GIDA (G›da Teknolojisi Derne¤i Yay›n›) THE JOURNAL OF FOOD (Published by the Association of Food Technology; Turkey) Cilt / Volume: 39 • Say› / Number: 4 • 2014 ‹ki ayda bir yay›mlan›r / Published bimonthly ISSN 1300 - 3070; ISSN 1309 - 6273 (GIDA on-line) Sahibi / Owner G›da Teknolojisi Derne¤i Ad›na / On behalf of the Association of Food Technology; Turkey Prof. Dr. A. Kadir HALKMAN Yönetim Kurulu Baflkan› / President of the Association Editörler Kurulu / Editorial Board

Danışma Kurulu / Advisory Board

Baş Editör/ Editor-in Chief Halkman, A. Kadir Ankara University, Turkey

Alichanidis, Efstathios Aristotle University of Thessaloniki, Greece Aran, Necla Istanbul Technical University, Turkey Art›k, Nevzat Ankara University, Turkey Baysal, Taner Ege University, Turkey Boyac›, ‹smail Hakk› Hacettepe University, Turkey Certel, Muharrem Akdeniz University, Turkey Draughon, Ann Tennessee University, USA Ekfli, Aziz Ankara University, Turkey El Soda, Morsi University of Alexandria, Egypt Fogliano,Vincenzo University of Napoli Federico II, Italy Ghosh, Bikash C. National Dairy Research Institute, India Gollop, Natan The Volcani Center, ARO, Israel Gökmen, Vural Hacettepe University, Turkey Griffiths, Mansel University of Guelph, Canada Gö¤üfl, Fahrettin Gaziantep University, Turkey Gümüflkesen, Aytaç Sayg›n Ege University, Turkey Güven, Mehmet Cukurova University, Turkey Heperkan, Dilek Istanbul Technical University, Turkey Ho, Chi-Tang The State University of New Jersey, USA Kaya, Mükerrem Atatürk University, Turkey Kaymak-Ertekin, Figen Ege University, Turkey Koçak, Celalettin Ankara University, Turkey Köksel, Hamit Hacettepe University, Turkey Morales, Francisco J. CSIC Instituto del Frío, Spain Mujtaba, Mustafa G. Florida Gulf Coast University, USA Ögel, Zümrüt Middle East Technical University, Turkey Özilgen, Mustafa Yeditepe University, Turkey Paalme, Toomas Tallinn University of Technology, Estonia Parlar, Harun Technical University of Munich, Germany Raspor, Peter University of Ljubljana, Slovenia Rezessy-Szabo, Judit M. Corvinus Universty of Budapest, Hungary fiahin, Serpil Middle East Technical University, Turkey Üstünol, Zeynep Michigan State University, USA Yetiflemiyen, Atila Ankara University, Turkey

Editörler / Co-Editors Çak›r, ‹brahim Abant ‹zzet Baysal University, Turkey Taban, Birce Ankara University, Turkey Tekin, Aziz Ankara University, Turkey Velio¤lu, Y. Sedat Ankara University, Turkey Yönetim Yeri Adres / Address Büyükelçi Sokak No: 18/1 Kavakl›dere/ Ankara Turkey Tel: (+90) 312 596 1180 • Faks: (+90) 312 317 8711 E-posta / E-mail: [email protected] URL: http://www.gidadernegi.org/dergi.asp Yayın Türü: Yayg›n süreli ve hakemli Basım Yeri / Printing House Sim Matbaac›l›k Ltd. fiti ‹vedik Organize San. Böl. Mat-Sit ‹fl Mrk. 1518. Sk. No:2/14 Yenimahalle / Ankara Turkey Tel : (+90) 312 230 22 09 Faks: (+90) 312 230 41 39 e-mail: [email protected]

Yayın Tarihi / Publication Date 15 08 2014

Bu dergi, uluslararas› CAB Abstracts, Citefactor, Index Copernicus, EBSCO, ULAKBİM (Yaflam Bilimleri) FAO Agris ve DOAJ veri tabanlar› kapsam›ndad›r. This journal is covered by CAB Abstracts, Citefactor, Index Copernicus, EBSCO, ULAKBİM (National Databases) FAO Agris and DOAJ database systems.

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İçindekiler / Content

Tuncer BÖ, Tuncer Y; Exopolysaccharide Producer Streptococcus thermophilus St8.01 Strain; A Potential Probiotic Culture / Ekzopolisakkarit Üreticisi Streptococcus thermophilus St8.01 Suflu; Potansiyel Probiyotik Kültür . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .195-202 Baysal T, Ergün AR, Akgün B, Karc› G, Kaplan N; Dondurma ‹flleminde Baz› ön Uygulamalar›n Çilek ve Mandalinan›n Kalite Özelliklerine Etkilerinin ‹ncelenmesi / Determining the Effects of some Pretreatments on the Quality Characteristics of Strawberry and Mandarin in Freezing Process . . . . . . . . . . .203-210 K›l›ç Y, Yüksekda¤ ZN, Yüksekda¤ H; Lactobacillus ve Bifidobacterium Cinsi Bakterilerin Beta Galaktosidaz Enzim Aktivitelerinin Belirlenmesi / Beta Galactosidase Enzyme Activities of Lactobacillus and Bifidobacterium Genus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .211-218 Tahmaz H, Söylemezo¤lu G; Farkl› Vinifikasyon Tekniklerinin Kalecik Karas› fiaraplar›ndaki Fenolik Bileflik ‹çeriklerine Etkisi / Effects of different Vinification Techniques on Phenolic Compounds In Kalecik Karasi Wines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .219-226 Kamilo¤lu S, Pasl› AA, Çapano¤lu E, Özçelik B; Kuru Meyvelerin Kuruyemifller ile Birlikte Tüketiminin Flavonoidlerin in vitro Biyoyararl›l›¤›na Etkisinin ‹ncelenmesi / Investigating the in vitro Bioavailability of Flavonoids During Consumption of Dried Fruits with Nuts . . . . . . . . . . . . . . . . . . . . . . . . . . .227-233 Büyüks›r›t T, Kuleaflan H; Fourier Dönüflümlü K›z›lötesi (FTIR) Spektroskopisi ve G›da Analizlerinde Kullan›m› / Fourier Transform Infrared (FTIR) Spectroscopy and Utilization in Food . . . . . . . .235-241 Arslan S, Erbafl M; Selüloz ve Selüloz Türevi Diyet Liflerin Özellikleri ve F›r›n Ürünlerinde Kullan›m ‹mkanlar› / The Properties of Cellulose and Derivatives Dietary Fibers and Useability in Bakery Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .243-250 Söbeli C, Kayard› C; Et Kalitesini Belirlemede Yeni Teknikler / New Techniques for Predicting Meat Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .251-258

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Editörden, Merhaba, GIDA Dergisi Y›l 2014; Cilt 39; Say› 4 [Temmuz-A¤ustos 2014] 2014 Haziran ortas›na gelmeden elektronik ortamda yay›mland›. Bir di¤er deyifl ile henüz 3. say› matbaaya gitmeden önce 4. say›m›z da haz›rland›. Çok muhtemelen meslektafllar›m›za bas›l› dergi olarak 39/3 ve 39/4 say›lar› Temmuz 2014 bafl›nda beraberce gönderece¤iz. GIDA Dergisi 2014 y›l›; Cilt 39; Say› 4'te 1 ‹ngilizce ve 4 Türkçe araflt›rma makalesi ile 3 derleme makalesi var. Dikkat çekebilir: ‹lk olarak 2013 y›l›; 38. cilt; 3. say›da DOI numaral› 3 makale yay›mlad›k. Devam›nda 4. say›da DOI numaral› 5 makale ve sonras›nda her dergide DOI numaral› 8 makale olmak üzere bu güne geldik ama bu say›da DOI numaras› alm›fl makale say›s› 7’dir. Yani bir eksi¤imiz bulunuyor. DOI numaras›, bir anlamda uluslararas› geçerli olan bir makale kimli¤idir. GIDA Dergisi olarak DOI numaras›n› çok yaklafl›k 1 y›l süre ile ticari bir kurulufltan ald›k. Sonra kendimizin do¤rudan DOI numaras› almas› gereklili¤i ortaya ç›kt›. Gereken baflvurular›m›z› yapt›k ama hemen arkas›ndan baflka bir sürece girdik. DOI numaras› ile ilgili bu süreç içinde birkaç aksama olacak gibi görülüyor ancak elimizden gelen gayreti gösteriyoruz. Önümüzde 05-07 Kas›m 2013 tarihlerinde Kufladas› Pine Bay Otelde yapaca¤›m›z 2. Uluslararas› G›da Teknolojisi Kongresi oldu¤unu ve http://www.intfoodtechno2014.org adresinde kay›tlar›n devam etti¤ini hat›rlatmak isterim. Kongre için bildiri özeti gönderilme süresi bitmifl de¤ildir ve bildiri özetleri gelmeye devam etmektedir. Sadece geç gönderilen bildiri özetleri kabul edilirse, yazarlar›n›n erken kay›t ödeme flans› azalmaktad›r.

Sevgi ve sayg›lar›mla, Prof. Dr. A. Kadir Halkman

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A Message from the Editor-in-Chief Hello, The forth issue of volume 39 of the year 2014 has been published in electronic media before the midst of June. In other words; forth issue has also been prepared before the printing of the third issue. We will very probably send the printed 39/3 and 39/4 issues together to our colleagues at the beginning of July 2014. In addition to 1 research article in English and 4 research articles in Turkish, there are 3 review articles in the forth issue of volume 39 of the year 2014. It may receive attention: We published 3 articles with DOI number in the third issue of volume 38 of the year 2013. Subsequently, there are 5 articles with DOI in the forth issue and thenceforth we have come with 8 articles with DOI in each issue, but the number of article with DOI number in this issue is 7. In other words, we have one absent. DOI number, in a sense, is an article identifier that is valid internationally. The FOOD Journal has received its DOI number from a commercial entity for approximately 1 year. Then, the necessity of getting the DOI number directly by ourselves had come out. We made our application, but we have immediately entered another process after that. It seems that there will be a few disruptions in this process regarding the DOI number, but we strive our best. I would like to remind you once again that there will be the 2nd International Congress on Food Technology at Pine Bay Hotel in Kufladas› on 05-07 November, 2013 and the registration has been continuing at http://www.intfoodtechno2014.org. The time for abstract submission for the Congress has not been finished yet and the submission of abstracts has been continuing. Only if late-submitted abstracts are accepted, the chance of early registration of their authors is decreased.

Best Regards, Prof. A. Kadir Halkman

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GIDA (2014) 39 (4):195-202 doi:

GD14015

Research / Araflt›rma

EXOPOLYSACCHARIDE PRODUCER STREPTOCOCCUS THERMOPHILUS ST8.01 STRAIN; A POTENTIAL PROBIOTIC CULTURE Banu Özden Tuncer1*, Yasin Tuncer1 Faculty of Engineering, Department of Food Engineering, Süleyman Demirel University, Isparta, Turkey

1

Received / Gelifl tarihi: 17.02.2014 Received in revised form /Düzeltilerek Gelifl tarihi: 21.03.2014 Accepted / Kabul tarihi: 24.03.2014 Abstract The aim of this study was to determine the probiotic potential of exopolysaccharide (EPS) producer Streptococcus thermophilus ST8.01 strain. This strain was able to survive at pH 3 and 1% bile salt. Viable counts were enumerated as 4.80±0.04 and 2.11±0.06 log cfu/mL after exposure to 0.4% phenol and gastric juice at pH 3, respectively. Strep. thermophilus ST8.01 was able to grow at 100 mg/L lysozyme and showed to have high autoaggregation (49.55±6.24%) and hydrophobicity abilities (67.23±7.16%). The ST8.01 strain was also found sensitive to most clinically important antibiotics. Results obtained in this study suggest that Strep. thermophilus ST8.01 strain may be used as a probiotic starter culture to produce dairy products. Keywords: Streptococcus thermophilus, exopolysaccharide, probiotic properties, antibiotic susceptibility

EKZOPOLİSAKKARİT ÜRETİCİSİ STREPTOCOCCUS THERMOPHILUS ST8.01 SUŞU; POTANSİYEL PROBİYOTİK KÜLTÜR Özet Bu çal›flman›n amac›, ekzopolisakkarit (EPS) üreticisi Streptococcus thermophilus ST8.01 suflunun probiyotik potansiyelinin belirlenmesidir. Bu sufl pH 3 ve %1 safra tuzunda hayatta kalma yetene¤ine sahiptir. %0.4 fenol ve pH’s› 3’e ayarlanm›fl mide suyu uygulamas› sonras› canl› hücre say›s› s›ras›yla 4.80±0.04 ve 2.11±0.06 log kob/mL olarak ölçülmüfltür. Strep. thermophilus ST8.01, 100 mg/L lizozim konsantrasyonunda geliflebilme ve yüksek otoagregasyon (%49.55±6.24) ve hidrofobisite (%67.23±7.16) yetene¤ine sahiptir. ST8.01 suflu ayn› zamanda klinik olarak önemli olan antibiyotiklere karfl› duyarl› bulunmufltur. Bu araflt›rmadan elde edilen sonuçlar, Strep. thermophilus ST8.01 suflunun süt ürünleri üretiminde probiyotik starter kültür olarak kullan›labilece¤ini düflündürmektedir. Anahtar kelimeler: Streptococcus thermophilus, ekzopolisakkarit, probiyotik özellikler, antibiyotik duyarl›l›k

* Corresponding author/ Yazışmalardan sorumlu yazar [email protected] ✆ (+90) 246 211 1734

(+90) 246 211 1538

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B. Özden Tuncer, Y. Tuncer

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INTRODUCTION

MATERIAL and METHODS

The relation between nutrition and good health has become more interesting during the recent years. Researches have increased to defining of food and food components with this driving force of increasing conscious of population. Probiotic products were defined as foods that contain live microorganisms providing positive effects on health of consumers (1). Especially popularity of dairy products with probiotic bacteria have increased day by day and still be demanded on the functional food market (2). Briefly probiotics are defined as "Live microorganisms which when administered in adequate amounts confer a health benefit on the host" by WHO/FAO (3). Lactic acid bacteria, particularly the Lactobacillus and Bifidobacterium spp. are known as probiotics most extensively studied and widely used in dairy products. Besides these species, member of the genera Enterococcus, Streptococcus and Propionobacterium are also esteemed as probiotics (4). Strep. thermophilus is a common dairy starter extensively used in the production of yogurts, cheese and some fermented milk products. Strep. thermophilus which is recognized as safe (GRAS), can survive in the gastrointestinal system; even though it is known that the ability to attach to intestinal epithelial cells is relatively weak and it could be damaged in gastric acidic condition (5-7). Also it has some inherent functional properties important for food industry such as organoleptic, technological and nutritional advantages (8). Besides these beneficial effects it is indicated that Strep. thermophilus can promote the health of human and animals. Different researchers reported that Strep. thermophilus can improve the intestinal microflora, prevent diarrhea induced by using antibiotics, reduce the lactose intolerance and the risks of certain cancers, ulcers, imflammation, stimulate the immune system, and also it can be used for curing some atopic dermatitis (9-17). In addition many Strep. thermophilus strains have most of the probiotic characteristics (resistance to bile salts, low pH degrees and gastric juice, hydrophobisity activitiy, etc.) desired for starter culture development processes (18, 19). The goal of this study was to determine some probiotic properties and antibiotic susceptibility of Strep. thermophilus ST8.01 which was characterized as exopolysaccharide (EPS) producer previously (20) isolated from homemade yogurt.

Bacterial Strain and Growth Condition Streptococcus thermophilus ST8.01 strain used in this study was previously isolated from homemade yoghurt and characterized as EPS producer (20). Strain ST8.01 was grown in M17 broth (Merck KgaA, Darmstadt, Germany) with 5% glucose (GM17) during the experiments. Stock culture of the strain was maintained at -20 °C in GM17 broth added with 15% glycerol. Bile Salt Resistance and Acid Tolerance Ability of tolerance for bile salts was determined based on the method of Gilliland and Walker (21) with slight modifications. Strep. thermophilus ST8.01 was inoculated (1 %, v/v) in GM17 broth supplemented with 0.3%, 0.5% and 1% (w/v) ox bile (Acumedia, Lansing, Michigan, USA) and without ox bile as a control incubated at 37 °C. Survival of the strain was determined on the GM17 agar as colony forming units (cfu) at 0. h and 24. h. Inhibition (%) was calculated with the following equation: Inhibition % = {[(initial cfu/mL) - (final cfu/mL)] / [initial cfu/mL]} x 100 (1) For detecting of low pH tolerance, the pH of the 2 mL volume of sterile phosphate-buffered saline (PBS) was adjusted to 1, 3 and 5 with 1M HCl and pH 7.2 as control. Overnight culture of ST8.01 strain was harvested by centrifuged at 3000 g (10 min, 4 °C) and washed once in sterile PBS. Pellet resuspended into PBS one-tenth of the culture volume was centrifuged. The 0.1 mL of suspension was added into 2 mL PBS at pH 1, 3 and 5 then incubated at 37 °C. Viable cells were enumerated at 0., 1., 2., 3. and 4. h on GM17 agar plates (22). Tolerance to Simulated Gastric Juice Sterile saline solutiun (0.5%, w/v) with pepsin (Sigma-Aldrich, St Louis, Missouri, USA) (0.3%, w/v) adjusted to pH 2 and 3 were used to simulate gastric condition. 30 mL overnight culture was centrifuged at 6000 g at 5 °C for 20 min. Pellet was washed twice with K2HPO4 (50 mM, pH 6.5) and then resuspended in 3 mL of the same buffer. 1 mL of resuspended culture was centrifuged at 12000 g (5 min, 5 °C) and resuspended in gastric juice (10 mL) pH 2 and 3. At the begining of incubation period and at the end of 3 h incubation

Exopolysaccharide Producer Streptococcus thermophilus... time viable cell counts were performed on GM17 agar (18). Inhibition (%) was calculated with equation 1.

The percentage of cell cerface hydrophobicity was determined using the following equation:

Resistance to Lysozyme and Survival in The Presence of Phenol

Antibiotic Susceptibility

The effect of lysozyme on the growth on Strep. thermophilus ST8.01 was examined by the method of Brennan et al. (23). The activated culture was inoculated (2%, v/v) into GM17 broth with and without 100 mg/L lysozyme (Sigma-Aldrich). Bacterial cells were counted on GM17 agar plates at 0., 3. and after 24 h incubation at 37 °C. Survival of the Strep. thermophilus ST8.01 in the presence of phenol was determined based on the method of Teply (24). Overnight culture was inoculated (2%) into 10 mL GM17 broth with and without 0.4% phenol and incubated at 37 °C. Viable cell numbers were determined at 0., 3. and 24. h of incubation on GM17 agar plates. Inhibition (%) was calculated with equation 1 for phenol treatment. Increase (%) was calculated with equation 2 for lysozyme treatment: Increase % = {[(final cfu/mL) - (initial cfu/mL)] / [final cfu/mL]} x 100 (2) Autoaggregation Assays Overnight culture was harvested by centrifugation at 7000 g (10 min, 20 °C) and washed with sterile saline (0.85% NaCl, w/v). Cell consantration was adjusted A660 nm = 0.3 within sterile saline and incubated at 37 °C for 60 min. The end of the incubation period suspension was centrifuged at 300 g (2 min, 20 °C) and absorbance of the supernatant (A60) was measured (25). Autoaggregation was calculated with the following equation: Autoaggregation = (A0 – A60) / A0 x 100

(3)

Hydrophobicity Bacterial culture was centrifuged at 5000 g for 15 min after growth in GM17 broth overnight. Pellet was washed twice in sterile PBS and resuspended in KNO3 (0.1 M, pH 6.2) then absorbance of the cell suspension was adjusted at A0 = 0.5-0.6 (600 nm). 1 mL of xylene was added to cell suspension and incubated at room temperature for 10 min. After this preincubation time suspension was vortexed (2 min) to mix the two phase system and incubated at room temperature. At the end of the 20 min, aquase phase was removed carefully and its absorbance was measured at 600 nm (A1) (26).

Hydrophobicity = 1 – (A1 / A0) x 100

(4)

Disc diffusion method was used to determine antibiotic susceptibility of Strep. thermophilus ST8.01 against vancomycin (30 µg, Oxoid Ltd, Wade Road, Basingstoke, Hants, UK), tetracycline (30 µg, Oxoid), streptomycin (300 µg, Oxoid), penicillin G (10 units, Oxoid), erythromycin (15 µg, Oxoid), ampicillin (10 µg, Oxoid), chloramphenicol (30 µg, Oxoid), gentamicin (120 µg, Oxoid), norfloxacin (10 µg, Oxoid) and sulphamethoxazole/ trimethoprim (1.25+23.75 µg, Oxoid). Inhibition zones were measured as diameter (mm) and results were expressed as susceptible, moderate susceptible and resistant by comparing with the interpretative zone diameters given by Clinical and Laboratuary Standards Institute (27).

RESULTS and DISCUSSION Bile Salt Resistance and Acid Tolerance Bile tolerance has been described as an important factor for the survival and growth of LAB in the intestinal tract (21). Probiotic strains must be resistant to bile salt at 0.3% if these are to be used for human beings (28). In our study Strep. thermophilus ST8.01 maintained the viability at three concentrations of bile salt (0.3%, 0.5% and 1%, w/v) after 24 h incubation. The highest inhibition percentage of this strain was detected as 38.34 at 1% bile salt (Figure 1). Similar to our results, Iyer et al. (29) showed that two Strep. thermophilus strains survived at 0.5%, 1% and 2% bile salt concentrations after 180 min. Conversely Vinderola and Reinheimer (18) informed that bile resistance of Strep. thermophilus strains was poor

Figure 1. Effect of bile salt concentrations on the viability of Strep. thermophilus ST8.01 at 37 °C for 24 h.

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B. Özden Tuncer, Y. Tuncer and most of the strains were inhibited at 0.5% bile salt. In addition some researchers indicated that many S. thermophilus were inhibited at 0.15% bile salt (30). Contrary of this statement Strep. thermophilus ST8.01 survived even after 24 h at 1% bile salt as specified by Mahmood et al. (31). The growth of Strep. thermophilus ST8.01 rapidly inhibited at pH 1 at the begining of the incubation and at the end of the two hours ST8.01 was completely inhibited. pH 3 represented less lethal environment to Strep. thermophilus ST8.01 than pH 1 but still cell viability was decreased during incubation and inhibition percentage of this strain was greater than >99.99% at pH 3. However, after 4 h at pH 5 Strep. thermophilus ST8.01 maintained its viability and the inhibition was detected at least (95.43%) for pH 5 among the low pH treatments (Figure 2). Previous studies showed that some Strep. thermophilus strains were dead when exposed to low pH degrees similar with our results (31-34). On the other hand Aswathy et al. (28) reported that most of the lactic acid bacteria isolates including Streptococcus grew at pH 5 and played a role during the fermentation of dairy milk and vegetables.

Vinderola and Reinheimer (18) reported that Strep. thermophilus showed to have very poor survival under simulated gastric conditions. Controversially, Pilar et al. (35) and Iyer et al. (29) reported that Strep. thermophilus maintained the viability under the simulated gastrointestinal stress condition. On the other hand the evidence of viable Strep. thermophilus in human feces and so the transit from the gastrointestinal tract were observed from the volunteers consuming the yogurt samples (6, 7). Based on these data and maintanence the vitality of the strain ST8.01 (2.11 log cfu/mL) when exposed to pH 3 with 0.3% pepsin suggested that it may survive when consumed with fermented milk product such as yogurt but to prove that food applications should be done at the further studies.

Figure 3. Tolerance to simulated gastric juice of Strep. thermophilus ST8.01 at 37 °C.

Resistance to Lysozyme and Survival in The Presence of Phenol

Figure 2. Effect of low pH on the viability of Strep. thermophilus ST8.01 at 37 °C.

Tolerance to Simulated Gastric Juice Gastric juice studies were done in 3 hours incubation for implementing residence time in stomach condition. Among the lactic acid bacteria Strep. thermophilus was known to show more sensitivity to simulated gastric juice (18). Strep. thermophilus ST8.01 showed significant decrease in the viable counts at pH 2 and pH 3 with 0.3% pepsin after 3 h incubation at 37 °C (Figure 3). For simulated gastric juice experiments at pH 2 and pH 3, inhibition percentage of this strain were calculated as >99.99%. Similar to our results,

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In this study we determined that Strep. thermophilus ST8.01 was grown even presence of lysozyme at the level of 100 mg/L (Figure 4). Increase percentages of control sample were calculated as 83.78% and 99.55% after first 3 h and 24 h incubation, respectively. For lysozyme experiment, increase percentages were exhibited as 69.80% and 95.32% after first 3 h and 24 h incubation, respectively. Lysozyme is used as attractive preservative to inhibit food spoilage bacteria which are harmful to human health. It is reported that using lysozyme at the levels up to 25 mg/L does not affect the growth of Streptococci and Lactobacilli. Vinderola et al. (36) determined that growth of the probiotic bacteria was not effected by the lysozyme treatment at the level of 25 mg/L and reported that high levels of lysozyme should be used for selecting probiotic starter bacteria.

Exopolysaccharide Producer Streptococcus thermophilus... On the other hand Xanthopoulos et al. (30) reported that some Strep. thermophilus showed resistance to 100 mg/L lysozyme.

surface (such as hydrophobicity) may affect the autoaggregation ability (40-42). The importance of autoaggregation ability for probiotics is that it might be necessary for their adhesion to the intestinal epithelial cells (43, 44). Based on the previous studies of Canzi et al. (45), Rahman et al. (46), and Kõll et al. (47), Strep. thermophilus ST8.01 has moderate autoaggregation but still this value might be considered to be higher then many of lactic acid bacteria. Hydrophobicity

Figure 4. Resistance to lysozyme of Strep. thermophilus ST8.01 at 37 °C.

Phenols can be occured in the intestines because of the deamination of some aromatic amino acids from the digested foods through the agency of the bacteria. Therefore tolerance to phenols is a desired probiotic characteristic (21, 37). In the present study, growth of the Strep. thermophilus ST8.01 inhibited after 24 h presence of 0.4% phenol but at the end of the first 3 hours of incubation vitality of the strain ST8.01 was still retain (Figure 5). After 3 hours incubation, inhibition percentage of phenol treated sample was calculated as 64.52%. Different researchers reported that some lactic acid bacteria showed high tolerance to phenol (0.2-0.5%) (38, 39) but Strep. thermophilus was known as very sensitive to this chemical (30).

Figure 5. Resistance to phenol (0.4%) of Strep. thermophilus ST8.01 at 37 °C.

Autoaggregation Ability Autoaggragation value of Strep. thermophilus ST8.01 was recorded as 49.55±6.24%. Among LAB various autoaggregation values were determined and it was indicated that the autoaggragation ability might be strain dependent. It was also reported that physochemical characteristics of cell

Determining of bacterial adherence to hydrocarbons was performed as described by Rosenberg (26) which is generally known as MATH or BATH test. Strep. thermophilus ST8.01 was showed 67.23±7.16% affinity to xylene. Previous studies indicated that lactic acid starter species have lower hydrophobicity (under 32%) than other lactic acid bacteria. However most of the probiotic bacteria among the lactic starters have higher affinity to hydrocarbons than 32% (18). Although studies for determining of hydrophobicity of Strep. thermophilus have been rarely done and it is reported that hydrophobicity of Strep. thermophilus varied from 24% to 98% depending on their source (48). In this study hydrophobicity value of Strep. thermophilus ST8.01 was also found higher than many of the Strep. thermophilus strains (67.23±7.16%) as similar with the study of Iyer et al. (29). Antibiotic Susceptibility Strep. thermophilus ST8.01 was assayed to 10 antibiotics using disc diffusion method. Strain ST8.01 exhibited complete susceptibility to nine antibiotics. This strain showed moderate susceptibility to only sulphamethoxazole/ trimethoprim. Previous studies confirmed that Strep. thermophilus is usually showed susceptibility to tetracycline, erythromycin, chloramphenicol, cephalothin, quinupristin/dalfopristin and ciprofloxacin while it has moderate susceptibility to high resistance to gentamicin, kanamycin and streptomycin (49-51). It is also reported that Strep. thermophilus used in yoghurt production has not been resistant to ampicillin and penicilin (31, 52), as strain ST8.01. Tosi et al. (53) determined that Strep. thermophilus strains can show atypic antibiotic resistance patterns. In their study some strains were shown resistant to tetracycline and clindamycin while some other resistant to

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B. Özden Tuncer, Y. Tuncer tetracycline and sensitive to erythromycin at the same time. The major problem is considered as the antibiotic genes might be transferred to pathogenic bacteria including Streptococci, Listeria and Enterococci in the gastrointestinal tract or in digested foods. For this reason complete susceptibility to clinically important antibiotics of Strep. thermophilus ST8.01 is adventageous.

CONCLUSION In this study, maintenance the vitality of Strep. thermophilus ST8.01 under some gastrointestinal stress conditions (bile salt, lysozyme, phenol and situmulated gastric juice); having autoaggregation and hydrophobicity abilities are required properties for probiotic cultures. Besides these features, if we consider rapid acidification, good proteolitic activity and good flavour compound production of EPS-producing Strep. thermophilus ST8.01 (20), this strain may be a candidate for probiotic starter culture.

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30.Xanthopoulos V, Ipsilandis CG, Tzanetakis N. 2012. Use of a selected multi-strain potential probiotic culture for the manufacture of set-type yogurt from caprine milk. Small Ruminant Research, 106 :145– 153. 31.Mahmood T, Masud T, Imran M, Ahmed I, Khalid N. 2013. Selection and characterization of probiotic culture of Streptococcus thermophilus from dahi. Int Food Sci Nutr, 64 (4): 494-501. 32.Haller D, Colbus H, Gänzle MG, Scherenbacher P, Bode C, Hammes WP. 2001. Metabolic and functional properties of lactic acid bacteria in the gastro-intestinal ecosystem: a comparative in vitro study between bacteria of intestinal and fermented food origin. Syst Appl Microbiol, 24: 218-26. 33.Maurad K, Meriem K. 2008. Probiotic characteristics of Lactobacillus plantarum strains from traditional butter made from camel milk in arid regions (Sahara) of Algeria. Grasas Aceites, 59: 210-224. 34.Khalil R. 2009. Evidence for probiotic potential of a capsular-producing Streptococcus thermophilus CHCC 3534 strain. Pol J Microbiol, 58: 49-55. 35.Pilar F, Paloma L, Angel LC, Carmen P, Teresa R. 2008. Probiotic strains: survival under simulated gastrointestinal conditions, in vitro adhesion to Caco-2 cells and effect on cytokine secretion. Eur Food Res Technol, 227: 1475-1484. 36. Vinderola CG, Mocchiutti P, Reinheimer JA. 2002. Interactions among lactic acid starter and probiotic bacteria used for fermented dairy products. J Dairy Sci, 85: 721-729. 37.Suscovic J. Brkic B, Matosic S, Maric V. 1997. Lactobacillus acidophilus M92 as potential probiotic strain. Milchwissenschaft, 52: 430-435. 38.Xanthopoulos V, Litopoulou-Tzanetaki E, Tzanetakis N. 2000. Characterization of Lactobacillus isolates frominfant faeces as dietary adjuncts. Food Microbiol, 17: 205-215. 39.Acharya MR, Shah R. 2002. Selection of human isolates of Bifidobacteria for their use as probiotics. Appl Biochem Biotech, 102-103: 81-98. 40.Kos B, Suskovic J, Vukovic S, Simpraga M, Frece J, Matosic S. 2003. Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92. J Appl Microbiol, 94. 981-87.

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B. Özden Tuncer, Y. Tuncer 41.Vlkova E, Rada V, Smehisova J, Killer J. 2008. Auto-aggregation and co-aggregation ability in Bifidobacteria and Clostridia. Folia Microbiol, 53: 263-269. 42.Todorov SD, von Mollendorff JW, Moelich E, Moelich E, Muller N, Witthuhn RC, Dicks LMT. 2009. Evaluation of potential probiotics properties of Enterococcus mundtii, its survival in boza and in situ bacteriocin production. Food Technol Biotech, 47: 178-191. 43.Asl›m B, Onal D, Beyatl› Y. 2007. Factors influencing autoaggregation and aggregation of Lactobacillus delbrueckii subsp. bulgaricus isolated from handmade yogurt. J Food Protect, 70: 223-227. 44.Collado MC, Meriluoto J, Salminen S. 2008. Adhesion and aggregation properties of probiotic and pathogen strains. Eur Food Res Technol, 226: 1065-1073. 45.Canzi E, Guglielmetti S, Mora D, Tamagnini I, Parini C. 2005. Conditions affecting cell surface properties of human intestinal bifidobacteria. Antonie Leeuwenhoek, 88: 207-219, 2005. 46.Rahman MM, Kim WS, Kumura H, Shimazaki, K. 2008. Autoaggregation and surface hydrophobicity of bifidobacteria. World J Microb Biot, 24: 1593-1598.

47.Kõll P, Mändar R, Smidt I, Hutt P, Truusalu K, Mikelsaar RH, Shchepetova J, Krogh-Andersen K, Marcotte H, Hammarström L, Mikelsaar M. 2010. Screening and evaluation of human intestinal Lactobacilli for the development of novel gastrointestinal probiotics. Curr Microbiol, 61: 560-566. 48.Flint SH, Brooks JD, Bremer PJ. 1997. The influence ofcell surface properties of thermophilic Streptococci on attachment to stainless steel. J Appl Microbiol, 83: 508-517. 49.Katla AK, Kruse H, Jhonsen G, Herikstad H. 2001. Antimicrobial susceptibility of starter culture bacteria used in Norwegian dairy products. Int J Food Microbiol, 67: 147-152. 50.Temmerman R, Pot B, Huys G, Swings J. 2003. Identification and antibiotic susceptibility of bacterial isolates from probiotic products. Int J Food Microbiol, 81: 1-10. 51.Asl›m B, Beyatli Y. 2004. Antibiotic resistance and plasmid DNA contents of Streptococcus thermophilus strains isolated from Turkish yogurts. Turk J Vet Anim Sci, 28: 257-263. 52.Hummel AS, Hertel C, Holzapfel WH, Franz CM. 2007. Antibiotic resistances of starter and probiotic strains of lactic acid bacteria. Appl Environ Microb, 73: 730-739. 53.Tosi L, Berruti G, Danielsen M, Wind A, Huys G, Morelli L. 2007. Susceptibility of Streptococcus thermophilus to antibiotics. Antonie Leeuwenhoek, 92: 21-28.

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GIDA (2014) 39 (4): 203-210 doi: 10.5505/gida.21939

GD13064-21939

Araflt›rma /Research

DONDURMA İŞLEMİNDE BAZI ÖN UYGULAMALARIN ÇİLEK ve MANDALİNANIN KALİTE ÖZELLİKLERİNE ETKİLERİNİN İNCELENMESİ Taner Baysal, Ahsen Rayman Ergün*, Berrin Akgün, Gözde Karcı, Nazmiye Kaplan Ege Üniversitesi, Mühendislik Fakültesi, G›da Mühendisli¤i Bölümü, Bornova, ‹zmir

Özet

Gelifl tarihi / Received: 27.09.2013 Düzeltilerek Gelifl tarihi / Received in revised form: 22.01.2014 Kabul tarihi / Accepted: 25.01.2014

Dondurma prosesinde; yenilebilir filmle (pullulan) kaplama, fleker flurubuna (%40’ l›k) dald›rma ve kalsiyum klorür (CaCl2) çözeltisine dald›rma ön ifllemlerinin ürün kalitesine etkilerinin incelendi¤i bu çal›flmada çilek ve mandalina örnekleri ay›kland›ktan ve y›kand›ktan sonra befl gruba ayr›lm›flt›r. Bu gruplar; kontrol grubu (hiçbir ifllem uygulanmam›fl), fleker flurubu, flurup+CaCl2, pullulan ve pullulan+CaCl2 gruplar›d›r. Meyve gruplar› ifllemler sonras›nda -18 ˚C’de dondurulmufl ve ard›ndan so¤uk hava deposunda (-24 ˚C), 15 gün süreyle depolanm›flt›r. Depolama süresince 0. ve 15. günlerde, örneklerde; a¤›rl›k ve s›z›nt› kayb›, titre edilebilir asitlik, pH, suda çözünür kuru madde, sertlik ölçümü analizleri gerçeklefltirilmifl ayr›ca renk ve duyusal özellikler incelenmifltir. Bu analizler sonucunda; a¤›rl›k kayb›, s›z›nt› kayb›, pH ve suda çözünür kuru madde de¤erlerinin flurup+CaCl2 ve pullulan+CaCl2 uygulamalar›nda daha iyi korundu¤u belirlenmifltir. Sonuç olarak meyvelere uygulanan, fleker flurubuna dald›rma ve yenilebilir film kaplamalar›n tek bafl›na uygulanmas›na k›yasla bunlara ek olarak CaCl2 eklenmesinin daha iyi sonuçlar verdi¤i ve bu ön ifllemlerin tüketime haz›r meyve kalitesi üzerinde olumlu etki sa¤lad›¤› gözlenmifltir. Anahtar kelimeler: Dondurma, çilek, mandalina, fleker flurubu, yenilebilir filmle kaplama, pullulan, CaCl2.

DETERMINING THE EFFECTS of SOME PRETREATMENTS on THE QUALITY CHARACTERISTICS of STRAWBERRY and MANDARIN in FREEZING PROCESS Abstract In this study, the aim is to evaluate the effects of pretreatments, such as an edible film coating with pullulan and the immersion of fruit into a sugar solution before the freezing process on the quality of the fruits. In order to investigate the advantage of immersion into a CaCl2 solution the pretreatments were combined; strawberries and mandarins were separated into five different groups after a washing and cleaning processes. These groups were: the control (without any pretreatments), sugar syrup, syrup+CaCl2, pullulan, and pullulan+CaCl2. After the different pretreatments, the fruits were frozen at -18 ˚C and stored at -24 ˚C for 15 days. The analyses were done after production and at the 15th day of freezing. The raw material and products were analyzed for weight and drip loss, titratable acidity, pH, soluble dry matter, hardness, colour, and sensory tests. According to the results of the analysis; the weight and drip loss, pH, and soluble dry matter properties were protected better in the group of syrup+CaCl2 and pullulan+CaCl2. As a result, better results were obtained by adding CaCl2 additionally to the application of sugar syrup and edible film coatings and these pre-treatments provided a positive impact on the quality of the fruits. Keywords: Freezing, strawberry, mandarin, sugar syrup, edible film coating, pullulan, CaCl2. *Yazışmalardan sorumlu yazar / Corresponding author; [email protected], ✆ (+90) 232 311 3042,

(+90) 232 342 7592

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T. Baysal, A. R. Ergün, B. Akgün, G. Karcı, N. Kaplan

GİRİŞ G›dalar aras›nda en kolay ve h›zl› bozulanlar, meyve ve sebzelerdir. Bunun nedeni di¤er g›dalara oranla yap›lar›nda %98’e ulaflabilen miktarlarda su içermeleridir. Dondurma ifllemi sayesinde g›dalar›n içerdikleri su, buz kristallerine dönüflünce bozulmaya yol açan mikroorganizmalar›n aktiviteleri durmakta ya da yavafllamakta böylece kimyasal ve biyokimyasal de¤iflimler asgariye indirilerek g›dalar›n en do¤al haliyle korunmas› sa¤lanmaktad›r. Bu özelli¤i ile dondurma ifllemi g›dalar›n kalite, tat, koku ve besin de¤erinin en iyi korundu¤u g›da saklama yöntemi olarak kabul edilmektedir (1). fieker ilavesi, meyveler için oksijen ile temas› engelleyerek, renk ve görünüflün korunmas›n› sa¤lamas› yönünden son derece önemli bir ön ifllemdir. Genellikle meyveyi kaplamak amac›yla oksijen iletimine ve esmerleflmeye karfl› bir bariyer olarak hareket eden, %30-60 konsantrasyon aral›¤›nda fleker fluruplar› kullan›l›r. Çeflitli çal›flmalar ön ifllem olarak flekerle muamelenin; dondurma ifllemi s›ras›nda tat, koku, renk ve besin de¤eri üzerine koruyucu etkisini göstermifltir (2). G›dalar›n raf ömürlerini uzatmak amac›yla dondurma ifllemi öncesi uygulanabilecek yöntemlerden biri de yenilebilir film ve kaplamalar›n kullan›lmas›d›r. Bu uygulama ile üründen su kayb› önlenebilece¤i gibi çevreyle gaz al›flveriflinin de engellenmesi sonucunda, solunum h›z› da yavafllat›labilmektedir. Özellikle son y›llarda, taze, dondurulmufl veya ifllenmifl birçok ürünün raf ömürlerini uzatmak ve kalitesini gelifltirmek amac›yla yenilebilir kaplamalar›n kullan›m› ile ilgili çal›flmalar daha yo¤un bir flekilde yürütülmektedir (3-5). Yenilebilir g›da filmleri üretiminde kullan›lan kaynaklar; jelâtin, kazein ve zein gibi proteinler ile selüloz ve dekstrin karakterli maddeler, alginat, mumlar, doymufl ya¤ asitleri, monogliseritler ve bunlar›n baz› türevlerinden oluflmaktad›r (6, 7). Pullulan filmler ekstraselüler mikrobiyel polisakkarit filmlerdir ve kokusuz, berrak bir film oluflturmaktad›rlar. Bu filmler, düflük ba¤›l nemde iyi bir oksijen bariyer özelli¤i göstererek, g›dan›n raf ömrünün uzat›lmas› amac›yla kullan›lmaktad›rlar. Yüksek ya¤ ve düflük su içeri¤ine sahip g›dalarda da ac›laflma ve oksidasyonun önlenmesi amac›yla kullan›l›rlar (8, 9). Ayr›ca, meyvelere dondurma öncesi uygulanacak ifllemler aras›nda çözündürme sonras› dokunun korunmas› amac›yla Ca+ ilave edilmesi de birçok çal›flmada araflt›r›lm›flt›r (10).

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Bu çal›flmada dondurma ifllemi öncesi çilek ve mandalina segmentlerine uygulanan; pullulan film ile kaplama, fleker flurubu ve kalsiyum uygulamas› gibi ön ifllemlerin, dondurulmufl meyvelerin kalite özellikleri üzerine etkisinin belirlenmesi amaçlanm›flt›r.

MATERYAL VE YÖNTEM Materyal Yerel marketlerden temin edilen çilek (Fragaria vesca, cv. Selva) ve mandalina örnekleri (C. Reticulata, cv. Satsuma) Ege Üniversitesi, Meyve Sebze ‹flleme pilot tesislerinde ifllenmifltir. Yöntem fieker flurubu, CaCl2 çözeltisi ve pullulan çözeltisi haz›rlanmas› amac›yla yap›lan ön denemeler sonras›nda kalite aç›s›ndan en olumlu sonuçlar›n elde edildi¤i fleker flurup konsantrasyonu (%40’l›k) seçilmifl ve Sakaroz (Merck Darmstadt, F.R, Germany) kullan›larak haz›rlanm›flt›r (11). Yap›lan literatür araflt›rmalar›nda %1’lik konsantrasyondan daha yüksek oranda kullan›lan CaCl2 çözeltisinin örnekte tat bozuklu¤una yol açt›¤› belirtilmifltir (12-14). Bu nedenle, çal›flmada CaCl2 (Merck, Darmstadt, F.R, Germany) çözeltisinin konsantrasyonu %1 olarak seçilmifltir. Hem çilek hem mandalina için 50 ml %2,6’l›k pullulan (P1-20 Hayashibara Co., Ltd., Okoyama, Japan), çözeltisi haz›rlanm›flt›r. Bu çözeltiye, Pullulan+ CaCl 2 gruplar›nda kullan›lmak üzere 0.5 ml %1’lik CaCl2 çözeltisi ilave edilmifltir. Çilek ve mandalina segmentleri; y›kama, ay›klama, kabuk soyma gibi ön ifllemlerinden sonra; pullulan, pullulan + CaCl2, fleker flurubu, fleker flurubu+ CaCl2 kaplama uygulamalar›na tabi tutulmufltur. Tüm kaplama uygulamalar› için meyveler 2 dakika (dk.) kaplama çözeltisinin içerisine dald›r›lm›fl ve yaklafl›k 20 ˚C’ de 30 dk. kurumaya b›rak›lm›flt›r. Kuruma ifllemi tamamland›ktan hemen sonra örnekler iki gruba ayr›lm›flt›r (150’fler gram). 0. gün analizleri yap›lacak örnekler dondurulmadan hemen laboratuvara al›n›rken di¤er örnekler dondurma ifllemi sonras›nda 15 gün boyunca depoda (-24 °C) muhafaza edilmifl ve 15. günde analizler gerçeklefltirilmifltir. Çileklerin dondurma iflleminde, bafllang›ç s›cakl›¤› 10˚C, mandalinalar›n ise bafllang›ç s›cakl›¤› 3.5˚C olarak ölçülmüfltür. Çilekler 25 dk., mandalina

Dondurma İşleminde Bazı Ön Uygulamaların... örnekleri ise 11 dk. süre ile IQF dondurucuda (Frigoscandia, Helsinborg, Sweden) dondurulmufltur. Merkez s›cakl›¤› -10 °C s›cakl›¤a ulaflt›¤› anda ürünler su geçirgenli¤i düflük ve ›s›l yap›flabilme özelli¤i olan polietilen pofletlere koyularak depolama (-24 °C) yap›lm›flt›r. 15 günün sonunda meyveler 4 °C’de buzdolab›nda çözündürülmüfl ve analize al›nm›flt›r. Uygulaman›n 0. ve 15. gününde ürünün °Briks, pH, asitlik, renk, a¤›rl›k ve s›z›nt› kayb›, sertlik ve duyusal özellikleri belirlenmifl ve her meyve için kalite özellikleri aç›s›ndan 0. gün ve 15. gün kendi içerisinde, ayr›ca günler aras›nda da karfl›laflt›rmalar yap›lm›flt›r.

tekstür aç›s›ndan s›ralama testine tabi tutularak duyusal olarak de¤erlendirilmifltir ve 0.05 güven aral›¤›nda istatistiksel analiz gerçeklefltirilmifltir (19). Ürünlerin fiziksel ve kimyasal analiz sonuçlar›n›n istatistiksel olarak de¤erlendirilmesi amac›yla ise SPSS 15.0 paket program› kullan›lm›flt›r. ANOVA uygulan›larak her bir örnek grubu Tukey testine göre 0.05 güven aral›¤›nda birbirleriyle karfl›laflt›r›lm›flt›r (20).

Yapılan Analizler ve Yöntemleri

Çilek ve mandalina örnekleri üzerine yap›lan a¤›rl›k ve s›z›nt› kayb› analizlerinin sonuçlar› Çizelge 1’de gösterilmifltir. 15. gün sonunda çilek örnekleri, kontrol örnekleri ile karfl›laflt›r›ld›¤›nda en az a¤›rl›k kayb› flurup+CaCl2 ve pullulan+CaCl2 uygulamalar›nda saptanm›flt›r. ‹statistiksel olarak ise a¤›rl›k kayb› aç›s›ndan flurup+CaCl2 grubuyla kontrol grubu aras›ndaki fark›n önemli oldu¤u bulunmufltur (P