Ultrases teknolojisinin balın kalitesi üzerine (HMF, enzim) etkisinin belirlenmesi ve sanayideki geleneksel ısıtma yöntemlerine alternatif yöntemlerin araştırılması
Investigating the effects of ultrasound on quality parameters of honey (HMF, enzymes) and evaluating alternative methods vs  conventional technologies
Yazar: MUSTAFA BAYRAKTAR
Danışman: PROF. DR. DİLEK BOYACIOĞLU
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2016
Konu: Gıda Mühendisliği = Food Engineering

Özet:
Bal, şeker ve su içeriğine ve ortam koşullarına da bağlı olarak belirli bir süre sonra kristalize olmaktadır. Kristalize olmuş balın viskozitesinin de artmasından dolayı akışkanlık özelliğinde azalma meydana gelir. Bu nedenle sanayide pompa ve makineler ile bir yerden bir yere aktarılması ve dolumu mümkün değildir. Diğer taraftan kristalize olmuş bal tüketiciler tarafından hem duyusal hem de görsel olarak kabul görmemektedir. Dolayısıyla bal, sanayide ısıl işlemler ile eritilerek tekrar akışkan hale getirilmekte ve daha sonra da kristalleşme süresini mümkün olduğunca uzatılabilmek için genellikle 70-80 ᵒC sıcaklıklarda pastörize edilmektedir. Bu prosesler balın içeriğinde doğal olarak bulunan enzimlerin diastaz hariç neredeyse tamamen yok olmasına ve istenmeyen HMF bileşenlerinin oluşmasına ve/veya artmasına sebep olmakta ve doğallığına zarar vermektedir.
Bu çalışmada balın kristalizasyonunun çözülmesi için sanayide uygulanan ısıl işlemlere alternatif yöntemlerin araştırılarak balın enzim değerlerini olabildiğince korumak ve HMF değerini minimum seviyede tutmak için araştırmalar yürütülmüştür. Bu kapsamda prob ultrases, ultrases su banyosu ve kontrol grubu olarak geleneksel (normal) su banyosu olmak üzere 3 farklı yöntem araştırılmıştır. Prob ultrases uygulamaları hem laboratuvar ölçekte 400 W güce sahip cihaz, hem de pilot ölçekte 2000 W güce sahip cihaz kullanılarak gerçekleştirilmiştir. Çalışmalar 4 çeşit bal (narenciye, geven, ayçiçek, pamuk) üzerinde uygulanmıştır. Pilot ölçekteki çalışmalarda 6 kg bal örneği kullanılırken, laboratuvar ölçekteki çalışmalarda 250 g ağırlığında bal numuneleri kullanılmıştır.
Laboratuvar ölçekteki çalışmalar 200 W, 300 W ve 400 W güç ile 0.5, 0.75 ve 1 döngü değerlerinde olmak üzere her bir bal çeşidi için 9 farklı proses koşulunda gerçekleştirilmiştir. Ultrases su banyosu 50 ᵒC ve 55 ᵒC olmak üzere iki sabit sıcaklıkta 45 kHz frekansta yürütülmüştür. Geleneksel su banyosu çalışmalarında yalnızca 50 ᵒC ve 55 ᵒC sabit sıcaklıklar kullanılmıştır. Prototip ölçekteki prob ultrases çalışmaları ise TÜBİTAK MAM pilot tesislerinde 2000 W, 1500 W ve 1000 W güçte yalnızca geven balında gerçekleştirilmiştir. 
Yapılan çalışmaların öncesinde ham balda ve sonrasında uygulanan işlemler  sonrası nem, diastaz, invertaz, HMF, renk, viskozite ve kristal miktarları incelenmiştir . Ayrıca balın tamamen sıvılaşması için geçen süre değerleri ve proses sırasında balın ulaştığı maksimum sıcaklık değerleri kayıt altına alınmıştır. Prosesler balın kristalleri tamamen çözülene kadar devam ettrilmiş ve kristallerin çözüldüğünün kanıtlanması için de proses öncesi ve sonrasında kristal ölçümleri DSC (Diferansiyel Taramalı Kalorimetre) ile gerçekleştirilmiştir. İstatistiksel analizler faktöryel desen üzerine kurulu olup, faktörlerin etkisi ve birbirleri ile girişim etkileri değerlendirilmiştir. 
Prob ultrases denemelerinde ortalama işlem süresi ortalama 9.5 dk iken ultrases su banyosu ve normal su banyosu prosesleri ortalama 201 dk sürmüştür. Ayrıca prob ultrases uygulamalarında balların sıcaklığı ortalama 68 ᵒC değerlerine ulaşmıştır.
Yapılan çalışmalar özellikle invertaz enziminin uygulanan işlemler arası farklılıktan oldukça etkilendiğini ortaya çıkartmıştır. Yapılan 2 farklı denemede her bir çeşit bal için invertaz enzimi miktarının güç, döngü faktörleri ve güç*döngü ineraksiyonundan p<0.05 istatistiksel önem düzeyinde etkilendiği saptanmıştır. Ayrıca viskozite miktarı üzerine de tüm bal çeşitlerinde p<0.05 önem düzeyinde güç, döngü faktörleri ve güç*döngü interaksiyonunun etkili olduğu bulunmuştur. Diastaz miktarında çok önemli farklılıklar gözlenmese de uygulanan işlem ile bal çeşidine göre değişkenlik gösterdiği saptanmıştır. Benzeri şekilde renk değerlerindeki değişimler için net bir yorum verilememekle birlikte bal çeşidi ve prosese göre değişkenlik gösterdiği bulunmuştur. Kristal miktarı tüm bal çeşitlerinde güç miktarına bağlı olarak değişmiştir (p<0.05). HMF miktarında çalışmada kullanılan tüm bal çeşitleri için p<0.05 önem düzeyinde güç faktörünün etkili olduğu belirlenmiştir. Nem içeriğinde de kristal haldeki ham bala göre bir düşüş yaşandığı bulunmuştur. Ancak bu azalma istatistiksel olarak önemli bir farklılık ifade etmemektedir (p<0.05) ve bal çeşitleri arasında değişkenlik göstermektedir.
Sonuç olarak ultrases prosesinin ülkemize özgü balların kalitesi üzerine etkileri belirlenmiş ve sanayide bal prosesinde uygulanabilmesi için önemli veriler elde edilmiştir.

Abstract:
Honey crystalizes after a while depending on its sugar and water contents and storage conditions. When honey is crystalized its mobility decreases, and it also becomes unacceptable to the consumers. It is not possible to pump and fill crystalized honey at the industrial scale. Besides, crystalized honey is often perceived to be an adultered product by consumers. Therefore; honey is heated to be liquefied and then pasteurized at 70-80 ᵒC to prolong recrystallization and thereby its shelf life. These applications nearly destroy all enzymes naturally occuring in honey (invertase, glucose oxidase etc.) except diastase and causes to the formation of undesired compunds such as HMF.
In the present study, alternative methods were investigated to liquefy honey, while protecting its enzymes and controlling increase in HMF as possibly. In this context, 3 different methods; probe ultrasound, ultrasound water bath, and conventional water bath (as a control group) were used. Probe ultrasound applications were both carried out at lab scale ultrasound (400 W) and pilot scale ultrasound (2000 W). Four different floral honey types, namely; citrus, astragalus, sunflower and cotton honey were selected as samples. 250 g honey sample was used in the lab scale trials while 6 kg each honey sample was used in pilot scale trials.
Lab. scale trials were carried out at 3 different power levels (400 W, 300 W, 200 W) and 3 different cycles (0.5, 0.75, 1) for four different honey types. Ultrasound water bath trials were applied at 2 different temperatures (50 and 55 ᵒC) with 45 kHz frequency while conventional water bath process was carried out at only 2 different temperatures (50 and 55 ᵒC). Pilot scale trials were only applied for astragalus honey at 3 different power levels (2000 W, 1500 W, 1000 W) at TUBITAK MAM pilot plant.
Before and after the treatments moisture content, diastase, invertase, HMF, color, viscosity and crystal contents of honey were analyzed. Besides, process time for complete liquefaction of honey and maximum temperatures reached during the process were also recorded for each type of honey. Decrystallization process was continued until the complete liquefaction of honey. Crystal content of honey was measured before and after the treatments by using DSC (Differential Scanning Calorimetry) for proving that honey was liqufied. Statistical anaylysis were achieved to investigate the effect of each factor and their interactions based on factorial design. 
Average process time for probe ultrasound appications were 9.5 minutes while ultrasound water bath and conventional water bath applications took about 201 min. Honey was reached to an average temperature of 68 ᵒC during probe ultrasound process.
It was observed that moisture content of liqufied honey was decreased compared to the crystalized honey but the difference was not significant (p<0.05) and it varied depending on the treatment and type of honey. The reason for a decrease in the moisture content in the liqufied honey is related to the glucose monohydrate formation while it was in the crystalline structure. Since honey is generally crystallizes in the monohydrate formation, only one molecule water is bounded to the glucose molecule and other water molecules are free. On the contrary, in the liqufied honey, glucose molecule is able to bound five water molecule to the five OH groups. Therefore, free water is less in the liqufied honey compared to crystallized one. For water bath and ultrasound water bath applications, moisture content did not change significantly (p<0.05).
On the other hand, distase number was not effected significantly (p<0.05) during prob ulrasound application. Diastase number of citrus honey was not used for the statistical calculations since the content of diastase was <1. Diastase number decreased between 0-27% after the application of lab scale prob ultrasound. Power of ultrasound was statistically important for diastase number of astragalus honey (p<0.05). It was found that power and power*cycle interaction was significantly imortant for the diastase number of cotton honey while only cycle was effective over diastase numbe of sunflower honey (p<0.05). For water bath and ultrasound water bath applications diastase number did nt show any statistically significant change (p<0.05).
Invertase was very sensitive to the different treatments. It was found that invertase was statistically related to the power, cycle and power*cycle interaction (p<0.05) for four types of honey used in this study.
It was found that 200 w, 0.5 cycle and 200 w, 0.75 cycle treatment did not change the invertase number of citrus honey while 400 w, 1 cycle caused he reduction of 92% of invertase number. It was also observed that invertase decreased from 40% to 80% for astragalus honey, from 40% to 90% for sunflower honey and form 15% to 80% for cotton honey. It is also observed for the water bath and ultrasound water bath applications that invertase was significantly decreased after the applications (p<0.05).
For HMF content, it was observed that HMF increased form 3.6 ppm to 5.0 ppm after prob ultrasund application. Power, cycle and power*cycle interaction was significantly important for HMF content (p<0.05). Fow water bath and ultrasound water bath applications HMF content did not change significantly (p<0.05).
Color results had a similar tendency to diastase results, of which highly depended on type of honey samples and treatments for both prob ultrasound and water bath applications including ultrasound system.
Likewise, viscosity was associated to the power, cycle factors and power*cycle interaction in all types of honey (p<0.05). For citrus and astragalus honey, viscosity was decreased approximately 60% after prob ultrasound application. It was less than 605 reduction for cotton honey while 22% reduction was observed sunflower honey.
Power, cycle and power*cycle factors was significantly important for crystal content of honeys.
For prob ultrasound, water bath and ultrasound water bath applications considered, power and cycle factors were significantly important for temperature of citrus honey while only cycle was significantly important for temperature of astragalus, sunflower and cotton honey (p<0.05).
For prob ultrasound, water bath and ultrasound water bath applications considered, cycle factors were significantly important for process time of citrus, astragalus, and sunflower honey (p<0.05). On the other hand, power, cycle and power*cycle interactions were significantly important for cotton honeyby means of process time.
It was also found that initial moisture content of honeys was not significantly important (p<0.05) for all process applied in this study.
Diastase number of prob ultrasound applied honeys was significantly different compared to water bath and ultrasound water bath applications. Diastase number of prob ultrasound applied sunflower and cotton honey was significantly higher than the water bath an ultrasound water bath applied honeys. It was related to the longer process time of water bat and ultrasound water bath applications.
It was also found that process type is significantly important for HMF content of honeys.
In conclusion, prob ultrasound process time was at least 10 times shorter compared to water bath and ultrasound water bath applications. However; when prob ultrasound is applied, as process time increases, temperature of honey reached increases significantly which causes reduction in ezyme content and an increase in HMF content. In order to prevent temperature increase, system that ultrasound is applied needs to be cooled from outside. Besides, new designed prob ultrasound which will enable to mix honey homogenously to enable ultrasound waves to touch every point of honey in order to shorten process time which will also keep the temperature under control. Available ultrasound systems does not allow to move prob around the medium. Therefore, a new prob ultrasound system needs to be designed in order to enable to application of prob ultrasound system to the industry.
It was also observed that as viscosity content of raw honey increases, process time needed for liqufection of honey increases. Therefore, viscosity of honey is related to the temperature increase and process time of honey which effects enzyme and HMF content. It was also found that as honey liquefy, viscosity of honey decreases accordingly.
Crystal content of honey also decreases as honey liquefies. Crystallization of honey is generally related to glucose monohydrate formation. Therefore, lower glucose monohydrate content, more liqufied honey.
It was for certain that invertase content of honey decreased accordingly with each process applied in this study. It means that temperature increase was significantly important for prob ultrasound, water bath and ultrasound water bath process applications. On the other hand, even if temperature increase was not higher than the prob ultrasound application, longer process time for ultrasound water bath and especially ultrasound water bath applications effected invertase number significantly (p<0.05).
On the contrary diastase number was not effected significantly from prob ultrasound, ultrasound water bath and water bath applications which made us question to rightness of standards which takes into account of diastase number to assesss the exposure of temperature. It was very obvious that even at high temperatures more than 70 ᵒC, diastase number was almost stable while invertase was almost disappeared.
Disconcertingly, it was also observed that HMF content was decreased compared to raw honey (unprocessed) after the application of some prob ultrasound process. It was thought that ultrasound waves either may break HMF into an another molecule or accelerate the process in order to make HMF to unite with some molecule in order to have a new compound.
The current research presented the effects of ultrasound process on the quality of different local honey types in Turkey and provided important information for the application of ultrasound process for the honey industry.