MATERIALS AND METHODS

This study was carried out in Dairy chemistry Laboratory, Food Industries and Nutrition Division, National Research Centre, Giza, Egypt, in the period from June-November, 2016.

Materials
Myrrh gum crystals source: Myrrh (Commiphora myrrha) belonging to family Burseraceous was purchased from Thailand as semi-white fine crystals.

Bacterial strains source: Seven pathogenic bacterial strains were obtained from Microbiological Resource Centre (Cairo MIRCEN). Listeria monocytogenes (598), Staphylococcus aureus (ATCC13565), Bacillus cereus (B-3711) and B. subtilis were used as Gram-positive bacteria. Escherichia coli O157:H7 (ATCC51659); Salmonella typhimurium (ATCC 25566) and Yersinia enterocolitica were used as Gram-negative.

Mold and yeast source: Strains of Aspergillus flavus (3357); Saccharomyces cerevisiae (Y2223) and Candida albicans were obtained from the Northern Regional Res. Lab. Illinois, USA (NRRL). However, Aspergillus niger; Penecillium roqueforti (J5) were obtained from Department of Microbiology, Swedish University of Agriculture Science Sweden.

Starter culture source: Strain of both Lactococcus lactis sub lactis and L. delbrueckii sub bulgaricus were obtained from Dairy Microbiology Laboratory, National Research Centre, Egypt.

Milk source: Fresh cow milk (12% TS and 4% Fat) as well as calf rennet, calcium chloride and commercial salt were purchased from Faculty of Agriculture, Cairo University, Egypt.

Experiments
Preparation of myrrh gum extract: Myrrh gum crystals were fine milled, sieved and dissolved in distillated water (1:10 w/v), then were strongly stirred and left for 6 h at room temperature (25°C).The aqueous extract was exposed to ultra-sonication at 160 W power, 20 kHz frequency with 50% pulse for 20 min (Sonic Ruptor 400, OMNI International the Homogenizer Company, Kennesaw, GA, USA). During sonication, samples were placed in cold water bath at 4°C to prevent overheating of emulsion.

Preparation of soft cheese samples: Traditional methods for preparing Egyptian soft cheese were applied as recently mentioned by Farag et al.¹⁵. After pasteurization, the cheese- milk was cultured and kept for 30 min at 32°C .Three concentrations of prepared MGE (5, 10 and 15 mL/1 L milk) were added before renneting to serve three treatments (T1, T2 and T3, respectively) in addition to control sample. After completed coagulation and forming homogenous curd, all manufacture steps were traditionally completed and cheese samples were stored at 7°C for 90 days. Cheese samples were chemically analyzed. Textural and sensorial evaluation were also assayed either fresh or during periodical storage periods (0, 30, 60 and 90 days).

Methods of analysis
Growth conditions of pathogenic bacteria: Stock cultures of Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Bacillus subtilis; Yersinia enterocolitica; Escherichia coli and Salmonella typhimurium were sub-cultured twice onto Tryptone Soya Agar (TSA) followed by incubation at 37°C/24 h. Cultures were prepared from subcultures and grown overnight in Tryptone Soya Broth medium (TSB, Oxoid, Basingstoke, UK) under optimal conditions for each microorganism.

Mold and yeast strains were cultivated in malt extract broth (Oxoid) then incubated at 25°C /72 h.

Antimicrobial assay using the disc diffusion method: The disc diffusion method according to Alabi et al.¹⁶, was applied as mentioned recently by Mohamed et al.¹⁷. The inhibition zones were measured in mm as recorded by Mohamed et al.¹.

Chemical analysis of soft cheese samples: All cheese samples were analyzed for their total solids (TS), total protein (TP), fat, soluble nitrogen (SN) contents as well as acidity percentage according to AOAC¹⁸. The pH values were determined using a digital laboratory pH meter (HI 93 1400, Hanna instruments) with glass electrode. Total Volatile Fatty Acids (TVFAs) contents expressed as 0.1 N NaOH/100 g cheese were also determined according to Kosikowiski¹⁹.

Textural properties assaying of soft cheese samples: Texture profile analysis (TPA) was performed on cheese samples using the double compression apparatus (Multi test 1d Memesin, Food Technology Corporation, Slinfold, W. Sussex, UK). Experiments were carried out as recently mentioned by Abbas et al.² using a compression test that generated a plot of force (N) versus time (s). A 25 mm diameter perplex conical-shaped probe was used to perform the TPA analysis at five different points on the sample surface. In the 1st stage, the sample was compressed by 80% of their original depth at a speed of 2 cm min^–1 during the pretest, compression and relaxation of the sample. From the force-time curve, the following parameters were determined according to the definition given by the International Dairy Federation²⁰.

Sensory properties evaluation of soft cheese samples: Sensorial properties of soft cheese samples were panel-evaluated by 20 of the trained staff members at Dairy Department of Science, National Research Centre, Egypt. Appearance and color scores were 20 points while flavor scores were 50 points and body and texture scores were 30 points. This scheme was suggested by the authors and recommended by the staff members to be more suitable for the product and to focus on the desirable property.

RESULTS

Assaying of Antimicrobial activity of myrrh gum extract (MGE): Obtained data indicated that the prepared MGE showed highly inhibition activity against the tested microorganisms as shown in Table 1.

The results revealed that Gram-positive bacteria were more susceptible than Gram-negative one. Majority of tested microorganisms were susceptible to the action of MGE. Bacillus cereus strain was the most resistant bacteria while Salmonella typhimurium and Yersinia enterocolitica strains were the most resistant bacteria while, Bacillis subtilis and E. coli O157:H7 followed by Listeria monocytogenes and Basilus cereus were the most sensitive strains.

Table 1:	Antimicrobial activity of Myrrh Gum Extract (MGE) against different microorganisms as diameter of inhibition zone (mm)

The most sensitive mold and yeast were Penicillium roquefortti and Saccharomyces cerevisiae, respectively. The most sensitive yeast was Saccharomyces cerevisiae. Myrrh extract had a highly inhibitory effect.

DISCUSSION

The results revealed that MGE had antimicrobial activity against the pathogenic bacteria and inhibited their growth as well as mold and yeast. The same trend was also observed by various researchers and by different means. Mohamed et al.¹, observed the same action of Dill and Caraway essential oils against pathogenic bacteria. Mohamed et al.¹², also reported that myrrh essential oil had highly antimicrobial action against pathogenic microorganisms which lead to preserve processed cheese samples and increased their shelf life to one year. While, Brieskorn and Nobel¹¹ and Dolara et al.¹⁰, also reported that the essential oils of Commiphora species such as myrrh are rich in furano sesquiterpenoids and sesquiterpenes which have been possess anesthetic, antibacterial and antifungal properties.

Chemical composition of soft cheese samples: The TS; TP; SN; Fat; TVFA and pH values as well as acidity percentage of all cheese samples are shown in Table 2. There were no paramount variations in the general chemical composition or other studied parameters between treated samples and control as shown in Table 2. Myrrh gum extract seemed to have any effect on the composition of cheese samples.

The obtained data revealed that there were no clear variations on the chemical composition of treated cheese samples and control. They indicated that the chemical composition of cheese samples lied in the normal ranges of Egyptian soft cheese^2,15,21. The level of acidity percentage and pH values had the normal trends which indicated that addition of MGE did not affect the starter culture activity. Mohamed et al.¹ mentioned also that essential oil of caraway and dill did not affected the activity of starter culture in Labenah samples.

Texture profile of soft cheese samples: Data listed in Table 3 reflected the textural properties of soft cheese samples. It could be noticed that there were no clear differences in the studied parameters either in Firmness, Gumminess or Chewiness.

The MGE did not affect the textural properties of resultant soft cheese.

These data were in agreement with that obtained by Abbas et al.², Mohamed et al.¹² and Foda et al.²¹. The concentration of Myrrh extract which was used in this study and the behavior of this material did not affect the textural properties. Logically, it was expected that myrrh may be increased the gumminess of samples because it have some gumming materials³ or indigenous gums and resins⁸ but the procedure that used in preparation and extraction was success in absent of this defect.

Table 2:	Chemical composition of soft cheese samples supplemented with different ratios of myrrh gum extract (MGE)
*TVFA: Total volatile fatty acids, expressed as 0.1 N NaOH/100 g of cheese, **SN: Soluble nitrogen, ***SN/TN: Soluble nitrogen/total nitrogen

Table 3:	Textural properties of soft cheese samples supplemented with different ratios myrrh gum extract (MGE)

Table 4:	Sensorial evaluation (Degree) of soft cheese samples supplemented with different ratios of myrrh gum extract (MGE)
A: Appearance, B: Body and texture, F: Flavor d: Degree

Sensorial behavior of soft cheese samples: Effect of using MGE in organoleptic properties of cheese samples is clear in Table 4. Soft cheese samples seemed to be natural and had no odd character either in the body and texture or flavor. No bitter taste was observed or gummy body and compact texture was noticed. Addition of MGE gave clean and acceptable acid taste.

So, Myrrh Gum Extract can be used in preparing soft cheese samples which had relative high moisture content (about 60%) and increase its shelf life without any risk in their chemical, textural or sensorial properties. This study recommended using of Myrrh gum extract as an antimicrobial agent in food sector and dairy products as natural, safe and effective material.

CONCLUSION

It could be concluded that the urgent need for natural and safe antimicrobial agents is still a requirement, however, myrrh is a natural and safe material which could be used as food preservative. The potential effect of myrrh gum extract (MGE) was observed as its antimicrobial effect against various examined-strains of pathogenic microorganisms. MGE did not affect the starter culture or development of acidity through 90 days of storage. Obtained results reveal that using 10 mL MGE L^–1 cheese milk in preparing soft cheese is more beneficial and gave the favorite acceptance. So, this study recommended its use in preparing cheese with high content of moisture, where the chemical; rheological and sensorial properties of samples did not impressed by its using.

SIGNIFICANCE STATEMENT

This study deals with using myrrh gum extract as a natural, safe and cheap preservative agent to produce healthy and safe soft cheese.