MATERIALS AND METHODS

Preparing materials: The present study was conducted during May and June months of 2018 from the apiary at Giza governorate which planting the marjoram crop from February to April months. Fifteen honey bee colonies which equal in strength and exposed to the routine work during the experimental period were used for this study to produce the marjoram bee honey for this work.

Physiochemical properties of marjoram bee honey samples: Determination of water content of honey was carried out by measurements of its refractive index value (ABBE WAY-IS) refractometer¹¹ at 20°C. The quantity of sugars (glucose and fructose) were performed by HPLC according to the method of Bogdanov et al.¹². The electrical conductivity was determined by the method of Vorwhol¹³, using EC meter model EN50081-1 at room temperature (2 g of honey sample was dissolved in 10 mL of distilled water and the results were expressed as ppm). The optical density and color of the honey samples were measured by using the relation between optical density and USDA standard as indicated by White¹⁴.

The honey sample was classified according to their botanical source based on the method of Von Der Ohe et al.¹⁵. Pollen classification as: mostly pollen (more than 45%), important minor pollen (10-20%) and minority pollen (less than 3%) by Andrade et al.¹⁶ physiochemical analysis was conducted in “Elements laboratory, Campus of research laboratories, FARP”, Faculty of Agriculture, Cairo University Research Park.

The goat milk was obtained from the experiment station and the Agricultural Research, Faculty of Agriculture, Cairo University. Then the goat milk was heated and maintained under refrigeration (4±1°C) until the experiment (maximum period 6 h). The samples of some marjoram Egyptian honeys were collected directly from different apiaries of different Egyptian governorate and the weight of each sample was approximately 500 g, stored at room temperature overnight before preparing the yogurts.

The starter culture (YC-X11) contain Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus and the probiotic culture of Lactobacillus casei EMCC761 was obtained from Cairo Mircen Microbiological Resources Center, Agriculture Faculty, Ain-Shams University, Cairo.

Characterization of raw materials: The raw goat milk used in preparing the yogurts was analyzed for total solids, fat, protein, fixed mineral residue, acidity and pH, while the samples of honey was analyzed for pH, total acidity, glucose, fructose content. All physiochemical measurements in goat milk was determined using standard procedures¹⁷.

For the marjoram honey sample, the concentration of glucose and fructose were determined by High Performance Liquid Chromatography (HPLC). Analytical stainless-steel column 4, 6 mm in diameter, 250 mm length, containing amine-modified silica gel with 5-7 μm particle size, according to the method of Bogdanov et al.¹².

Preparation of yogurts: Goat milk was pasteurized (90°C/10 min), then milk was cooled to 42°C which is the optimum temperature for culture inoculated at a concentration of 3% for the starter culture consisting of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus and a concentration of 1% for the probiotic culture of Lactobacillus casei EMCC761 defined according to previous testing to guarantee a minimal final count of approximately 7 log CFU mL^–1.

Fifteen different goat yogurt formulations were prepared using different concentration of honey (0, 5, 10, 15 and 20%) in a volume of 225 mL for each sample. The fermentation process was performed at 42°C for 4 h and the end point of yogurt fermentation was based on verification of clot firmness and pH value, which should reach a maximum of 4.6. Subsequently, the product was cooled to 4±1°C and the clot was broken by manual stirring with a glass rod. Then, the honey samples were added to the different formulations (w/v). All yogurt formulations were prepared in triplicate. The yogurts were subjected to physicochemical and microbiological analyses at different time points during 14 days of refrigerated storage. The physiochemical analysis of yogurt was performed depending on standard procedures¹⁷.

Microbiological analysis of yogurts: The raw goat milk was assessed for microbiological quality parameters like total count (T.C), coliform and mold and yeasts. All of these analyses were performed according to standard procedures. The analysis as curtained total count <10³ CFU mL^–1 and coliform counts <10 CFU mL^–1 and mold and yeast counts <1 CFU mL^–1, confirming the goat milk as being suitable for human consumption and for use as raw material for preparing yogurts.

For the lactic acid bacteria counts at each pre-established time, DE MAN, ROGOSA, SHARPE (MRS) broth medium were used. The MRS agar plus 0.5 g L^–1 cysteine were used for counting L. casei EMCC761 N and the starter bacteria group (S. thermophilus and L. bulgaricus), respectively by MRS agar. The counts were expressed as the log of the colony forming units per mL of yogurt (log CFU mL^–1).

Organoleptic properties: The sensory analysis was established for different yogurt formulations on the 14th day of refrigerated storage. The yogurts were subjected to tests of acceptance and relative preference. The panelists were recruited from master’s degree students, laboratory technicians non-trained panelists from Department of Dairy Science, Faculty of Agriculture, Cairo University, Giza, Egypt.

All sensory evaluation assays were performed with the same panelists who worked in individual with controlled temperature and lighting conditions. Each panelist was served 50 mL of each yogurt formulation on a small white glass at 6°C coded with a random number immediately after being taken out of the refrigerated storage. The acceptability of appearance, colour, flavor, taste, texture and overall acceptance were evaluated on a nine-point and the scale ranging from 9 as like very much to 1 as dislike very much¹⁸.

Rheological parameters measurements: Rheological measurements of milk inoculated with bacteria (viscosity, shear stress and torque) were carried out in triplicates over temperature of 25°C using a concentric cylinder Brookfield Programmable viscometer (Model DV-II; Brookfield Engineering Laboratories, USA) with UL adaptor and ULA spindle over a shear rate of 6.1 sec^–1. WinGather version 1.1 (Brookfield Engineering Laboratories, Inc.) software was used to collect, store and plot the data on a personal computer connected to the viscometer.

Statistical analysis: The two-way statistical analysis of variance (ANOVA), mean separation and correlation required subprogram of MSTAT microcomputer statistical program¹⁹ was applied to evaluate physiochemical and microbiological parameters of goat yogurt fortified by some Egyptian marjoram honey samples. Simple and multiple linear regression analysis were applied and the student t-test was used to test mean at p<0.05.

RESULTS AND DISCUSSION

Physiochemical parameters of marjoram honey: The physiochemical properties of marjoram (Majorana hortensis) bee honey samples were determined. As shown in Table 1 there were clear significant differences in all tested parameters.

Table 1:	Physiochemical parameters of the marjoram honey samples

Table 2:	Total solids (T.s) and protein contents in the yogurts made from goat milk with marjoram

The water content (moisture %) was 18.10% in marjoram honey where the Electrical Conductivity (EC) was 0.011%, also the pH value was 3.84 in marjoram honey. The obtained data showed that, the glucose content was 28.00% (marjoram honey) and the fructose content was recorded 37.50 in marjoram. The Optical Density (O.D.) of honey samples were measured in Table 1, the O.D. was 0.25 OD in tested samples of marjoram bee honey. The moisture values which obtained in this study were similar to those found in South Asia honey²⁰: from 15.3-21.7 g/100 g and in North African honey (from 14.6-21.8 g/100 g)^21,22,1.

The E.C. values were in the same range as those reported by other authors in Burkina Faso honey^23-25. The sugar content (glucose and fructose sugars) of bee honey samples are similar to those found with Escriche et al.²⁶ which detected the levels of glucose (from 27.8-31.9 g/100 g) and fructose (38.3 and 42.7 g/100 g). While pH value was agree with that obtained by Rateb²⁷.

Physicochemical analyses of yogurts: The mean values of the physicochemical parameters (Total solid, protein content %) of goat yogurts formulations were presented in Table 2. According to obtained data the yogurt formulations containing 20% added marjoram bee honey presented higher total solids content, due to the higher amount of honey contained. The same effect was observed during storage period 14 days. The protein content in yogurt decrease with increase the marjoram honey, due to the high amount of honey which contain low protein concentration (0.3 g/100 g honey).

During the storage period, pH of yogurt gradually decreased in the treatments may be due to post acidification. pH of yogurt can be decreased due to accumulation of lactic acid which had been produced by yogurt starter and other living micro-organisms during storage also noted that low pH, in yogurt formulations containing added marjoram honey compared to control (0% honey).

The acidity of bee honey comes from the naturally occurring organic acids in its composition²⁰. The initial pH of the assessed yogurt formulations continuously decreased until the 14th day of storage and the yogurt samples containing added marjoram bee honey showed lesser pH that was proportional to the honey amount add during preparation as shown in Fig. 1a. At the same time, the acidity also increased during storage period in all yogurt tested formulations (Fig. 1b).

It was confirmed that, these changes in pH and acidity values observed in yogurt formulations containing added marjoram bee honey could be related to the presence of extra fermentable compounds in bee honey namely sugars also addition of bee honey seemed to stimulate the lactic acid metabolism and consequently the acidification of yogurt formulations. This increased in the acidity values in yogurts containing marjoram bee honey may be associated with the presence of prebiotic oligosaccharides in bee honey which may promote the growth and the metabolic activity of lactic acid bacteria.

Organoleptic analyses of yogurts: Sensory analysis is often used to assess the flavor, appearance, texture and other attributes of food products as a function of processing parameters²⁸. The results given inTable 3described the effect of mixing marjoram honey with goat’s yogurt on the sensory evaluation. Yogurt with no addition of honey had the highest scoring points of color and appearance whether in fresh or after 7 and 14 days of the cold storage as shown in Table 3.

In contrast to the color and appearance evaluation, body and texture attributes of goat yogurt fortified by honey had negatively affected on body and texture. Panelists recorded the lowest scores of body and texture for sample 4 (15 %) and sample 5 (20%).

The greatest effect of supplementation of goat’s yogurt with marjoram honey on the sensory characteristics was observed in flavor. In Egypt, the majority of consumers don’t prefer the goaty flavor.

Table 3:	Effect of adding marjoram honey to goat’s yogurt on sensory evaluation at room temperature

Fig. 1(a-b):	Mean values of (a) pH and (b) Acidity of goat yogurt containing L. casei EMCC671 and marjoram honey at different concentrations, during 14 days of refrigerated storage

The panelists showed that adding honey to goat’s yogurt mask the goaty flavor with good sweetened one which increased the overall acceptability. The sensory evaluation showed that lower sensory acceptability was recorded for goat’s yogurt stored for two weeks than the fresh products.

Moreover the added marjoram honey may had rendered the goat yogurt containing L. casei EMCC 671 more acceptable and attractive due to the presence of poly saccharides also the highest acidity observed in goat yogurts containing added marjoram honey did not affect negatively on the sensory acceptance due to honey sweetness that in combination with the higher acidity may provide an enjoyable flavour to yogurts this was the same result obtained by Baraka et al.²⁹.

Microbiological count analyses: The results of the viable counts of the starter bacteria group (composed of S. thermophilus and L. bulgaricus) YC-X11 and L. casei EMCC671 in goat yogurt containing or not containing marjoram bee honey during refrigerated storage were presented in Fig. 2 the counts of yogurt starter bacteria group YC-X11 at the first day of storage were approximately 8.8 log CFU mL^–1 and decreased to approximately 7.1 log CFU mL^–1 and 6.2 log CFU mL^–1 on the 7th and 14th days of storage. The starter bacteria group presented a linear decrease in counts during the period in all yogurt formulations; however, these counts were higher in yogurts containing marjoram honey in 7th-14th day of storage.

The decreases in starter bacteria counts had been an expected behaviour during yogurt storage which was due to high acidity produced by microbial fermentation. Also a similar behavior of sharp reduction in counts of L. casei EMCC671 occurred from 7th-14th day of storage in all yogurt formulations as shown in Fig. 2b.

Fig. 2(a-b):	Viable cell counts of bacteria starter group (a) Yc-X11 and (b) L. casei EMCC671 in goat yogurt containing or not marjoram honey at different concentrations during storage under cooling

Fig. 3:	Average viscosity/10 (cP), shear stress (D/cm2) and torque (%) of yogurt sample (yog 0% control, yog 5%, yog 10%, yog 15%, yog 20%), respectively

Rheological characteristic: Bee honey is considered a high-viscosity fluid, also a pseudo-plastic offering greater resistance and higher viscosity for yogurts. Furthermore, the addition of marjoram bee honey increase total solids content and increase the consistency of the product but adding the honey led to decrease in the yogurt viscosity that was directly proportional to the amount of honey add.

Table 4:	Mean, LSD value and coefficient of variation (Co. Var.) of rheological parameters of goat yogurt with marjoram honey

A corroding to Shaker et al.³⁰ who reported that yogurt is an emulsion of fat and protein suspended in water. Honey is a very concentrated solution of sugars. If you put honey and yogurt in contact, you create osmotic pressure because the number of solutes in the honey is much higher than the number of particles suspended in the yogurt. Osmosis causes the water to flow towards the greater concentration of solutes - the honey. The reason it gets kind of liquid in between the bee honey and the yogurt is because the honey is slow to dissolve.

At complete clotting pH 4.6±2, sample fortified by 20% marjoram honey showed the minimum values for viscosity (20.667 cP), shear stress (17.76 D cm^–2), torque (26.73 %) while the control sample without marjoram bee honey (yog 0%) had higher viscosity (1097.66 cP), shear stress (67.36 D cm^–2) and torque (85.60%) than both yog 5,10 and 15%, respectively as shown in Fig. 3.

Table 4 showed that, addition of marjoram bee honey in 20% v/v in goat yogurt recorded the lowest viscosity shear stress and torque value. The rheological parameters values in control sample was higher than 5 times as much as the value s recorded after marjoram honey addition in high concentration. The significant influence statistically found was for the concentration of bee honey add.

CONCLUSION

The addition of marjoram honey in goat yogurt containing the Probiotic L. casei EMCC671 has a positive effect on some of physical and rheological characteristics of the product during the 14 days of refrigerated storage. The L. casei EMCC 671 counts in all goat yogurt formulations remained in adequate amount during storage (>6 log CFU mL^–1) which had several health benefits to the consumer.

However, the formulation containing marjoram honey presented the highest counts of L. casei EMcc671 and yogurt starter YC-X11, indicating a growth promoting effect for starter bacteria. Moreover, the addition of marjoram honey affect directly on the acid profile of the goat yogurt during cooled storage, without negatively affecting its acceptance and its organolyptic properties. Otherwise, the yogurts containing marjoram honey presented the best sensory acceptance according to their score card. Finally, the results of this study presented a successful incorporation of both the Probiotic L. casei EMCC671 and the marjoram as ingredients of a new goat dairy product with satisfactory nutritional and sensory quality due to masking of goaty flavor, as well as added market value.

SIGNIFICANCE STATEMENT

This study confirmed that the addition of marjoram bee honey to the goat yogurt has led to the prolongation of the period of conservation also led to an increase in the survival of the yogurt and the increase of the period of vitality for Lactobacillus casei.

ACKNOWLEDGMENT

The author would like to thank Dairy Technology Department, Faculty of Agriculture, Cairo University and Ashwak Hassan assistant lecture at Dairy Department, Faculty of Agriculture, Cairo University for their efforts.