MATERIALS AND METHODS

Study area: The study was conducted in laboratories of Dairy and Food Science, Department of Nutrition, NRC, Egypt and Department of Food Science and Nutrition, Collage of Health Science, Kuwait, from January, 2018-March, 2019.

Materials
Essential oils source: Nigella sativa (Black seed) oil and Thymus vulgaris oil were obtained from squeezing and extracting natural oils unit, National Research Centre, Giza, Egypt.

Milk: Fresh buffalo’s milk (6% fat and 16% TS) was obtained from the Faculty of Agriculture, Cairo University, Giza, Egypt.

Bacterial strains
Starter culture: Lactobacillus delbrueckii sub sp. bulgaricus and Streptococcus thermophilus (as yogurt starter culture), for Labneh preparation, were obtained from stock cultures of the Dairy Microbiology Laboratory of Egyptian National Research Center, Dokki, Egypt. While Lactobacillus acidophilus N4495 strain was obtained from Chr. Hansen Denmark.

Pathogenic strains: The tested pathogenic strains: Bacillus cereus (ATCC 33018), Salmonella typhimurium 9027, Staphylococcus aureus (ATCC 20231) were obtained from the stock cultures of the Agricultural Research Centre in Giza, Egypt. E. coli O157:H7 (ATCC 6933) and Listeria monocytogens V7 and Yersinia enterocolitica subsp. enterocolitica ATCC9610TM were obtained from Liofilchem S.r.l., Italy. Helicobacter pylori and Klebsiella pneumonia were obtained from the stock cultures of Microbiology Lab of Dairy Science, National Research Centre, Dokki, Egypt. Saccharomyces cerevisiae Y-2223 (provided by the Northern Regional Research Laboratory Illinois, USA), Aspergillus niger and Penicillium roqueforti J5 (obtained from Department of Microbiology, Swedish University of Agricultural Sciences).

METHODS

Antimicrobial activity of Thymus vulgaris and Nigella sativa oils: Before the incorporation of essential oil into the plain skimmed yogurt, T. vulgaris and N. sativa oils antimicrobial activity was estimated against some food pathogenic and spoilage organisms by the agar well diffusion assays according to Sabina et al.¹⁷. The test was conducted against: Bacillus cereus, Salmonella typhimurium, staphylococcus aureus, E. coli O157:H7, Listeria monocytogenes and Yersinia enterocolitica subsp. Enterocolitica, Helicobacter pylori and Klebsiella pneumonia, separately. Each organism was cultured in brain heart infusion broth and then diluted in sterile saline solution. The nutrient agar plates were surface inoculated with 0.1 mL of the diluted cultures. The wells (8 mm in diameter) were cut from the agar and inoculated with essential oil. The plates were kept for 1 h at room temperature to allow the diffusion into the medium a nd then incubated at 37°C for 18 hrs. After incubation, all plates were examined for any zone of growth inhibition around the wells. The experiment was repeated two times and the results (zone of inhibition, mm) were expressed as average values^18,19. Helicobacter pylori were grown under anaerobic conditions, using the Anaerobic Jar (Oxoid, Ltd., Basingstoke, UK). In the case of each of A. flavus, A. niger and Penicillium spore suspension (10⁶ spores mL^–1) were prepared and 100 μL of each was spread on potato dextrose agar (PDA) dishes. After absorption, the cork borer was used to bore. The dishes were incubated for 5 days at 25°C. Also Saccharomyces was spread on potato dextrose agar and incubated for 5 days at 25°C (PDA) dishes. Visible inhibition zone around bore was followed according to Freire et al.²⁰.

Minimal inhibitory concentrations (MICs): The Minimum Inhibitory Concentrations (MICs) of extracts of N. sativa and Thymus vulgaris oil were determined against pathogenic bacteria, mold and yeast strains following the micro-dilution and disk diffusion method, using 96-well micro-titer plates^21,22. Minimal Inhibitory Concentrations (MICs) were calculated based on the degree of the growth (count) of each organism with oils and that was the lowest oil concentration that resulted in an 80% reduction in growth compared the oil-free growth control. A 100 μL (volume) of different concentrations was added to the wells and inhibition was measured, after incubation of the plates. Tween 80 (0.5%) as well as a mixture of Tween 80 (0.5%) and ethanol (1%) were assayed and considered as the negative controls, while also oils show no inhibition for any of the tested bacterial strains were recorded as negative results.

Manufacturing of acidophilus labneh flavored with thyme and black cumin: Acidophilus Labneh was made according to the method described by Ozer²³, Abd El-Sattar et al.²⁴ and Hatice and Cakmakc²⁵ with some modifications. Standard buffalo's milk 4% fat was used for the manufacture of Thyme and N. sativa Acidophilus Labneh. Buffalos milk 4% fat was heated 85°C for 20 min and then cooled to 42°C, mixed with 1.5% salt, Thymes and N. sativa (25 μg/100 mL) and inoculated with (3%, v/v) S. thermophilus and Lactobacillus delbrueckii spp. bulgaricus (1:1) and (1%) Lactobacillus acidophilus, that made three separate treatments. The mixtures (control and 3 treatments) were incubated at 40°C until uniform coagulation was obtained. The resulting acidophilus Labneh was divided into 4 batches as follow:

Control: S. thermophilus and Lactobacillus delbrueckii spp. Bulgaricus (1:1) and (1%) Lactobacillus acidophilus

Then the 4 batches were transferred to cloth bags and hung at 7°C overnight in the refrigerator to allow whey drainage. The resulting coagulation scooped into plastic cups (250 mlL stored at 7°C and analyzed for microbiological and organoleptic properties when fresh and after 7, 15, 21 and 30 days of storage. The whole experiment was repeated in triplicates.

Microbiological analysis: The samples of Labneh were microbiologically examined for each treatment after 0, 7, 15, 21 and 30 days of cold storage. Twenty-five grams of Labneh samples were added aseptically to 225mL of sterile solution (2% w/v) of sterile Buffered Peptone Water (BPW) and homogenized. Lactobacillus acidophilus was enumerated using MRS agar medium and plates were incubated anaerobically using anaerobic gas pack Jar at 37°C for 48 hrs according to Collins²⁶, Gillil and Walker²⁷. Total Aerobic Colony Count (TACC), using plate count agar (Oxoid), molds and yeast count, using acidified potato dextrose agar (Mu96, Himedia, Mumbai), were carried out in acidophilus Labneh samples during storage period according to FDA²⁸. Streptococcus thermophilus and Lactobacillus delbrueckii spp. bulgaricus enumerated using modified M17 and MRS agar and the plates were incubated at 37°C for 48 hrs according to Harrigan and McCance²⁹, El Kholy et al.³⁰, Shenawy et al.³¹, Dave and shah³².

Sensory evaluation of acidophilus labneh with thymes and black cumin: Fresh and cold stored (4-7°C) acidophilus Labneh samples were evaluated for their sensory properties according to the score card recommended by Keating and Randwhite³³. Sensory evaluation was carried out by 10 panelists, researchers at the Department of Dairy, National Research Center, Dokki, Egypt. The score card includes, flavor (50 points), body and texture (40 points) and appearance (10 points).

RESULTS

Chemical composition of thyme essential oil: The compounds identified in thyme essential oil are presented in Table 1. GC analysis of the oil resulted in the identification of 23 constituents, accounting for 98.83% of the oil. The chemical analysis of the oil showed that P-Cymene (30.87%), Thymol (30.72%), Y-Terpinene (8.98%), Bornyl acetate (6.54%), β-Caryophyllene (3.56%), Linalool (2.23%) and Carvacrol (1.87%).

Fatty acid composition of the fixed oil of Nigella sativa: The fatty acid profile of Nigella sativa fixed oil is presented in Table 2. It is obvious from the results that linoleic acid, oleic acid and palmitic acid were major fatty acids amounting to 59.94, 19.07 and 12.19%, respectively. Stearic acid and arachidic acid were present in percentages of 3.14 and 2.77% of the fixed oil, respectively. Similarly, linolenic acid and lauric acid were also present with percentages of 1.54 and 1.17%, while myristic acid was in meager proportions of 0.18%. It is evident from the results that sum up of saturated and monounsaturated fatty acids were i.e., 19.45 and 19.07%, respectively, while polyunsaturated fatty acids were 61.48%.

Thymus vulgaris and Nigella sativa Antimicrobial Activity: Results of antibacterial activity of the T. vulgaris essential oils (0.1%), shown in Table 3, reveal high antibacterial activity against Yersinia enterocolitica, Klibsiella pneumonia, Salmonella typhimurium, Listeria monocytogenes and S. aureus, in descending order, then N. sativa and oils in combination. Furthermore, the results in Table 3 indicated that Bacillus cereus, E. coli and H. pylori were less sensitive against the thyme and N. sativa oils compared with the other tested bacterial species. On the other hand, results of antifungal activity, in Table 4, clear that T. vulgaris and N. sativa oils showed higher antifungal activity in mixes than in single oils against Penicillium roqueforti, Aspergillus flavus, Aspergillus niger and Saccharomyces cerevisiae, in descending order.

Minimal inhibitory concentrations of T. vulgaris and N. sativa (MICs): Results of MICs as shown in Table 5, revealed that MIC of thyme oil for Yersinia enterocolitica, Klebsiella pneumonia, E. coli O157:H7, A. Niger and A. Flavus was 10.0 μg/100 mL.

While, MIC for S. aureus, Salmonella typhimurium, Bacillus cereus, Listeria-monocytogenes and Saccharomyces cerevisiae was 25 μg/100 mL and MIC for Penicillium roqueforti was 20 μg/100 mL. On the other hand, MIC of N. sativa oil for Yersinia enterocolitica, Salmonella typhimurium and Listeria monocytogens was 20 μg/100 mL. MIC for Klebsiella pneumonia, S. aureus, Bacillus cereus, E. coli O157:H7, A. Niger and A. Flavus was 30.0 μg/100 mL. Meanwhile, MIC for Penicillium roqueforti and Saccharomyces cerevisiae was 25 μg/100 mL.

Microbiological analysis of acidophilus labneh flavored with thyme and N. Sativa: Results in Table 6 indicated a very slight decrease in TPC in the acidophilus Labneh samples, especially those not treated with oils (control), then samples that treated with an oily mixture and then came the other treatments which showed ~1 log cycle decrease after the thirty days of cold preservation. Meanwhile, results did not show any growth of the fungus and yeast in all oily treatments except in the non-oil treated samples (control) as mold appeared after 21 days. On the other hand, yoghurt starter culture bacteria, S. thermophilus and L. bulgaricus, were slightly decreased in control (<1 log cycle) than in the oily treatments (>1-1.5 log cycle), where T₃ (12.5 μL/100 mL of each of the mixed oils) was better than others T₁ and T₂ (25 μL/100 mL of each oil) as shown in Fig. 1a-c.

Fig. 1(a-c):

Viability count of L. acidophilus as probiotic, (b) S. thermophylus starter and (c) L. bulgaricus the starter culture in plain (control) and treatments of flavored labneh at during storage

A similar trend was shown by the probiotic bacteria, L. acidophilus as the counts decreased very slightly, particularly, in the control (<0.5 log cycle) than in the oily treatments (>1-1.5 log cycle), where T₃ was better than others, T₁ and T₂ as shown in Fig. 1c.

Sensory evaluation of the acidophilus labneh flavored with thyme (TV) and N. sativa (NS): Data given in Table 7 showed that TV and NS mix oils (T₃) flavored Labneh was tastier and highly preferred compared with the non-oily Labneh as control, the only theme (T₁) and the only N. sativa (T₂) oily Labneh even after 30 days of storage at 4°C. Also, the concentrations of TV or NS oils (25 μg/100 mL for each oil, separately or 12.5 μg /100 mL of each in the mix) played a big role in determining the sensory quality of the Labneh. Besides, results indicated the longer the shelf life of the oily Labneh as the non-oily samples were unaccepted mainly for flavor after 21 days.

DISCUSSION

The compounds identified in thyme essential oil were identified as 23 constituents, accounting 98.83% of the oil. The chemical analysis of the oil showed that P-Cymene , Thymol, Y-Terpinene, Bornyl acetate, β-Caryophyllene, Linalool and Carvacrol and present at the highest fractions as these findings are in agreement with Eqbal and Aminah³⁴ Sartoratto et al.³⁵. Also linoleic acid, oleic acid and palmitic acid were major fatty acids than Stearic acid and arachidic acid while polyunsaturated fatty acids were 61.48%. This finding came in accordance with reports published by Mamun and Absarr⁹.

The high antibacterial activity of the thyme essential oils against Yersinia enterocolitica, Klebsiella pneumonia, Salmonella typhimurium, Listeria monocytogenes and S. aureus, this is in accordance with Sartoratto et al.³⁵, Roy et al.³⁶, Teixeira et al.³⁷ and Valizadeh et al.³⁸. The high antimicrobial activity of thyme essential oil originates from the high content of thymol (64%), gamma-terpinene (9%), para-cymene (6%)] and carvacrol (5%)^39,40. The antimicrobial action of N. sativa was originating from the phenolic, thymoquinone and melanin compounds which are considered as powerful active compounds^41-43. Bacillus cereus, E. coli and H. pylori were less sensitive against the thyme and N. sativa oils compared with the other tested bacterial species and these verifications may be attributed to oil extraction methods, amount of ingredients plant area and season of collection^44,45.

Thymes and N. sativa oils showed higher antifungal activity in mixes than in single oils. This finding is interesting because the growth of these surface organisms on the fermented dairy products leads to a rapid deterioration in appearance, taste and general quality of the product and the use of these oils limits this phenomenon. At the same time results revealed that the antifungal activity of thyme and N. sativa essential oil showed weaker activity than their antibacterial activities at the same concentration 0.1%. In this respect several types of research indicated high antifungal activity of thyme essential oil against different fungal species and inhibited Aflatoxins (AFs) production^46,47.

MIC of thyme required to prevents visible growth of Yersinia enterocolitica, Klebsiella pneumonia, E. coli O157:H7, A. Niger and A. Flavus were less than that required for S. aureus, Salmonella typhimurium, Bacillus cereus, Listeria monocytogens and Saccharomyces cerevisiae. Studies by Maksimov⁴⁸, Bussmann et al.⁴⁹ and Lambert et al.⁵⁰ evaluated the Minimum Inhibitory Concentration (MIC) of different plant species for their antibacterial properties against Gram-positive and Gram-negative bacteria. Whereas, Kokoska et al.²² reported that Thymoquinone N. sativa constituent exhibited potent growth-inhibiting activity against gram-positive bacteria, with MICs ranging from 8-64 μg mL^–1. On the other hand, results for MIC obtained by Darah, et al.⁵¹ revealed that methanol extract of Wedelia chinensis leaves against similar Gram-positive and Gram-negative pathogenic bacteria were 3.12-6.25 and 25 μg mL^–1, respectively.

The mold and yeast in all oily treatments showed no growth except in the non-oil treated samples (control) as mold appeared after 21 days. The findings indicated that adding thyme or N. sativa oils did not significantly affect the total bacterial count, while they remarkably affected mold and yeast growth, in Labneh during storage. These results were following Abd El-Sattar et al.²⁴, while results were inconsistent with Rahim et al.⁵² for black pepper. Yogurt starter culture bacteria, S. Thermophilus, L. bulgaricus and probiotic L. acidophilus were slightly decreased in control than in the oily treatments where T₃ (mixed oils) was better than others T₁ and T₂. The behavior of the growth of each of S. thermophilus, L. bulgaricus and L. acidophilus as well, was similar to that found by Abd El-Sattar et al.²⁴ and Moritz et al.⁵³. However, this advantage may turn into liability if that antimicrobial action extends to inhibit the activity of the starter bacteria (S. thermophilus and L. bulgaricus) and probiotic bacteria (L. acidophilus) that fortunately did not appear. These organisms should remain active even after the Labneh or probiotic dairy product have been manufactured and marketed for as long as possible, until the expiration date, in accordance with Schrezenmeir and Michael⁵⁴, Ezema⁵⁵, Hatice and Cakmakc²⁵.

Mix oils (T₃) flavored Labneh was tastier and highly preferred compared with control, the only theme (T₁) and the only N. sativa (T₂) oily Labneh even after 30 days of storage at 4°C. Good sensory properties were recorded for many probiotics fermented dairy products as reviewed by Okda et al.⁵⁶ and Ebojie⁵⁷. Moreover, adding plants or its oils, like thyme and N. sativa, may elongate the shelf life of the milk products and produce high-quality products with good sensory properties as recommended by Desalegn⁵⁸, Wael et al.⁵⁹, Asala et al.⁶⁰, Heba et al.⁶¹ and Fouad et al.⁶².

From this study at lab scale, it was possible to produce a new functional structure dairy product that contains thyme and N. sativa oils, in combination with yogurt starter culture and acidophilus bacteria that result in a valuable product with high nutritional, health and sensory properties. Therefore, the study recommends producing and applying this product at the industrial, nutritional and health levels for health and common good.

CONCLUSION

The current investigation indicated that using the flavoring property, phytochemical constituents, the antibacterial and antifungal potential of Thymus vulgaris and N. sativa oils, besides using probiotic bacteria, was a successful trial for preparing a good flavored functional fermented dairy product. Also, the present findings suggested that adding a mix of Thymus vulgaris and N. sativa oils into acidophilus probiotic yogurt "Acidophilus Labneh" formulation could improve the sensory quality and potential functionality of this new product.

SIGNIFICANCE STATEMENT

This study explores the potential synergistic effect of using T. vulgaris and N. Sativa oils in combination with yogurt-acidophilus bacteria for preparing a new dairy product that could be beneficial in dairy field and people's health. This study will help the researcher to uncover the critical area of preparing functional dairy products that many researchers were trying to explore. Thus, a new theory on these plant extracts-microbe combinations and possibly other combinations, may be arrived.