be viable, active and abundant in the product. The popular fermented milk products are Yoghurt, Labneh, Acidophius milk, Rayeb and Butter milk, etc. (Ahmed et al., 2014).

Yoghurt is a classical fermented milk product which consumed all over the world mainly in northern European countries, Balkans, Middle- East and Indian sub-continents (Tamine, 2002).

Yoghurt is manufactured by the addition of healthy bacteria and live cultures to milk as Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (Steinkraus, 1997 and Tamine and Robinson, 2007). The main reason pointed out for yoghurt and other fermented dairy products

Corresponding author: Dr. MOHAMED, E.A.S.

E-mail address: bataahmed_2011@yahoo.com

Present address: Animal Health Research Institute, Beni-Suef Branch, Dokii, Egypt.

(Guarner et al., 2005; McKinley, 2005; Álvarez-León et al., 2006 and Cueva and Aryana, 2008).

consumption is the cultural, as improves lactose digestion and eliminates the symptoms of lactose intolerance, high in protein, calcium, phosphorus, magnesium, potassium, riboflavin and vitamin A Soft white cheese is processed from cow's or buffalo's milk or a mixture of them according to the Egyptian cheese-making technology. Production may be artisanal or industrial, depending on whether the cheeses are manufactured with raw thermized (heated below pasteurization level) or pasteurized milk (Robinson and Tamime, 2002).

Laban Rayeb is one of the fermented milks product consumed by different ages in Egypt and other countries, for its highly nutritive value and therapeutic properties (Sayed, 2012).

The genus Staphylococci includes over 30 species, with 18 of these species and subspecies are of potential hazard in food poisoning as they produce either coagulase, heat stable nuclease or enterotoxins. S. aureus subsp. aureus is coagulase positive and the most are common enterotoxigenic species (Le Loir et al., 2003 and Loncarevic et al., 2005). S. aureus is a common pathogen associated with serious community and hospital acquired diseases and has been considered for long as the important food- borne pathogen and made a major problem of public health (Pesavento et al., 2007 and Morandi et al., 2009).

S. aureus is a desiccation tolerant organism as it has the ability to survive in dry and stressful environments and can grow in wide range of temperatures (7° to 48.5° C; optimum 30 to 37°C), pH (4.2 to 9.3; optimum 7 to 7.5), and sodium chloride concentration up to 15% NaCl. It is existing on human nose, skin and inanimate surfaces such as clothing and surfaces (Chaibenjawong and Foster, 2011).

S. aureus produces 15 enterotoxins (Atichou et al., 2004). The five classical enterotoxins (SE, type A, B, C, D and E) which queried for 95% of staphylococcal food poisoning (SFP) cases and the new types of SEs (SEG- SEO) responsible for other cases (Jay et al., 2005).

SEs are highly resistant to environmental conditions such as heat, freezing and drying (Le Loir et al., 2003). They are also resistant to lowered pH and proteolytic enzymes such as pepsin or trypsin that rendering them completely functional in the gastrointestinal tract after ingestion. They also belong to superantigens family, which subverted the immune system of the host by targeting the innate and adaptive responses (Argudín et al., 2010).

The invention of a large number of S. aureus in the food (>10⁶ cfu/g or ml) is verificated that the isolated S. aureus strain is able to produce the enterotoxin (Najera-Sanchez et al., 2003). Dairy products with a level of enterotoxin as low as 0.5 ng /g or ml are frequently involved in SFP cases (Bergdoll, 1991 and Vernozy-Rozand et al., 2004).

SFP symptoms as nausea, abdominal cramps, diarrhea and a characteristic projectile vomiting are appeared within 1-6 h from ingestion of contaminated food, depending on individual susceptibility and ingested toxic dose and the clinical signs disappear within 24-48 h. Deaths occur rarely and specifically in the very young or elderly (Jay et al., 2005).

A concentration of 10⁵ bacteria/g in foods is sufficient for toxin production and induction of disease (Kluytmans and Wertheim 2005, Paciorek et al., 2007 and Fooladi et al., 2010).

SEA and SEB consider the most FP agents (> 60%) in USA and England (Kluytmans and Wertheim 2005). As associated with human contamination (mainly food manipulators) and SEC and SED are associated with animal contamination (mainly cows and pigs) (Un Lee et al.,1998).

Enterotoxins have been detected by various methods but the PCR technique has the ability of detecting specific gene sequences by DNA amplification (Mclauchlin et al., 2000; Mehrotra et al., 2000; Sharma et al., 2000 and Omoe et al., 2002). PCR is much faster and can be applied to detect SEs in most kinds of food such as milk and cheese products irrespective of the available conventional techniques (compared with animal tests) (Shijia et al., 2016).

New SEs (SEG, SEH, SEI and SEJ) and its genes were reported through the 1990s, (Zschock et al., 2005). More recent data resulting from partial or complete genome sequence analyses have led to the description of further ‘‘new’’ se genes: sek, sel, sem, sen, seo, sep, seq, ser and seu. (Omoe et al., 2002 and Letetre et al., 2003). The role of these new SEs has not yet been explained in FPcases (Vernozy-Rozand et al., 2004 and Boerema et al., 2006).

Probiotics are known as live microorganisms that have alternative effect to antibiotics in the treatment of foodborne diseases as it provide a health benefit to the host when administered in adequate amounts. (FAO/WHO, 2001 and Sanders, 2003). It's important to analyze probiotics stability in their characteristics during manufacture and storage and to ensure that they are retained in different types of foods (Lee and Salminen, 1995 and Huis In’t Veld and Shortt, 1996).

Probiotics have a public health significance against many diseases such as pathological allergic including atopic eczema and rhinitis, diarrhoea, necrotizing enterocolitis inflammatory bowel disease and viral infection (Robles Alonso and Guarner, 2013). Also they have benefit effect in lactose digestion, control intestinal infections and balance the intestinal mucosal barrier, immunogenic effects, shortening the duration of diarrhea and other health effects (Salminen et al., 1998 and Pal and Jadhav, 2013).

Dairy products consider as probiotic carrier foods especially fermented milk products (Bergamini et al., 2005). Lactobacillus and Bifidobacterium genera are the most known probiotic microorganisms (Prasad et al., 2000).

Lactobacilli and bifidobacteria are recognized as a good example for health-promoting constituents of the microflora. Lactobacilli has a health importance in gastrointestinal disturbance as reduce constipation, infantile diarrhea, traveller's diarrhea, irritable bowel syndrome (IBS), lactose-intolerant individuals and resist infections such as Salmonellae. Bifidobacteria also has benefit effect as stimulate the immune system, inhibit pathogen growth, help to restore the normal flora after antibiotic therapy, produce B vitamins, reduce blood ammonia and blood cholesterol levels (Gibson, 2002).

PH is an important factor which can dramatically affect bacterial growth, Lactobacillus spp. as a probiotic can tolerate a wide range of pH (1-9) and grow well at acidic pH 1-5 (Chowdhury et al., 2012).

Food containing such LAB should contain at least 10⁷ live microorganisms per g or ml at the time of consumption, in order to benefit the consumer (Ishibashi and Shimamura, 1993 and Hathout and Aly, 2010).

Probiotic bacteria possess antagonistic activity against numerous food-borne disease organisms such as S. aureus, Salmonella spp., E. coli, L. monocytogenes and Cl. perfringens (Millette et al., 2007). Lactobacillus acidophilus consider as one of the body’s primary defense mechanisms against Candida as it has protection effect against pathogenic yeast infections (Mercenier et al., 2003). As well as, many researches indicated that L. acidophilus is the most popular species of probiotic bacteria produces substances that slow or prevents the growth of Candida (Yang, 2000 and Mohamed et al., 2010).

Lactic acid bacteria have antagonistic effect due to producing some substances such as organic acids (lactic, acetic, propionic acids), carbon dioxide, hydrogen peroxide, diacetyl, low molecular weight antimicrobial substances and bacteriocins (Quwehand and Vesterlund, 2004).

Therefore, this study was planned on the impact of probiotics on S.aureus growth in some dairy products.

MATERIALS AND METHODS

I-1- Collection and handling of the samples:

A total of 180 samples of yoghurt (small and large scale), soft white cheese (Tallaga and Feta) and rayeb milk (small and large scale) 30 of each were arbitrarily collected from different localities as dairy shopes and markets in Beni-Suef city, Egypt. The collected samples were delivered as soon as possible to the laboratory in an insulated ice-box and examined in the same day.

I-2-a- Enumeration and isolation of Staphylococcus aureus.

It was performed on Baird parker agar plate according to APHA, (1992).

1-2-b- Identification of the isolated Staphylococcus organisms.

Microscopical and biochemical examination were done according toQuinn et al. (2002).

1-3- Detection of enterotoxins genes of Staphylococcus aureus.

Nine pairs of primers have specific sequence were supplied for detection five enterotoxins genes of S.aureus as Sea,Seb SecSed andSee, according to Mehrotra et al. (2000).

1-4- Impact of probiotic bacteria on the growth and survival of Staphylococcus aureusin vitro:

1-4-a- Bacterial strains:

 The bacterial strains used in this study were S.aureus which obtained from this work after the identification by multiplex PCR, Lactobacillus acidophilus and Bifidobacterium bifidum which were obtained from The Animal Health Research Institute (Dokii, Egypt).

1-4-b- Media used for growth of the bacteria:

Probiotic strains were propagated in de Man Rogosa and Sharpe (MRS) broth supplemented with 0.05% L-cysteine hydrochloride (Sigma, Buchs, Switzerland) at 37⁰C for 24 h under an atmosphere of 5% CO₂ for L. acidophilus and anaerobically for B.bifibum. S.aureus strain was propagated in 10 ml of Brain–Heart Infusion broth (CM1135B, Oxoid) at 37⁰C for 24 h.

1-4-c- Suspension inoculations for bacterial strains:

S.aureus counts were adjusted at concentration of 10⁸cfu/ml, while probiotic strains were adjusted at concentration of 10⁹cfu/ml.

1-4-d- Tallaga cheese manufacturing:

Cheese was prepared according to the method outlined byAbou-Donia (1986)as the sodium chloride was added at levels of 6% w/v and the samples were refrigerator storage and examined each two days.

1-4-e- Rayeb-milk manufacturing:

Rayeb-milk was prepared according to the method outlined by Sayed (2012) and Ramesh and Arun (2013)and the samples were refrigerator storage and examined each day.

1-4-f-Chemical analysis of manufactured products:

The pH of all cheese and rayeb samples was measured using (AD 111 digital pH meter 609) at 25±1 ^oC APHA (1974).

The salt percentage (sodium chloride content) in cheese samples was measured by the standered method described byAPHA (1992).

1-4-g-Enumeration of probiotic strains was performedaccording toSouza and Saad (2009).

1-4-h- Enumeration of S.aureuswas done according toRoberts and Greenwood (2003).

RESULT

Figure 1: Statistical analytical results of the examined samples based on their S.aureus count /ml or g.

Figure 2: Frequency distribution of the examined samples based on their S.aureus count /ml or g.

Figure 3: Incidence of enterotoxins genes of Staphylococcus aureus.

Figure 4: pH of control and probiotic Tallaga cheese during production and refrigerator storage.

Figure 5: Salt content (NaCl %) of control and probiotic Tallaga cheese during production and refrigerator storage.

Figure 6: Impact of L.acidophilus on S.aureus organism in Tallaga cheese during production and refrigerator storage.

Figure 7: Impact of B.bifidum on S.aureus organism in Tallaga cheese during production and refrigerator storage.

Figure 8: PH of control and probiotic rayeb milk during production.

Figure 9: Impact of L.acidophilus on S.aureus count (growth) in rayeb milk during production and refrigerator storage.

Figure 10: Impact of B.bifidum on S.aureus count (growth) in rayeb milk during production and refrigerator storage.

DISCUSSION

The high contamination rate of dairy products with S. aureus is mainly due to use of poor quality milk, environmental contamination as unclean hands of worker either suffering from diseased or apparent healthy carriers, unsanitary production and marketing practices (Araujo et al., 2002).

In this study, it is obvious in Figure (1) that the occurrence of S.aureus organism was 66.7, 23.3, 80, 36.7, 80 and 30 % of the examined yoghurt (small and large scale), soft white cheese (Tallaga and Feta) and rayeb milk (small and large scale) samples with mean values of 5.7X10³±3.2X10³, 9.3X10²± 3.9X10², 4.3X10⁴±2X10⁴, 5.1X10³± 1.7X10³, 3.6X10⁴ ±1.3X10⁴ and 5.5X10²±2.7X10², cfu/ml or g., respectively. The highest frequency distribution 60, 76.7, 80, 63.3, 50 and 70 % lies within the ranges of 10²-<10⁴, <10², 10² - <10⁶, <10², 10²- <10⁴ and <10², respectively Figure (2).

Several investigators had higher results of S.aureus in the small scale yoghurt samples as Oksuztepe et al. (2007) and Abou El-Makarem (2013).

 Nearly similar counts of S.aureus in the large scale yoghurt samples were postulated by Abdel-Fattah (2007), while higher values were observed by Abou El-Makarem (2013) and Ahmed et al. (2014), but lower results were obtained by El-Bessery (2001).

Similarly, the counts of S.aureus in the Tallaga cheese samples were previously found by Aiad (2013), however lower results were reported by Ghada et al. (2004) and Amer et al. (2005).

The dropping of S.aureus counts in the Feta cheese samples were detected by Al-Tahiri (2005) and El Sayed et al. (2011).

The lower rate of contamination in the small scale rayeb milk samples were observed by Shawer (2013) and Tarekgne et al. (2015), but in the large scale rayeb milk lower count were demonstrated by El-Bessery (2001) and Shawer (2013).

In spite of pasteurization destroy S. aureus pathogen, thermostable SEs can resist heat treatment and still spirit with their biological activity which may cause severe health hazard (Balaban and Rasooly 2000). Also, the presence of less than 10⁶ staphylococcal counts in the products doesn’t help in enterotoxin production(Tatini, 1973 and Robinson, 2002).

The obtained results in Figure (3) regarded that 60% of the examined S. aureus strains were enterotoxigenic by PCR technique as carried one or two se- genes and See gene consider the highest frequency incidence 3 (60%) then Seb and Sec 1(20% of each) but the Sea and Sed could not be detected.

Higher values were recorded by Morandi et al. (2007) and Mathenge et al. (2015), but lower results were indicated by Arcuri et al. (2010) and Kav et al. (2011).

Probiotics are live organisms that produce benefits to health of the consumer or host when ingested through their ability to implantation on the intestinal microflora, resist gastric acidity and bile digestion (Sfakianakis and Tzia, 2014). Also, probiotics are added with concentrations of 10⁷–10⁸ cfu/gm or ml. as adjunct cultures to food and products, if no longer participating or participating in the fermentation, they able to reach a concentration of10⁸-10⁹ cfu/g or ml after the fermentation happened (Vinderola et al., 2011).

The mechanism of lactic acid bacteria "LAB" in the controlling of microbial and pathogens growth is their production of lactic acid in addition to other antimicrobial compounds which exerted inhibition action on the growth of pathogens (Tadesse et al., 2005).

Lactobacillus acidophilus had antagonistic actions against Staphylococcus aureus as it secretes hydrogen peroxides which partially responsible for antimicrobial interaction rather than the produced amounts of acid (Gilliand and Speck, 1977).

The result obtained in Figure (4) showed that after 30 days refrigerator storage control Tallagacheese had pH value "4.8", L.acidophilus Tallaga had "4.0" and B.bifidum Tallaga cheese "4.3".

From the data presented in Figure (5) it is evident that the salt content (NaCl%) nearly similar in L.acidophilus Tallaga cheese, B.bifidum Tallaga cheese  and control one during production and after 30 days refrigerator storage.

The results given in Figure (6) showed that L.acidophilus  strain had inhibitory effect on S.aureus organism viability in Tallaga cheese  as decreased its count from 6.7 x10⁸ to 9.0x10³ cfu/g through a month storage in the refrigerator than control one that decreased from  6.7 x10⁸ to 3.0x10⁷cfu/g and the results were in harmony with  the results obtained by Arques et al. (2005) , Charlier et al. (2009) Amer (2011) and Abou El-Makarem (2013)., but did not agree with that postulated by Abd El-Gawad et al. (2014).

In Figure (7) it was found that B.bifidum strains had antimicrobial effect against S.aureus pathogen growth in Tallaga cheese as lowering its count from 6.8 x10⁸to 1.6x10⁴ cfu/g through a month storage in the refrigerator than control one that decreased from 6.7 x10⁸ to 3.0x10⁷ cfu/g and this agree with Arques et al. (2005), Charlier et al. (2009) and Abou El-Makarem (2013) .

The antimicrobial properties of yoghurt samples were mostly higher than these of the cell free and these activities may due to lactic acid production, decreasing in pH and also other antimicrobial compound that may be present in the yoghurt (Hassan et al., 2013).

The data illustrated in Figure (8) showed that after 15 days refrigerator storage control rayeb milk had PH values "3.2", L.acidophilus "2.9" and B.bifidum rayeb milk "3.0" and these result agree with that revealed Abd El-Gawad et al. (2014).

It is evident from the results which presented in Figure (9) that L.acidophilus  strains had antibacterial effect on S.aureus organism viability in the rayeb milk  as lowered its count from 7.8 x10⁸to 1.0x10³cfu/ml through 15days storage in the refrigerator than control one that decreased from 8.0 x10⁸to 2.5x10⁷cfu/ml and these data was nearly similar to that obtained  by  Arques et al. (2005) , Charlier et al. (2009), Kaboosi (2011) and Abou El-Makarem (2013), but did not agree with that revealed by Abd El-Gawad et al. (2014).

It is clear from the data showed in Figure (10) that B.bifidum strains had inhibitory effect against S.aureus growth in the rayeb milk as decreased its count from 7.7 x10⁸to 2.1x10³cfu/ml. through 15days storage in the refrigerator than control one that decreased from 8.0 x10⁸to 2.5x10⁷cfu/ml and the results were nearly similar to those recorded by Arques et al. (2005), Charlier et al. (2009), Kaboosi (2011) and Abou El-Makarem (2013).

Finally, it is clear the importance of probiotics to health of the consumers when ingested in the dairy products and we advise that all dairy products must be produced with addition of probiotics at a certain number to produce a good quality and safe guard dairy product.

CONCLUSION

It was concluded from this work that some of dairy products as yoghurt, soft cheese and rayeb milk sold in Beni-Suef city, Egypt were of bad quality as heavy microbial contaminated with S.aureus that rendering them threating to public health and may causes many diseases .

Also, the information which given in this work demonstrated that for improvement the microbiological quality of dairy products probiotics strains as L.acidophilus and B.bifidum may be used as it's have antagonistic affect against many species like S.aureus strain so, these confirm the health benefits derived from the human consumption of fermented dairy products.

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