Antifungal Activity of Lactobacillus plantarum and Sage Extract on Aspergillus Fumigatus in Yogurt

Aspergillus fumigatus is a common fungus that causes dairy products contamination. The aim of this study was to evaluate in vitro antifungal activity of Lactobacillus plantarum and sage extract on the growth of Aspergillus fumigatus in yogurt during 1, 7, & 14 days of storage at 4°C. Two different groups of yogurt samples were prepared. The first one consisted of the plain yogurt with L. plantarum + A. fumigatus (control), and sage yogurt with L. plantarum + A. fumigatus, and the second group contained plain yogurt + A. fumigatus (control), and sage yogurt + A. fumigatus. Both groups were investigated for pH, total titratable acidity (TTA), viable cells counts of L. plantarum and inhibition of A. fumigatus growth during refrigerated storage. The pH values of all samples were decreased non-significantly (p>0.05) during storage whereas TTA ranged between 1.6% 1.7% at the end of storage. The presence of sage in yogurt increased significantly (p<0.05) the viability of L. plantarum compared to control over one week of storage. Sage alone or in a combination with L. plantarum showed moderate growth inhibition of A. fumigatus in yogurt compared to their respective controls. L. plantarum and sage extract could be used as antifungal activity against A. fumigatus in yogurt.


Introduction
Fermented milk has been consumed for thousands of years. It has been known to possess several nutritional strengths and characteristics that make it a valuable contribution to the dietary patterns of human's life [1]. In addition, it has a reputation of being highly nutritious and even therapeutic. Therefore in the past few decades, researchers on fermented milk such as yogurt have increased rapidly [2].
Lactic acid bacteria (LAB) such as Lactobacillus plantarum is one of the most diverse species that are generally used as probiotics because of their specific healthy properties in the food [3][4][5]. This bacteria is widely used in the fermented food's and beverages while participating in both sensory qualities of the food and the prevention of spoilage. They have antagonistic effect against various pathogenic and toxigenic organisms [6][7][8].
Yogurt is one of the best-known of foods that contain probiotics. The main action of probiotics can be summarized as a reinforcement of the intestinal mucosal barrier against pathogenic microorganisms [9]. LAB in yogurt can produce several compounds these including organic acids i.e. lactic acid, acetic acid, citric acid, and hippuric acid [10]. These organic acids can inhibit the growth of fungi or lower the level of toxic metabolites produced such as mycotoxin [11]. Aspergillus fumigatus is one of the most important opportunistic pathogen producing aspergillosis. It was found frequently present in different types of food included soft cheese, freeze-dried soup, juices, regular tea and sweet biscuits [12].
Salvia officinalis (Sage) is a perennial hardy sub-shrub native to Mediterranean regions and has been widely used in the preparation of many foods and traditional medicine in the Arab world because of its flavoring and seasoning properties [13]. Sage herbs have been possess a number of biological activities for the treatment of a diverse diseases including antiseptic, antibacterial [14,15], antioxidant [16], inflammatory [17,18], antiviral [19,20], antitumor [21], and anti-mycobacterial [22].
Probiotics are safe and widely used in fermented milk to increase immune response and prevent diseases. It is possible to use probiotics as bio-preservatives in yogurt to control the growth of the pathogenic fungi species associated with food spoilage thus reducing the risk of foodborne Illness, prolonged shelf-life, advanced the safety of foods, and increasing nutritional value and therapeutic properties of food. Therefore, the aim of this study was to estimate the in vitro Lactobacillus plantarum and S. officinalis (sage) effects on the growth of Aspergillus fumigatus in yogurt.

Materials and Chemicals
Pasteurized full cream milk (Al-marai, Saudi Arabia) was purchased from the supermarket. Sage leaves (S. officinalis) were purchased from a local store in Saudi Arabia. Lactobacillus plantarum (EMCC 1027), and Aspergillus fumigatus was obtained from Egypt microbial culture collection (EMCC), Cairo, Egypt. All agar and chemicals used in the present study were purchased from Micro master, Maharashtra, India.

Water Extraction of Sage
Sage water extract was prepared according to Xavieret al. [23] with some modification. 10 gram of sage was mixed with 90 mL of distilled H 2 O. The mixture was incubated in a water bath at 100°C for 5 min before filtered to clear supernatants. Sage water extract was used freshly in the production of herbal yogurt.

Preparation of Yogurt
Yogurt was prepared according to shori and Baba, [24] with some modification. Four types of yogurt samples were prepared, namely plain yogurt with L. plantarum + A. fumigatus (control), sage yogurt with L. plantarum + A. fumigatus, plain yogurt + A. fumigatus (control), and sage yogurt +A. fumigatus. Sage yogurt with L. plantarum + A. fumigatus was prepared by mixing 85 mL of pasteurized full cream milk with 10 mL of sage water extract and 5 g of starter culture containing Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus and L. plantarum in the ratio of 1:1:4). Full cream milk powder (4g) was added to adjust the milk solid content. The mixture was mixed carefully and placed in water bath at 42°C. The pH was determined every 30 min using pH meter until the pH of yogurt reached 4.5. Then, the yogurt was taken out and cooled down before 1 mL of spore suspensions of A. fumigatus (containing a spore number of ~10 5 spores/ml) inoculated into the yogurt. Yogurt sample was kept in the refrigerator at 4°C for two weeks. Plain yogurt with L. plantarum + A. fumigatus was prepared in a similar way except adding distilled water instead of sage water extract.
Plain-and sage yogurt with A. fumigatus were prepared using the same method above except the yogurt starter culture containing L. delbrueckii subsp. bulgaricus and S. thermophilus in the ratio of 1:1.

Determination the pH and Total Titratable Acid (TTA)
Yogurt was diluted with distilled water (1:1) [25]. The mixture was homogenized and the pH reading was taken using a digital pH meter (Mettler-Toledo 320). The total titratable acid (TTA) was determined by titration with 0.1 of NaOH. A sample of yogurt (1mL) was mixed with 9 mL distilled water and 2-3 drops of phenolphthalein [25]. Titration was carried out under continuous moving until the change of a stable pink color. TTA (%) was calculated as follows: where V is the volume of NaOH required to neutralize the acid and 0.009 represent the weight of lactic acid (g) neutralized by 1ml 0.1N NaOH.

Determination the Viable Cell Count of L. plantarum and the Growth Rate of A. fumigatus in Yogurt
Spread plate method was used for the bacteria counts and fungal growth. Properly diluted yogurt (1 mL) was transferred to the MRS agar (for L. plantarum) and PDA agar (for A. fumigatus) plates and mixed by lope. The MRS plates were sealed by parafilm and incubated in an inverted position at 37°C for 48 h [26] whereas PDA plates incubated at 28°C for 7 days [27]. The viable cell count of bacteria was calculated as follows: " colony forming unit (CFU)/mL= (Number of colonies formed X dilution factor of the sample)/1mL of sample".

Statistical Analysis
The experiments were performed in a total of three different batches of yogurt (n=3). All assays were performed in duplicates and the results were expressed as mean± S. E. M (standard error of the mean). One way analysis of variance (ANOVA), followed by Duncan's post hoc test for mean comparison were carried out using SPSS 19.0. The criteria for statistical significance was p<0.05.

Changes in the pH and TTA of Yogurt
The changes in the pH and TTA in yogurt samples were showed in Figures 1& 2. The pH values of all samples were decreased non-significantly (p>0.05) from 4.5 to 4.3 after 14 days of storage ( Figure 1). Sage yogurt in the presence of L. plantarum +A. fumigatus showed the lowest TTA (1.1±0.08%) among the yogurt samples at first day ( Figure  2). However, prolonged storage for two weeks increased (p<0.05) TTA to 1.7±0.17%. Other yogurt samples showed TTA ranged from 1.5% to 1.7% during 14 days of storage.

Effects of Sage Extract and L. Plantarum on the Growth Rate of A. fumigatus in Yogurt
The growth rate of A. fumigatus in yogurt samples both in the presence and absence of sage extracts and L. plantarum was shown in Table 1. The addition of sage extract in yogurt either with or without L. plantarum showed positive effects (medium growth) on the inhibition of A. fumigatus compared to control (heavy growth) over 14 days of storage. Sage yogurt showed low growth of A. fumigatus at day 7 of storage. L. plantarum in yogurt had no effects on the inhibition of A. fumigatus growth (Table 1).

Discussion
Preservatives are essential to prevent food spoilage and subsequently consumers' infections by harmful pathogenic [28]. Recently, the consumers required to reduce the use of synthetic preservatives [29] thus the preserving ability of probiotic bacteria that naturally occurring in food has been increasingly employed over the recent years. Probiotics have inhibition activity against food-contaminated pathogenic microbes via the production of organic acids, enzymes, and other secondary metabolites [8,30]. Several studies found that most fungi are sensitive to organic acid i.e. lactic, acetic and phenyllactic acids [8,11,30]. Lactic acid bacteria have been widely used in food as bio-preservatives which display a lot of benefits including extended shelf-life, improving food safety, and antimicrobial activity against broadspectrum of pathogenic microbes [6].
In the present study, the decline of pH in yogurt samples over 14 days of storage occurred as a result of organic acid (i.e. lactic acid) accumulation which produced by yogurt starter culture as secondary metabolites [26]. Although the addition of sage in yogurt has significantly affected the viability of L. plantarum (Figure 3), however, this did not influence the post-acidification of yogurt. A similar observation has been seen by Shori and Baba [9,31] who found there were no effects of Allium sativum and Cinnamomum verum water extracts on the yogurt postacidification.
Sage extract presented significant effect on the viability of L. plantarum at first week of storage ( Figure 3). This finding is in disagreement with Bachir et al., [32] who reported that S. officinal is extract inhibited the growth of L. plantarum with inhibition zone range from 2-2.5 at 10, 50 and 100 ppm concentration. On the other hand, several studies have showed that medicinal plants have positive effects on the growth of probiotics in yogurt and other dairy products [10,24,[33][34][35][36].
Lavermicocca et al. [37] reported that some strains of L. plantarum have antifungal activity related to organic acids, cyclic dipeptides and low molecular mass metabolites that produced by the bacteria. Moreover, Magnusson et al. [38] have tested the antifungal activity of a large number of Lactobacillus isolates from a different environment. A number of those isolates was showed strong growth inhibition against A. fumigatus. Lactobacillus delbrueckii and Lactobacillus rhamnosus showed significant antifungal activity against A. parasiticus and A. flavus [39]. The present study has demonstrated moderate inhibition of A. fumigatus growth by sage yogurt both in the presence and absence of L. plantarum (Table 1). Abu-Darwish, et al. [40] observed that S. officinalis essential oil exhibited growth inhibition against A. fumigatus. The antimicrobial activity depends on their chemical composition [41]. The chemical composition of the sage essential oil has been studied [42,43]. The major components of sage essential oils are α-thujone, 1, 8-cineole and camphor which they known for their antimicrobial activity [44,45]. Further study is needed to evaluate and characterize the chemical composition of sage yogurt with antimicrobial components.

Conclusion
The presence of sage had no effect on the postacidification of yogurt but significantly influenced the viability of L. plantarum during one week. In addition, sage extract alone or in a combination with L. plantarum contributed to preventing A. fumigatus contamination in yogurt. Therefore, sage yogurt with or without L. plantarum could have a potential effect on preventing yogurt spoilage by A. fumigatus. This mechanism can be applied for preventing yogurt spoilage by A. fumigatus during storage. Moreover, further study is needed to understand the mechanism of antifungal activity by L. plantarum and sage extract in yogurt and the possibility of using it as biopreservatives.