Egypt. J. ofAppl. ScL, 26 (12B) 2011 343-360

ANTIBACTERIAL ACTIVITY OF SOME NATURALPRESERVATIVE MATERIALS AND THEIR EFFECTS ON

CHARACTERISTICS OF YOGHURTElalfy, M.B.; M.E. Shenana; A.M. Abd El-Aty and E.S. Elkhtab

Food Science Department (Dairy), Faculty of Agriculture, Benha University, Egypt

ABSTRACT

This research aimed to study the antibacterial activity of some naturalpreservative materials (rosemary, garlic, onion, black cumin, thyme, parsley,celery essential oils, Kombucha, propolis ethanolic extract 20% and propoliswater extract 20%) compared with the antibacterial activity of some chemicalpreservatives which may be used in dairy products and their effects oncharacteristics of yoghurt. The antibacterial activity of these preservativematerials was studied on some spoilage and pathogenic bacteria (Bacillussubtilis subsp spizizenii ATCC 6633, Escherichia coli ATCC 14169,Streptococcus pyogenes ATCC 21060 and Staphylococcus aureus ATCC29737), through, the inhibition zone and the minimum inhibitory concentration(MIC). The results indicate that the inhibition zone for thyme recorded the highestlevel ranging from 16 mm for Str. pyogenes to 24 mm for Staph. aureus followedby celery and rosemary. While, the inhibition zone of propolis water extraction(20%) and onion essential oil recorded the lowest. The effect of using somenatural preservative materials in yoghurt manufacture on the keeping quality,sensory evaluation, chemical composition, microbiological analysis andrheological properties of the obtained product was evaluated. The resultscleared that the Kombucha (3 & 4%), ethanolic extraction of propolis (0.1%),black cumin essential oil (0.1%) and parsley essential oil (0.05 & 0.1%) canproduce yoghurt with good organoleptic, rheological properties and prolongedshelf life for 28 days.

INTRODUCTIONOne of the most important trends in the food industry today is the

application of natural food additives. On the other hand, many naturallyoccurring compounds found in plants have been shown to possessantimicrobial functions and serve as a source of antimicrobial agents againstfood borne pathogenic microorganisms. Herbal spices have been added to foodsince ancient times, not only as flavoring agents, but also as folk medicine andfood preservatives. In addition to imparting characteristic flavors, certainspices and herbs prolong the shelf life of foods via their bacteriostatic orbactericidal activity; also preventing rancidity through their antioxidantactivity. Essential oils and their components are becoming increasingly popularas natural antimicrobial agents to be used for a wide variety of purposes,including food preservation, complementary medicine and natural therapeutics.

344 Egypt. J. ofAppl ScL, 26 (12B) 2011

At present, essential oils are used by the flavouring industry for flavourenhancement and for their antioxidant effect, while the potential use of theseoils as natural antimicrobial agents has been less explored (Cosentino et al.,2003). In recent years, there has been a constant search for alternative andefficient agents for food preservation aiming a partial or total replacement ofantimicrobial chemical additives and hence reduce the risk of bacteria andfungi in food and play important role for enhancing shelf-life of foods andcontrolling food pathogenic.

Dairy and dairy products considered to be the most likely foods overall theworld. Fermented milks also considered to be most public dairy products. Yoghurtis one of the major likely fermented milks. Although yoghurt has many desirableproperties, it is still prone to deterioration, especially at ambient temperature,within a matter of days.

There are so many ways for prolonging the keeping quality of yoghurt.Nevertheless, yoghurt become impalatable within 1 -2 weeks, for this reason analternative preservation processes such as adding of either herbs or essentialoils for more prolonging shelf life of such dairy products. One of the mostaccepted ways to extend the shelf-life of sensible 'products the use of bio-preservatives (Burt, 2004 and Draughon, 2004).Therefore, the present investigation aims to study the following objectives:

1- The antibacterial activity of some natural preservatives against somepathogenic and spoilage bacteria compared with the antibacterialactivity of some chemical preservatives used in the dairy field.

2- Producing yoghurt contains some natural antibacterial and flavouringagents and studying the chemical, microbial and sensory qualities alongthe refrigeration storage periods.

MATERIALS AND METHODS

1. Materials1.1 Milk:

Fresh mixed cows and buffalos' milk (1:1) was obtained from the herd ofFaculty of Agriculture, Moshtohor, Benha University. The chemicalcomposition of milk was ~ 3.2% fat, 3.4% protein, 5% lactose and 0.7% ash.1.2. Yoghurt starter:

Dried yoghurt starter culture consists of Lactobacillus delbreuckii subsp.bulgaricus and Streptocuccus thermophilus was obtained from Chr. Hansen,Copenhagen, Denmark. The obtained starter culture activated and added at a rateof 2g/100g for all the treatments.1.3. Spoilage and pathogenic bacterial strains:

Spoilage and pathogenic bacterial strains Le. (Bacillus subtilis subsp.spizizenii ATCC 6633, E. coli ATCC 14169, Streptococcus pyogenes ATCC21060 and Staphylococcus aura-is ATCC 29737) were obtained from

Egypt. J. ofAppl ScL, 26 (12B) 2011 345

Microbiological Resource Center (MIRCEN), Faculty of Agriculture, AinShams University, Cairo, Egypt.1.4. Natural preservatives:

1.4.1. Rosemaiy, parsley, black cumin and celery essential oils wereobtained from Kato Aromatic Company, Giza, Egypt.

1.4.2. Garlic and onion essential oils were obtained from Al-Neenaea Companyfor drying food products, Menofia Qovernorate, Egypt.

1.4.3. Propolis was purchased from local market which collected from thehybrid honey bee colonies at Qualiobia Governorate, Egypt. Storedseparately in the freezer until used.

1.4.4. Kombucha solution was prepared at Food Science Department, Facultyof Agriculture, Moshtohor, Benha University, Egypt.

1.5. Chemical preservatives:Sodium nitrate, sodium nitrite, potassium nitrate, potassium nitrite and

hydrogen peroxide were obtained from El-Nasr pharmaceutical CO. Adwic,E0018111, Egypt1.6. Standard disc 0.6 mm diameter was obtained from El-Nasr pharmaceutical

CO. Adwic, E0018111, Egypt.2. Methods:2.1. Extraction of propolis:

Extraction was prepared according to the method of (Tosi et a/., 2007)2.2. Preparation of essential oils emulsion:

Essential oils emulsions were prepared according to the methoddescribed by (Ismail and Pierson, 1990).2.3. Estimation of antibacterial activity:

Antibacterial activity was measured using agar diffusion method(Kirbey-Bauer method) as described by (Tosi et al., 2007). Minimuminhibitory concentration (MIC) was determined by the microdilution agar platemethod of (Moreira et al, 2005).2.4. Manufacture of yoghurt:

Yoghurt was manufactured according to the method described byTamime (1978). The fresh mixed milk 1:1 cow's: buffalo's was divided into 8portions, 5 kg each. The 1st portion was served as yoghurt control, the other 7portions were used for manufacture of yoghurt with different types of naturalpreservatives ie Kombucha at a rate of 3% TI, Kombucha at a rate of 4% Ta,ethanolic extraction of propolis at a rate of 0.1% TS, black cumin essential oilat a rate of 0.1% T4, parsley essential oil at a rate of 0.05% T5, parsley essentialoil at a rate of 0.1 % T6 and garlic essential oil at a rate of 0.1 %T7, respectively.Each portion was heat treated up to ~ 85°C for 30 min, cooled to 42°C,inoculated with 2 g/100 g (w/w) of starter culture, dispensed into plastic cups(120 g each) fitted with press-on-lids and incubated at 40 - 42°C till the pHreached 4.6 the time required for coagulation of each treatment was recorded.

346 Egypt. J. ofAppl. ScL, 26 (12B) 2011

The produced yoghurt was stored at ~5°C and analyzed for its properties Le.(chemical, microbiological, penetrometer reading, curd synersis and sensory).3. Methods of analysis:3.1. Chemical analysis of yoghurt:3.1.1. Protein, fat, and total solids of milk and yoghurt were determined

according to the methods of International Dairy Federation (IDF), 1993,1991a and b, respectively.

3.1.2. Titratable acidity of yoghurt treatments was determined according to themethodology- of AOAC (1990).

3.1.3. Total volatile fatty acid contents (TVFA) were determined by the directdistillation method as described by Kosikowski (1997).

3.1.4. Acetaldehyde content was determined according to the method describedby Lees and Jago (1969).

3.2. Microbiological examination:3.2.1. Lactic acid bacteria (LAB) was enumerated according to Elliker et al.

(1956).3.2.2. Colifonn bacterial count was determined by plating suitable dilution on

violet red bile agar (VRBA) medium as suggested by APHA (1992).3.2.3. Yeasts & moulds count was determined according to IDF (1990).3.3. Physical properties:3.3.1. Penetrometer reading:

Penetrometer reading was measured using the penetrometer supplied bySetamatic MKVI controller. The results were expressed as penetrometerreading (0.1 mm/s) according to Dixon and Parekh (1980).3.3.2. Curd synersis:

Curd synersis was determined according to the method of Dave and Shah(J 998). Curd synersis is calculated as the percent weight of the separated wheyduring the determination time (120 min).3.4. Sensory evaluation:

Organoleptic evaluation was done by 10 experienced Food ScientistsStaff at Food Science Department, Moshtohor, Faculty of Agriculture, BenhaUniversity, according to the methodology of IDF (1995).3.5. Statistical analysis:

Statistical analysis for the obtained data was carried out according to themethod described by Clarke and Kemp son (1997).

RESULTS AND DISCUSSION

Antibacterial activityA- Inhibition zone:

The data obtained in Table (1), indicate the inhibitory effect of the naturalpreservatives (rosemary, garlic, onion, black cumin, thyme, parsley, celeryessential oils, Kombucha, propolis ethanolic extract 20% and water extract 20%),and chemical preservatives (tfcOa 250 ppm, sodium nitrate 200 ppm, potassiumnitrate 200 ppm, sodium nitrite 200 ppm and potassium nitrite 200 ppm) on the

Egypt. J. ofAppl. SeL, 26 (12B) 2011 347

tested bacteria (B. subtilis, E. coli, Str. pyogenes wd Staph. aureus). The diameterof inhibition zone proportionally lead to the antibacterial effect of the addedpreservatives.Table (1). Inhibition zones (mm) of natural and chemical preservatives on some

spoilage and pathogenic bacteria.

Preservative materialsTested bacterial strains

B.subtilis

£'coli

Str.pyogenes

Staph.aureus

1 -Natural essential oils or preservativesRosemaryGarlic *Onion *Black cuminThymeParsleyCeleryKombucha extinctEthanolic extraction of propolis (20%)Water extraction of propolis (20%)

151412NI221321181511

21NININI22NI1621NINI

1522NI1416NI21NI20NI

201171524NI2116118

2- Chemical preservatives:Hydrogen peroxide (250 ppm)Sodium nitrate (200 ppm)Sodium nitrite (200 ppm)Potassium nitrate (200 ppm)Potassium nitrite (200 ppm)

8NINININI

10NINININI

NININININI

NININININI

"Concentration of garlic and onion essential oils is 10% of the stock oil.NI:No Inhibition

Whereas, Garlic and onion essential oils were diluted with Tween 80 to10% concentration.

The results indicate that the inhibition zone for thyme recorded the highestlevel followed by celery and rosemary on all tested bacteria, while, the inhibitionzone of propolis water extraction 20% and onion essential oil recorded the lowestlevels. These results are in agreement with Smith-Palmer et aL (1998), Vardar-Unlii et al. (2003), Misic et al. (2008) and Snoussi et al (2008) who reported thatthe antimicrobial activity test with fractions of the essential oils showed that theactivity was mainly observed in those fractions containing thymol. AlsoImelouane et aL (2009) evaluated the essential oil of thyme for its antibacterialactivities against Gram positive and Gram negative pathogenic bacteria i.e. Staph.aureus, Streptococcus sp., Pantoa sp., and!?, coli. El-Gayyar etaL (2001) reportedsimilar results with respect to rosemary on E. coli which showed inhibition zoneranging from 23 to 30 mm. Also the inhibition diameters of rosemary essential oilwere 15, 21, 15 and 20 mm for B. subtilis, E. coll, Str. pyogenes and Staph.aureus, respectively. The inhibition action for rosemary essential oil is due to themain compounds present in this oil Le, 1,8-cineole, a pinene, camphor... etc.which have been their antimicrobial effects (Viljoen et aL, 2003 and Fu et aL,2007).

348 Egypt. J. ofAppl ScL, 26 (12B) 2011

On the other hand, hydrogen peroxide with a concentration of 250 ppmonly affects on U, subtilis and E. coli as it recorded 8 and 10 mm, respectively.Individually hydrogen peroxide and sodium bicarbonate are known to possessantimicrobial activity against oral microorganisms (Miyasaki et al., 1984 andMiyasaki et al, 1986).B- Minimum inhibitory concentration (MIC):

The minimum inhibitory concentration is defined as the minimum levelof essential oil concentration that causes 90% reduction in the growth ofmicrobial colonies than the control.

Table (2) shows the minimum inhibitory concentration of testedpreservatives (rosemary, garlic, onion, black cumin, thyme, parsley, celeryessential oils, Kombucha, propolis ethanolic extract 20%, propolis water extract20% and H2O2) on the spoilage and pathogenic tested microorganisms (Bacillussubtilis subsp. spivzenu ATCC 6633, E. coli ATCC 14169, Streptococcuspyogenes ATCC 21060 and Staphylococcus aureus ATCC 29737. Minimuminhibitory concentration (MIC) was studied on the preservative materials that giveinhibition zones. From the obtained data it was observed that the MIC of propoliswater extract 20% on Staple aureus recorded the highest level followed byKombucha. On the other hand, the effect of garlic essential oil on B. subtilis needslow MIC. These results are in agreement with Tosi et aL (2007) who reported thatthe MIC dependent on propolis composition and botanical origin.

Regarding to hydrogen peroxide (H2O2) the minimum inhibitoryconcentration (MIC) was 50 ppm for both B. subtilis and E. coll The resultswere in agreement with Miyasaki et al., 1986 who studied the minimuminhibitory concentration (MIC) of H2O2 and NaHCOs individually and incombination on Gram negative oral bacteria. The tested bacteria exhibited MICvalues from 5 to 500(i mol/1 for H2O2.Table (2). Minimum inhibitory concentration (ppm) of natural and chemical

preservatives on the tested bacteria.

Preservative materialsTeasted bacterial strains

B. subtilis £coli

5.pyogenes

Staph. aureus

1 -Natural essential oils or preservativesRosemaryGarlicOnionBlack cuminThymeParsleyCeleryKombucha extractEthanolic extraction of propolisJ20%)Water extraction of propolis (20%)

210020600-

30004000200040000

10012000

3000---

6000-

300040000

--

1000400

-40004000

-3000

-1000.

35002500

-70005000

-500050000200078000

2- Chemical preservative:Hydrogen peroxide 50 50 _

-

Egypt. J. ofAppl. Sci, 26 (12B) 2011 349

Characteristics of yoghurtCoagulation time

Coagulation time of the manufactured yoghurt with adding differenttypes of natural preservatives was tabulated in Table (3). Concerning to theobtained data it was clear that the control yoghurt recorded the lowestcoagulation time. However, T2, 1(, and Ty recorded longer coagulation timewith 74.41% increase coagulation time than the control followed by TS and T]which recorded 65.69% and 33.72% respectively.

The variation of coagulation time could be attributed to the effect ofadded natural preservatives on the activity of lactic acid bacteria and on theability of producing acid which led to slow rate of acid development andprolonged the time of coagulation, similar results were obtained by Olasupo etal. (1996).Table (3): Coagulation time of yoghurt as affected by some natural

preservatives.Treatments

Coagulation Time h :min

% of increase

Cont.

2:52C

0.00

TI3:50b

33.72

T2

5:00a

74.41

T3

3:03C

6.39

T4

3:00°

4.65

T5

4:45a

65.69

T6

5:00a

74.41

T?

5:00"

74.41Cont = co ntrolT3 = Propolis 0.1%T6 = Parsley 0.1%

T,= Kombucha3%T4 = Black cumin 0.1%T7 = Garlic 0.1%

T2 = Kombucha4%Ts = Parsley 0.05%

Chemical composition of yoghurt:Tifratable acidity (TA):

Table (4) shows the titratable acidity of yoghurt produced with somenatural preservatives and stored at ~ 5°C. There was a higher acidity recordedfor the control yoghurt when fresh and during storage period up to 14 days thanthe treated yoghurt. This could be attributed to the antimicrobial activity of theadded natural preservatives which were found to exhibit biological activitiesagainst several species of bacteria and fungi (Gayoso et a/., 2005 and Rouatbietal.,20Q7).

Kombucha 4% (T2) and 3% (Ti) recorded the lowest titratable aciditywhen fresh and at the end of storage period (28 days) followed by black cumin0.1% (T4), parsley 0.05% (T5), parsley 0.1% (T6), propolis 0.1% (T)), andgarlic 0.1% (T7)compared with control. The variable acidity of the differenttreatments may be due to the effect of different natural preservatives on theLAB count. Control and treated yoghurt with garlic 0.1% (Ty) were valid onlyfor 14 days and then spoiled, also garlic treatment (T?) had the lowestantibacterial effect. The obtained results are in accordance with that obtainedby Marques et al. (2008) who reported that oregano had the highestantibacterial properties, followed by chitosan and garlic. On the other hand, itcould be noticed that titratable acidity for the control and the different

350 Egypt. J. ofAppl ScL, 26 (12B) 2011

treatments were increased all over the storage period and this could beattributed to the activity of starter culture which were confirmed by manyworkers (Ismaiel et a/., 2006 and El-Nagar et aL, 2007).Table (4) Chemical composition of yoghurt produced by some natural

preservative during storage period at 5°C.

Properties

acidity %

Fat%

Totalsolids%

Protein %

Ash%

TVFAcontent

Acetalde-hyde

content

Storageperiod(days)Fresh

7142128

Fresh7142128

Fresh7142128

Fresh7142128

Fresh7142128

Fresh7142128

Fresh7142128

Cont

0.80a

0.86 a0.90abspoiledspoiled3,32a3.36a3.43abspoiledspoiled13.79a14.10a14.38aspoiledspoiled3.51a3.82a3.93a

spoiledspoiledp.84ab0.89a0.92abspoiledspoiled4.93e6.33cd7.66e

spoiledspoiled1.93ab3.15a1.18c

spoiledspoUed

Ti

0.71 cd

0.78e0.81 cd0.83 ab0.85b3.20cd3.20bc3.30cd3.38a3.43b13.75a13.91ab14.25ab14.10a13.97a3.27a3.67a3.81a3.73a3.67b0.85a0.88ab0.89ab0.92a0.93b535a6.65td8.20de9.80bc12.10b1.78ab2.77a1.48b1.14b0.91c

T,

0.68 d

0.72 f0.76 d0.78 b0.79 c3.15d3.18c3.25d3.30a3.36c13.74a13.77b14.01b14.01a14.01a3.45a3.68a3.85a3.78a3.54c0.78b0.82b0.89ab0.88a0.91d4.93a5.93d7.13e8.86c

12.80ab1.82ah3.19al.S7bl.OSb0.90c

T3

0.75ab

0.81 c0.87 be0.85 a0.87ab3.29ab3.30ab3.37abc3.37a3.51a13.82aI4.12a14.3 la14.04a14.06a3.958

3.84a3.90a3.84a

3.62b0.83ab0.87ab0.89ab0.88a0.94a7.70b9.23b10.50b11.33a14.05a1.72ab3.59a1.46b1.06b0.97c

T4

0.74abed

0.81 c0.85 be0.86 a0.89 ab3.29ab3.32a3.39ab3.39a3.51a13.78a14.11a

14.23ab13.91 a13.92a3.57a3.79a3.93a3.99a3.68b0.84a

0.86ab0.90ab0.87a0.91c7.26bc8.80b9.80b

10.73ab13.25ab1.56b4.01a1.65bl.SOa1.261)

T5

0.74 bed

0.80 d0.84 be0.87 a0.89 ab3.2Sabc3.25abc3.36bc3J8a3.51a13.34b13.70b14.13ab14.20a14.06a3.27a3.71a3.86a3.77a

3.71b0.85a0.88ab0.87b0.90a0.92c6.60b7J5c8.75cd10.26ab12.25ab1.86ab2.90a1.55b1.69ai.26b

Tt

0.74abed

0.80 d0.85 be0.88 a0.92 a3.32a334a3.44a339a3.54a13.66a13.80ab14.30a14.06a14.08a3.50a3.76a3.96a3.77a3.83a0.82ab0.85ab0.87b0.90a0.91c6.76cd8.56b9.73bc10.73ab13.70ab1.97a3.88a2.06a1.82a1.45a

T,

0.79abc

0.86b0.93aspoUedspoiled3.29ab3J2a3.41abspoiledspoiled13.75a13,92ab14.30aspoiledspoiled3.60a3.71a3.88a

spoiledspoiled0.82ab0.85ab0.89abspoiledspoUed15.60a17.13a18.903spoiledspoiled1.95a4.00a2.14a

spoUedspoiled

LSD(5%)

0.055

0.0060.0550.0580.0580.0770.0950.0770.1150.0580.2680.2950.2320.3730.3820.3190.2740.23203940.0810.0550.0550.0550.0580.0060.5390.9801.0001.2051.7230.3281.1400.2510.3250.141

Cont = control Tj= Kombucha 3 % T2 = Kombucha 4%T3 = Propolis 0.1% T4 = Black cumin 0.1% Ts = Parsley 0.05%T6 = Parsley 0.1% T7 = Garlic 0.1%

Statistical analysis of titratable acidity indicated significant differencesbetween all treatments and control along the storage period.Fat content:

Results in Table (4) shows the fat content of yoghurt produced withadding some natural preservatives when fresh and during the storage period at

Egypt. J. ofAppt. ScL, 26 (12B) 2011 351

5°C. The results in general revealed that there was no pronounced differencesbetween control and different treatments of yoghurt either when fresh or in thedifferent storage period.

As storage period progressed the fat content of the control and alltreatments were slightly increased. This slight increase related to the slightincrease in total solids due to the loss of moisture during the storage period.

Similar trends were obtained by Badawi et al (2004) and El-Sonbaty etaL (2008).

Control yoghurt and that treated with garlic (T?) were spoiled after 14days of storage. The prolonged shelf-life of other treatments (Tj to Te) could beattributed to the effect of added natural preservatives. These results wereconfirmed by Burt (2004) and Draughon (2004).

Statistically, there were significant differences in fat % between all thetreatments.Total solids %:

Results in Table (4) recorded the total solids-of yoghurt produced withadding some natural preservatives during storage periods at ~5°C.

It could be noticed from the presented results that there were no effect onthe total solids content among the treatments of the same storage period, andthere was a slight increase either for the control or for the other treatments dueto the storage. This could be attributed to the loss of moisture during storage.These results are in agreement with those obtained by Badawi et aL (2004),Kebary et aL (2004), EI-Nagar et al (2007) and El-Sonbaty et al. (2008) whoreported that there was a slight increase in total solids of yoghurt due toevaporation of some moisture during storage.

According to statistical analysis of total solids, there were no significantdifferences between all treatments with exception of T2.Protein content:

Table (4) shows the protein content of yoghurt produced with addingsome natural preservatives during storage period at 5°C. It could be observedslight differences between all treatments when fresh. While, the protein contentof yoghurt was slightly increased during storage period. The slight increaseobtained could be related to the slight increase of the total solids among thedifferent treatments during storage, hence it could be attributed to the lake oflost moisture along the storage due to evaporation. These results are inagreement with Kebary et al. (2004), Mahmoud (2005) aad EI-Nagar et aL(2007).

Statistical analysis of protein content cleared that there were nosignificant differences between all treatments and among the storage periods.Ash content:

Concerning ash content of yoghurt produced with adding some naturalpreservatives during storage period at 5°C are presented in Table (4). Theresults cleared that there was no pronounced effect on the ash content due to

352 Egypt. /. ofAppl. ScL, 26 (12B) 2011

the added natural preservatives when fresh and after storage period, and thiswas in accordance with the results of Kebary et aL (2004) and Badawi et al.(2004). On the other hand, there was slightly increase in ash content byadvancing the storage period. The slight increase related to changes in the totalsolids which could be attributed to the loss of moisture by advancing thestorage period. These results are in accordance with El-Nagar et al. (2007).

There were no significant differences between treatments for .ash content,however there were significant differences for TS and Tg with 28 days ofstorage.Total volatile fatty acids (TVFA):

Table (4) represent the total volatile fatty acids values of yoghurt madewith adding some natural preservatives during storage period at 5°C.

There was pronounced differences among yoghurt treatments when freshand all of them that recorded a pronounced increase than the control. Thisindicated that TVFA contents were affected by the type of added naturalpreservatives. The highest value was recorded by yoghurt made with garlicessential oil which recorded 216.43% increase than the control while the lowestvalues were recorded for Kombucha. During the storage period the TVFAvalues were gradually increased. These results are in agreement with the resultsobtained by Ismaiel et al. (2006).

Statistical analysis of TVFA values clears that there was significantdifferences between all yoghurt treatments.Acetaldehyde content:

The main effective compound in yoghurt flavour is acetaldehyde, whichrealized during the metabolism of microorganisms especially lactic acidbacteria.

The effect of adding some natural preservatives on acetaldehyde contentof yoghurt when fresh and during storage period at 5°C are presented in Table(4). Acetaldehyde values were slightly higher in control than the othertreatments except Te and Ty, the lowest values were recorded for T4 (1.56mg/100 g), and the highest value was recorded for T6 (1.97 mg/100 g), whichmay be attributed to the effect of added essential oils and natural agents andtheir effect on retarding the growth of lactic acid bacteria and hence delayedacid and acetaldehyde production. Similar results were recorded by Ismaiel etal. (2006).

Acetaldehyde content increased within the first week and then there wasnoticeable decrease in acetaldehyde content by progressing storage of yoghurt.This may be due to the demonstrated ability of numerous lactic acid bacteria toconvert the acetaldehyde to ethanol (Salama, 1993 and El-Nagar etal, 2007).

Egypt. J. ofAppL ScL, 26 (12B) 2011 353

The data of acetaldehyde contents were analyzed statistically and theanalysis revealed that there were significant differences between all thetreatments.MICROBIOLOGICAL EXAMINATION:Lactic acid bacteria (LAB):

Fig. (1) indicated the count of LAB of different yoghurt treatments madewith different natural preservatives. It was observed a little decrease in LABcount in all the treatments than the control. This may be attributed to thevariation in the affected compounds of the nataral preservatives (AbouDawood, 2002 and Ismaiel etal., 2006). A little increase in LAB counts wasclear till 7 days and started to decrease due to the increase of acidity, theseresults were in agreement with Barrantes et al. (1994).

Concerning the coliform bacteria, it was not drtected in all the yoghurtsamples, which due to the sanitation conditions duing making of yoghurtexperiment. Also yeast & mould were not detected in all yoghurt sampleseither when fresh or during storage period except few samples had < 10 cfii/g,which may be due to the post contamination during the storage.

From the microbiological side of view it could be concluded that thenatural preservative materials in this stidy have a little effects on LAB, and canextend the shelf life of yoghurt up to X days. These results are closely agreedwith Abou Dawood (2002) and Ismaie et al. (2006).

Fresh 'diys 14 days 21 daysStorage period

Fig. (1): Effect of some natua preservatives on ^la^&cJdcmof yoghurt durii> storage period.

28 days

354 Egypt. J. ofAppL ScL, 26 (12B) 2011

RHEOLOGICAL PROPERTIES:A- Firmness:

The penetrometer data in Table (5) indicate the penetrometer of yoghurtmade with adding different natural preservatives.

It was clear that adding of different natural preservatives may affect thephysical characteristics of yoghurt. Among the different treatments, there wereslight differences between, the treatments of yoghurt firmness when fresh. Byadvancing storage there were slight increase for the curd firmness, which wasdue to somewhat slight increase in the product's dry matter. These results arein agreement with El-Nagar (2002) and Shenana et al. (2007).

Significant differences were observed for penetrometer reading eitherbetween treatments when fresh and during storage periods.b- Curd synersis (wheying off):

Data in Table (5) showed the curd synersis of manufactured yoghurtmade with different natural preservatives. The obtained data cleared that wheyoff of fresh samples were varied depending on the added materials. The highlevel of wheying off was recorded for Ta when fresh and during storage.

By advancing storage, there was a noticeable decrease in the synersis for allsamples until the end of storage. These results agree with Basyouny et a/. (2002)and El-Nagar (2002).

Statistical analysis of curd synersis data cleared that there were nosignificant differences either between the treatments or among storage up to 28days.Table (5) Rheological properties of yoghurt produced by some natural

preservative during storage period at 5°C.penetrometer reading (0.1 mm/s)

Storageperiod(days)Fresh

7142128

Cont

250f

240"

252f

spoiledspoiled

T,

251e

242g

236"245'255"

T2

250f

239'230s

240f

251'

CurdsFresh

7142128

35J2bc

36.971

31.08C

SpoiledSpoiled

38.90"38.67"34.67"33.67"b

34.09"

45.98"42.681

37.82"37.24"34.36"

T3

255'253"

245a

251"266"

T4

257"250C

243"252C

259C

Ts

255C

246d^237C

254"

268"

T«

253d

243f

2308

253"255"

T7

258*251"

236"spoiledspoiled

LSD5%

0.0050.5770.0050.0060.006

vnersis 120 min (g/ lOOg)38.58°38.64s

33.06C

34.45'b33.44"

40.59"40.09"36.87"37.62"32.39"

38.41U

38.23"32^89°35.J9*"33.13*

36.20bc

39.06"33.22°32.95"33.07"

38.37"37.08"32.48*SpoiledSpoiled

4.7238.0991.9933.9292.095

Cont = control Tj= Kombucha 3% T2 = Kombucha 4%T3 = Propolis 0. 1% T4 = Black cumin 0.1% T5 = Parsley 0.05%T6 = Parsley 0.1% T7 = Garlic 0.1%

Egypt. J. ofAppL ScL, 26 (12B) 2011 355

ORGANOLEPTIC PROPERTIES:The sensory profile data of the manufactured yoghurt are shown in Table

(6). The total high score was recorded for TI and T2, either when fresh orduring storage period.

The high score of TI and T2 is due to addition of Kombucha to theproduct as a bio-preservative. The Kombucha contain different volatilecompounds which give more flavour Le. acetic acid, folic acid, gluconic acid,glucoronic acid, usnic acid, and lactic acid. Most of leaking was of T? flavourdue to using of garlic which may be reduces the yoghurt flavour.Table (6). Organoleptic properties of yoghurt produced by some natural

preservative during storage period at 5°C.Properties

Flavour

(30)

Odour

(20)

After taste

(10)

Body&texture

(30)

Appearance

(10)

Total

(100)

Storageperiod (days)

Fresh

7

14

21

28

Fresh

7

14

21

28

Fresh

7

14

21

28

Fresh

7

14

2j28

Fresh

7

14

21

28

Fresh

7

14

21

28

Cent.

2536'

26.20"

2650H

Spoiled

Spoiled

17.43""

IS-loV

18.16*

Spoiled

Spoiled

7.83'

8.16b

8.20"

Spoiled

Spoiled

2630'

26.96*

27.70'

Spoiled

Spoiled

9.00"

9.00'

9.00"

spoiled

spoiled

85.92'

8848"c

8956"'

Spoiled

Spoiled

T,

27.15'

28.05*

28.25'

28.75'

2830"

18.00'

19.00' _

19.00'

19.00"

1850*

8.60'

9.00'

9.00'

9.00*

830*

26.15'

26.95"

27.55'

27.50*

27.00'

9.00"

9.00'

9.00'

9.00'

9.00'

88.90*"

92.00'

92.80"

93.25"

91.10*

T2

27.26"

27.96"

28.50'

28.50"

2850*

18.16*

18.86'

18.83*

19.00"

19.00"

8.50'

9.00'

9.00'

8.95"

830'

26 JO"

27.23'

27.86"

28.00"

27.00"

9.00"

9.00'

9.00'

9.00*

9.00'

89.22"

92.05"

93.19*

93.45*

90.55"

T,

26.63ab

2736"

28.03"

27.83"

24.75"

17.80*

18.56*

18.83*

18.93*

18.65*

8.40*

8.83*

9.00'

8.91"

830"

2633"

27.13'

27.73'

27.91"

27.00"

9.00"

9.00"

9.00"

9.00"

9.00"

88.16*

90.88*

92.59"

9258a"

V.W

X,

25.%"*

26.70'

27.16b

2736"

27.00*

17.80*"

1836*

18.50*

18J6b

18.300'

8.06"

8^3l

8.56ab

8.56a"

8JO*

26JO*

27.00*

27.40"

27.66*

27.12"

9.00*

9.00'

9.00'

9.00"

8.75b

87.12"

89J9"

90.62b

91.14"

89.4711

T,

25.45'

26.20"

27.001*

27.ir

26.70*"

le-so"1

18.00**

18JO*

18.00"

18J5"

7.65'

8.00"

8.00"

8.50"

7.92*

26.20'

26.95*

2755"

27.50*

26.951

7^5b

850"

8.00"

8.00*

8.00'

83.65*

87.65°

88.85*

89.12'

87.82'

Tj

25.00'

25.86"

26J3"

26.73'

26.12"

H-OO1""

17.43'

17^6b'

18.41"

I7.75C

7.43'

7.70"

7^6"7.9011

7.75b

2640"

27.06"

27.53"

27.93"

27.12"

6.73'

7.16'

7J3'

7J3'

7.00"

82.56*'

85J1"

86.91*

8830'

85.74"

T,

2330"

24.50'

2533'

Spoiled

Spoiled

16J6"

16-M»"

17.10c

Spoiled

Spoiled

6^3"

6.93'

6.90'

spoiled

spoiled

263.V

26.96'

2736*

spoiled

spoiled

9.00'

9.00'

9.00"

spoiled

spoiled

8132'

83.89"

85.69*

Spoiled

Spoiled

LSD

(5%)

0.730

0363

0.607

0567

1.671

0.730

0.778

0.921

0495

0558

0483

0531

0.648

0511

0377

0561

0342

0.661

1.133

0.970

0391

0333

0333

0431

0.182

1.883

, 1344

1.655

1554

L 1472

Cent = controlT3 = Propolis 0.1%T6 = Parsley 0.1%

TI= Kombucha 3%T4 = Black cumin 0.1%T7 = Garlic 0.1%

T2 = Kombacha 4%T5 = Parsley 0.05%

356 Egypt, /. ofAppl ScL, 26 (12B) 2011

From statistical analysis of flavour, it was clear that there was nonesignificant differences between T\d T2 among the storage up to 28 days. Butthe significant differences were observed between the rest of treatments alongthe storage periods.

With regard to body & texture the sequence of yoghurt samples had thesame observation for flavour, either when fresh or during intervals of thestorage periods. The organic acids in added Kombucha help in formulation ofyoghurt consistency. These results are similar to Ismaiel et al (2006), Shenanaetal., 2007 and Milanovic etcd, (2008).

From the statistical analysis of the body & texture data there were nosignificant differences between all the treatments during storage period.

REFERENCES

Abou Dawood S.A.I. (2002). Sensitively of yeast flora of Labneh to spices.Egyptian J. Dairy Sci., 30: 35.

AOAC (1990). Official Method of Analysis. 15th Ed, Association of OfficialAnalytical Chemists INC., Arlington, Virginia, USA.

APHA (American Public Health Association) (1992). Standard Methods forthe Examination of Dairy Products. 16th ed. American Pub. HealthAssociation, Washington.

Badawi RJVL; K.M.K. Kebaiy and S.A. Hussein (2004). Impact of substitutingnon-fat dry milk with pectin on cow's milk yoghurt quality. The 9Egyptian Conf. for Dairy Sci. & Tech "mill:, and dairy products for ahealthy future" Dokki, 9-11 October, Cairo, Egypt.

Barrantes E.; A. Y. Tamime; D.D. Muir and A.M. Sword (1994). The effect ofsubstitution of fat by microparticulate whey protein on the quality ofset-type, natural yoghurt. Journal of the Society of Dairy Technology,47:61-68.

Basyouny A.E.; H.A. Abd El Rahman and N.H.A. Assem (2002). Production ofyoghurt-like using Hull-less barley as substitution material. Proc. 9th Int.Conf. Food Industries Quality Control., 617-635.

Burt S. (2004). Essential oils: their antibacterial properties and potentialapplications in foods: a review. Int. J. Food Microbiol., 94:223.

Clarke G.M. and R.E. Kempson (1997). Introduction to the design and analysisof experiments. Arnold, a Member of the Holder Headline Group, 1st

Edt., London, UK.Cosentino S;; A. Barra ; B. Pisano ; M. Cabizza ; F.M. Pirisi and F. Faunas

(2003). Composition and antimicrobial properties of Sardinianjuniperus essential oil against food-borne pathogenic and spoilagemicroorganisms, J. Food Prot., 66: 1288-1291.

Egypt. J. ofAppl. ScL, 26 (12B) 2011 357

Dave, R.I. and N.P. Shah (1998): Ingredient supplementation effects onviability of probiotic bacteria in yoghurt. J. Dairy Sci, 81(11): 2804-2816.

Dixon, B.D. and J.V. Parekh (1980): The use of cone penetrometer for testingthe firmness of butter. J. Texture Studies, 10(4): 421-434.

Draughon F.A. (2004). Use of botanicals as biopreservatives in food. FoodTechnol, 58: 2-20.

E!-Gayyar M. ; F.A. Draughon ; D.A. Golden and J.R. Mount (2001).Antimicrobial activity of essential oils from plants against selectedpathogenic and saprophytic microorganisms. Journal of FoodProtection, 64: 1019-1024.

Elliker, P.R. ; A.W. Anderson and G. Hannesson (1956): An agar culturemedium for lactic acid streptococci and lactobacilli. J. Dairy Sci., 39:1611-1612.

El-Nagar G.F. (2002). Effect of exopolysaccharide-produced by lactic acidbacteria on rheological and sensory properties of low-fat yoghurt.Annals of Agriculture Science, Moshtohor, 40: 335-350.

El-Nagar G.F. ; M.B. El-Alfy ; M.F. Younis and A.A. Atallah (2007). Use ofbacteriocins produced by some LAB as a natural preservative inyoghurt. Annals of Agric. Sc., Moshtohor, 45: 1497-1510.

El-Sonbaty A. H.; K. M. K. Kebary ; R.M. Badawy and A. E. Hweda (2008).Effect of stabilizers on the quality of stirred yoghurt. Egyptian J. DairySci., 36: 185-195.

Fu, YJ.; Y.G. Zu ; L.Y. Chen ; X.G. Shi; Z. Wang ; S. Sun and T. Efferfii(2007): Antimicrobial activity of clove and rosemary essential oilsalone and in combination. Phytotherapy Res., 21(10): 989-994.

Gayoso C.W. ; E.G. Lima ; V.T. Oliveira ; F.O. Pereira ; E.L. Souza ; I.O.Lima and D.F. Navarro (2005). Sensitivity of fungi isolatedonychomycosis to Eugenia cariophyllata essential oil and eugenol.Fitoterapia, 76: 247-249.

EDF (1990). International Dairy Federation, Milk and milk products.Enumeration of yeasts and moulds colony count technique at 25°C.IDF 94 B.

IDF (1991a). International Dairy Federation, Milk and milk productsDetermination of fat content. General Guidance on the Use ofButyrometric Methods IDF: 151.

IDF (1991b). International Dairy Federation, Yoghurt: Determination of totalsolids content IDF: 151.

IDF (1993). International Dairy Federation, Milk: Determination of nitrogencontent IDF: 20B.

358 Egypt. J. ofAppl. Sci, 26 (12B) 2011

IDF (1995). International Dairy Federation, Sensory evaluation of dairyproducts Reference method. IDF: 99k

I mclouane, B.; H. Ahmadi ; J.P. Wathelet; M. Ankit; K. Khedid and A.Elbachiri (2009). Chemical composition and antimicrobial activity ofessential of thyme (Thymus vulgaris) from Eastern Morocco. Int. J.Agri.Biol.,11:205-208.

Ismail, A. and D.M. Pierson (1990). Inhibition of growth and germination of C.botulinum 33 A, 40B, and 1623 E by essential oil of spices. J. FoodSci., 55:1676.

Ismaiel, A.M. ; S. Harby and A.S. Salem (2006). Production of flavouredLabneh with expended shelf life. Egyptian J. Dairy Sci., 34: 59-68.

Kebary, K.M.K. ; S.A. Hussein and R.M. Badawi (2004). Impact offortification of cow's milk with a modified starch on yoghurt quality.Egyptian J. Dairy Sci., 32: 111-124.

Kosikowski, F.V. (1997): Cheese and fermented Milk Foods. Printing BrookTonalds, 3rd Ed., 1058pp. New York 14817, USA.

Lees, G J. and G.R. Jago (1969). Methods for the estimation of acetaldehyde incultured dairy products. Aust. J. dairy Tech., 24:181.

Mahmoud S.F. (2005). Influence of substitution non-fat dry milk with locustbean gum on cow's milk yoghurt quality. Minufya J. Agric. Res., 30:77. " •

Marques, A. ; S. Encarnacao ; S. Pedro and M.L. Nunes (2008): In vitroantimicrobial activity of garlic, oregano and chitosan. World J.Microbiol Biotechnol, 24(10): 2357-2360.

Milanovic S.D,; E.S. Loncar; S.D. Mirjana ; V.M. Radomir ; N.T. Midorag;D.I. Mirela and G.D. Katarina (2008). Low energy Kombuchafermented milk-based beverages. BIBLID, 39: 37-46.

Misic, D.; I. Zizovic ; M. Stamenic ; R. Asanin ; M. Ristic ; S.D. Petrovic andD. Skala (2008): Antimicrobial activity of celery fruit isolates andSFE process modeling. Biochemical Engineering Journal, 42(2): 148-152.

Miyasaki K.T. ; M.E. Wilson ; H.S. Reynolds and RJ. Genco (1984).Resistance of ActinobacUlm actiiwmycetemc&mi&ais sod differentia'susceptibility of oral Hoemophilus species to the bactericidal effectsof hydrogen peroxide. Infect. Immun., 46: 644-648.

Miyasaki K.T.; RJ. Genco and M.E. Wilson (1986). Antimicrobial propertiesof hydrogen peroxide and sodium bicarbonate individually and incombination against selected oral Gram-negative, facultative bacteria.J. Dent. Res., 65: 1142-1148.

Egypt. J. ofAppL Sci., 26 (12B) 2011 359

Moreira 1VLR.; A.G. Ponce ; C.E. Del Valle and S.I. Roura (2005). Inhibitoryparameters of essential oils to reduce a food-borne pathogenic.Lebensmittel Wissenschaft und Technologic, 38: 565-570.

Olasupo N.A. ; S.M. Akinsanya ; O.F. Oladele and M.K. Azzez (1996).Evaluation of nisin for the preservation of nono-a Nigerian fermentedmilk product. J. Food Processing and Preservation, 20: 71-78.

Rouatbi, M.; A.Duquenoy and P. Giampaoli (2007). Extraction of the essentialoil of thyme and black pepper by superheated steam. Journal of FoodEngineering, 78: 708.

Salama, F.M.M. (1993). Chemical and organoleptic properties of lactosehydrolyzed milk. Egyptian J. Dairy Sci., 21: 273.

Shenana, M.E. ; G.F. El-Nagar ; S. El-Shibiny and S.M. Abdou (2007).Preparation and use of whey protein/carrageenan particulate in makinglow-fat yoghurt. Egyptian J. Dairy Sci., 35: 185-193.

Smith-Palnier, A.; X Stewart and L. Fyfe (1998). Antimicrobial properties ofplant essential oils and essences against five important food-bornepathogenic. Letters in Applied Microbiology, 26: 118-122.

Snoussi, M. ; H. Hajlaoui ; E. Noumi ; D. Usai ; L.A. Sechi ; L. Antonio ;S.A.L. Zanetti, and A. Bakhrouf, (2008): In vitro Antivibrio spp.activity and chemical composition of some Tunisian aromatic plants.World J. Microbiol Biotechnol, 24(12): 3071-3076.

Tamime, A.Y. (1978). The manufacture of yoghurt from hydrolyzed milk. XXInternational Dairy Congress, Paris, p.832.

Tosi, E.A. ; R. Edmundo M.E. Ortega and A.F. Cazzoli (2007). Foodpreservative based on propolis: Bacteriostatic activity of propolispolyphenols and flavonoids uponjE. cott. Food Chemistry, 104, 1025-1029.

Vardar-Unlu, G. ; F. Candan ; A. Sokmen ; D. Daferera ; M. Polissiou ; M.Sokmen ; E. Donmez and B. Tepe (2003): Antibacterial andantioxidant activity of the essential oil and methanol extracts ofThymuspectinatusFisch. et Mey var. pectinatus (Lamiaceae). Journalof Agricultural and Food Chemistry SI, 63-67.

Viljoen, A.; S.V. Vuuren ; E. Ernst; M. Klepser; B. Demirci; H. Baser andB.E Van Wyk (2003): Osmitopsis asteriscoids (Asteriaceae) - theantimicrobial and essential oil composition of a Cape-Duch remedy. J.Ethnopharmacol., 88: 137-143.

.„

360

^k U jib

ikaUJ!

Egypt . J. ofAppl. ScL, 26 (12B) 2011

*JiaUJ J j-11 o^wl UjlSJl

jliiS Jjfr Oxi » t (jR

IJ* U

, - *&l j jll Aj£ - 2jj

j" •— y

il jju AjjiljJ aj t*'^ .(^jfl JU^ ,J ->•'•'•••' jB AjjLuSJI 2U.4UB jljJU 4jjlL«

L&« (_>aij-Vl j JUjil Aji-iJI AjjjjSall tliV -Ji t>ia«J t> (J J JJ' ^L"

subtttis subsp spizizenii ATCC 6633, Escherichia coli ATCC 14169,Streptococcus pyogenes ATCC 21060 and Staphylococcus aureus ATCCduj J gi'iill da^ajl . V'- j-iSjJ JSi j AlaAJI Aaklall ia.U*« AjJjJ J U ^> 29737)

1—ljjS.la (Jt ^» ^ "I j^j Civjl jp ^VinnJI A^LouJI (j-> dlLa.jJ J*-' CJ?^ ' JJC- jH

Staphylococcus )vjjSj.» Y £ (Streptococcus pyogenes ATCC 21060)

uj j- JS Aolj (oMreiis ATCC 29737

.A^UJI ^J (JsVl (%Y .)

Aj*.nLl! A,h°lUJI Jjj! aift jilj Ajjljj aj

. i V>< j Aja.jlj.ijll j

(%£-r) LSj l JL.

Ajjla-iSJI

J a

u j

w i

Cuj j

AJILI A-u-aJl j Aja.jlj.ijl! AjaAill J« CjlL-oll Joa. ( jUj tij! (% . 4 1 - . 4

Y A .Jia.