Fındığın Depolanması Sırasında Bazı Küfler Tarafından Oluşturulan Lipaz Etkisinin İncelenmesi

Abstract

Bu çalışmada fındık mikroflorasını oluşturan Aspergillus niger ve Trichothecium roseum küflerinin tek ve kombine olarak fındık üzerinde lipaz enzimi oluşturmaları ve bunun sonucunda fındık yağında meydana gelen değişiklikler incelenmiştir. Bu küflerin tek ve kombine lipaz aktivitelerinin belirlenmesi amacıyla fındıklar % 80, % 85, % 90 olmak üzere 3 farklı bağıl nem ortamında ve nemi artırılarak 25°C sıcaklıkta 4 hafta depolanmıştır. Depolama boyunca küflerin lipolitik aktivitesine bağlı olarak meydana gelen trigliserid (TG), di ve monogliserid İle serbest yağ asidi (FF A) miktarları, nem ve pH tayinleri yapılmıştır. Uygun olmayan koşullarda lipolitik aktiviteye bağlı olarak FFA miktarında yükselme meydana gelmesi prensibiyle, küfler tarafından lipaz oluşturulması ve bunun etkileri ince tabaka esasıyla IATROSCAN cihazı kullanılarak belirlenmiştir. A. niger küfü ile aşılanan fındık numunelerinden elde edilen analiz verilerine göre ortam bağıl nemi % 90 olduğunda örnek nemi yükselmiş, pH değeri ise önemli miktarda düşme göstermiştir. % 90 bağıl nem ortamında lipaz etkisiyle yağların parçalanması nemi artırılmış fındık numunelerine göre daha az; % 85 ve %80 bağıl nem ortamında depolananlara göre daha fazla gerçekleşmiştir. Fındık mikroflorasında yer alan küflerden olan T. roseum'un lipaz oluşturma özelliğine ait literatür verisine rastlanmamıştır. Yapılan çalışmada fındık numunesindeki pH değerlerinin değişiminde T. roseum küfünün etkili olmadığı, pH'da önemli bir azalma meydana getirmediği saptanmıştır. T. roseum'un lipaz aktivitesi gösterebilmesi için aynı sıcaklıkta nemin etkili bir parametre olduğu nemi artırılmış fındık numunesinde yüksek miktarda TG parçalanmasıyla görülmüştür. A. niger ve T. roseum küflerinin birlikte aşılanması durumunda fındıktaki lipaz oluşumunda sinerjist etkinin söz konusu olduğu, T. roseum'un A. niger'in lipaz oluşturmasını bir miktar arttırdığı saptanmıştır.

Key Words: Hazelnut, lipase activity, Aspergillus niger, Trichothecium raveum, storage conditions Due to the fact that hazelnut is very rich in terms of nutrients, and that it is one of our outstanding foreing currency resources, it represents a huge potential for creating commercial activities in our country's economy. Turkey bears the status of the largest hazelnut producer and exporter throughout the world. Types of Turkish hazelnuts contain 60-80 % fat and 15-19 % protein. Fat content of Turkish hazelnuts differs according to the regions they are cultivated (ÖZDEMİR, 1997). Essential fatty acids minerals (iron, calcium, zinc, potassium); vitamins Bi, B2, Bo and vitamin E contribute to the nutritional values hazelnut provides (AYFER, 1996). Hazelnut is inclined to deteriorate and lose its quality quickly during drying, storing, transportation and process stages. High-content fat in its composition is important during storing and process stages. Fats are sensitive to external factors such as heat, light, water and the atmosphere and to microorganisms such as bacteria and mould. Fats deteriotae chemically due to diversified changes when they are subject to the effect of such factors during their storage period. Shelf life of hazelnut depends much on the auto-catalytic reactions or microbial contamination (KTNDERLERER and JOHNSON, 1992). Enzymes lipase, esterase, polyphenol oxidase and peroxidase are found to exist in cotyledons in kernels (KEME et al, 1983). These enzymes cause deterioration via contamination at certain stages such as storing and transportation, starting as soon as the hazelnut blossoms into fruit. Certain mould causes lipase. Enzymatic activity plays a significant role in hazelnut preservation (BONVEHI and ROSUA, 1996). Free fatty acids are released from triglycerides under the effect of moisture through lipases. Free fatty acid is a significant criterion in determining the quality of hazelnut, which effects the taste of hazelnut on its own. Free faty acid causes a bitter taste and soapy aroma in hazelnut. Lipase activity depends on factors such as moisture, water activity, temperature and pH (BONVEHI and ROSUA, 1996). It is very important for hazelnut to contain low moisture level at the initial stage of storing and during the storage period so that no loss of quality occurs in hazelnut, since enzymatic reactions are triggered in high- moisture environments and deterioration accelerates (BAŞ, 1990). VUl Hazelnut is much less effected from mould contamination thanks to its thick shell, when compared to other products. However, mould can develop and cause deterioration and mycotoxin formation in the hazelnut (EKE and GÖKTAN, 1987). Eke (1986) studied the microflora formation in hazelnut. The mould found mostly in hazelnut is Trichothecium roseum, followed by A. flaws, Penicillium commune, Eurotium spp., P. brevicompactum, P. crustosum, P. janczewskii, A. niger, A. versicolor, Trichoderma harzianum, Cladosporium spp. and Rhizopus spp.. Aspergillus niger dealt with in this study is very commonly found and very important in foodstuff. It may be black or brownish-black or purple-brown in colour. While most spoil the foodstuff, certain types are used in preparation of certain foodstuff (such as sitric acid, gluconic acid, enzyme preparations) (FRAZDER, 1967). They cause spoilage and deterioration in foods such as hazelnut, peanut, corn, sweet potato, apple, grape, peach, strawberry, tomato and muskmelon (PITT and HOCKING, 1985). A. niger needs a temperature of minimum 6-8 °C, optimum 35- 37 °C and maximum 45-47 °C. Minimum water activitiy at 43 °C necessary for the development of A. niger is 0.78-0.83 (BEUCHAT, 1978). A. niger is a significant industrial lipase source (GODFRESTEN, 1993). T. roseum most-found mould in hazelnut microflora, is pink in colour and it also grows, apart from hazelnut, in fruits such as apple and peach, certain vegetables and grains including wheat, corn and barley (FRAZIER, 1978). Minumum water activity for T. roseum growth is 0.90 at 22-25 °C (BEUCHAT. 1978). The temparatures under which it grows range from minimum 15 °C to an optimum 25 °C to a maximum 35 °C (SAMSON and HOEKSTRA, 1988 ). This study covers the formation of lipase enzyme by moulds. A. niger and T. roseum, individually and in combination, forming the hazelnut microflora and certain changes occuring in hazelnut fat as a result of such formation. In order to determine the individual and combined lipase activities of these moulds hazelnuts were stored for a period of 4 weeks at 25 °C, under three different relative humidity environment, 80 %, 85 %, 90 % respectively, while increasing the moisture of the hazelnut. Triglyceride, di and monoglyceride and free fatty acid (FFA) amounts, humidity and pH levels formed during the storage period were recorded. Based on the principle where an increase in FFA amount is achieved depending on the lipolytic activity under inconvenient conditions, the formation of lipase by the moulds and its effects have been found out by employing an IATROSCAN device under a thin layer basis. Moulds used in the inoculation of hazelnut in the study ( A. niger + T. roseum ) were isolated from hazelnuts procured from the markets in the microbiology laboratories of the Food Engineering department of Istanbul Technical University. Moulds which were isolated and identified were kept in horizontal agar in pure culture form (MEA) at +4 °C until they were used in analysis process. Hazelnuts used in the research were 1996-crop raw nuts of Tombul type, cultivated in Giresun region and were supplied from Karin A.Ş.. Hazelnuts were preserved under +4 °C until an analysis was made. IX As a first step in the study the said moulds were isolated and identified, then they were examined whether such moulds had lipolytic activity. In order to find out the lipase activity "Turbidimetric Method" was used among other lipase-idetification methods (REED, 1975). In this method where tributyrin was used as substrate a clear zone has been measured which was formed in tributyrin agar as a result of lipolysis and which should be at a length of, at least, 20 mm. as the indicator of lipolytic activity. Height of the clear zone has been measured to be 20 mm and 30 mm, for A. niger and T. roseum, respectively, and these moulds were found to have lipolytic activity. Isolated and identified moulds were incubated in horizontal MEA at 25 °C and spor suspensions of 106 unit/ ml for these moulds at the end of the 7th day. Hazelnuts were ground in blender in a manner to be homogenous and they were filled into phials of 150 ml, each phial holding 50 gr ground hazelnut. These hazelnut samples were sterilised for 15 minutes at 121 C provided that the caps of phials were closed tightly. Sterilised hazelnut samples were stored for a period of 28 days under three different relative humidity levels, that is, 80 %, 85 % and 90 %, at 25 °C. BaCİ2 for 90 % relative humidity level (Merck), KC1 for 85 % relative humidity level (Merck), (NtL^SC^ for 80 relative humidity level (Merck) prepared salt solutions were filled into capped and airtight glass jars of 100 ml and they were sterilised for 15 minutes at 121 °C. Hazelnut samples inoculated with spor suspension with 1 ml 106 unit/ml concentration were placed into jars containing salt solutions and were stored for a period of 28 days at 25 °C. Moisture, pH, FFA, TG values were found for hazelnut samples within the jar and the samples were taken at 2nd, 4th, 6th, lO*,^*, 14th, 16th, 20th, 24th and 28th days. The same process was applied to the hazelnut samples, moisture of which was increased. At certain days during the storage period hazelnuts were left for 30 minutes in a water bath of 100 °C following sampling for moisture and hazelnut fat was extracted with a hand-type press prepared for this purpose. The hazelnut fats put into the test tubes with caps were left 15 minutes in water bath of 100 °C and nitrogen gas was added under 0.5 atmospheric pressure for 1-2 minutes. The tubes were kept in a refrigerator until a measurement was made in the TLC equipment after they were capped. As per the analysis data obtained from the hazelnut samples inoculated with A. niger the moisture of the sample was recorded to increase when the relative humidity of the environment was 90 %, however pH value experienced a sharp fall. Under relative humidity environment of 90 % the decomposition of the fats with the lipase effect were experienced to be less when compared to the hazelnut samples with their moisture level increased and to be higher than the ones stored in an environment with 85 % and 80 % levels of relative humidity. moisture was an important factor at the same temperature in order for T. roseum to exhibit any lipase activity when a high level of TG decomposition was witnessed in a hazelnut sample with an increased level of moisture. When A. niger and T. roseum moulds were inoculated together, it was observed that synergist effect on the formation of lipase in the hazelnut was created and T. roseum increased the formation of lipase by A. niger to a certain degree. 25 20 15 2? u_ 10 - FFA %80 Relative Humidity - FFA %85 Relative Humidity FFA %90 Relative Humidity 4 12 16 20 Storage time, day Figure 1. Relationship between the storage period and FFA for Aspergillus niger at RH 80%, RH 85% and RH 90%. XI -FFA 10 12 14 Storage time, day Figure 2. Relationship between the storage period and FFA for Aspergillus niger at moistured hazelnut samples -FFA 6 12 Storage time, day Figure3. Relationship between the storage period and FFA for combined at90RH%.