Investigation the effect of pulsed electric field treatment on Kluyveromyces marxianus of Doogh

Introduction: Swelling is one of the main disadvantages of Doogh (Iranian yoghurt drink).  Kluyveromyces marxianus is the main cause of Doogh spoilage and its swelling. Investigations showed the yeast count of Doogh samples more than permitted standard level (Mehraban Sangatash et al., 2011). Other researches also confirmed the presence of mold and yeast contamination in Doogh samples in Zanjan (Mir Alizadeh et al., 2017). A major yeast causative spoilage of yogurt and its products is Kluyveromyces marxianus (Viljoen 2003). One of the non-thermal methods of food processing involves the use of pulsed electric field operations. The use of pulsed electric field causes inactivation of microorganisms without adverse effects especially on flavor and nutritional value (Wang et al., 2018). In this research, the effect of pulsed electric field treatmenton the Kluyveromyces marxianus in Doogh was investigated. Quality of Doogh samples was also determined.
Material and methods: The yeast Kluyveromyces marxianus (PTCC 5188) was purchased from Iranian Research Organization for Science and Technology. After activation, cells were incubated in YDP agar at 4°C. For preparation of inoculum, a complete loop of pure yeast colony was transferred to a 250 ml Erlenmeyer flask containing 100 ml of inoculum (5% lactose, 0.3% yeast extract and 0.5% peptone in distilled water) and then placed in a shaker incubator (30°C, 150 rpm, 24 h). Thereafter, yeast cells were isolated by centrifugation at 4500 g for 10 minutes (three times rinsing with deionized water) (Mohammadzadeh et al., 2014). Then, a concentration of 10⁵ cells per ml was prepared (Ebrahimi et al., 2013). Preparation of Doogh samples was done according the following steps. 40% yogurt, 0.7% salt and 59.3% water was mixed (130 rpm). Mixing was continued for 2–3 minutes to prepare a uniform Doogh samples (Foroughinia et al., 2009). In this study, electric field strength (1, 2, 3 and 4 kV / cm) and pulses number (50, 100, 150, 200 and 250) were used. The control sample did not undergo pulsed electric field operation.
 To determine the amount of protein, fat, acidity and dry matter, standard method of Iranian National Standards Organization was used (Iranian Institute of Standardization and Industrial Research; Doogh – Specifications and test method, ISIRI 2453). The pH of the Doogh samples was measured by digital pH meter 744 Metrohm at ambient temperature. Serum separation of Doogh samples were determined according to the following steps. Experimental tubes with specified dimensions (1.4 cm diameter, 16 cm in length) were used to determine the serum separation percentage. These tubes were marked down to a height of 10 cm, this height of 10 cm was considered as100%. The samples were then poured into the tube to the marking line and, after capping, positioned completely vertically and in specific time intervals, the line formed between the two phases was measured, and each milliliter was equal to 1% of the phase separation rate (Foroughi Nia et al., 2009).
The flow behavior test was measured at a shear rate range of 11- 321 per second using a Spindle SC4-18 LVDV III Ultra rotary viscometer from Brookfield, USA. Morphological study of yeast cells was performed before and after applying pulsed electric field treatmentusing scanning electron microscope with magnification of 15000-5000.
Results and discussion: Doogh had 2.1% protein, 0.98% fat, 6.9% dry matter, acidity=43, pH about 4.2 and apparent viscosity of 10.92 mPa.s.  Evaluation of the yeast population of Kluyveromyces marxianus in different Doogh samples indicates that the yeast population is reduced under conditions of application of pulsed electric field operation. The highest yeast population belonged to the control sample (p <0.05) and application of pulse electric field operation reduced the yeast population and in conditions of pulse electric field operation with electric field strength of 3 kv /cm and pulses number= 200, the log of the yeast population reached about 1.9 ± 0.01. In the conditions of pulsed electric field operation 4 kv / cm and pulses number=250, yeast population continued to decrease, reaching to 1.65 ± 0.01. Statistical analysis showed that there is a significant difference between serum separation of Doogh samples under different conditions of pulse electric field operation including the electric field strength and the number of pulses used (p <0.05). Pulse electric field operation increased percentage of serum separation and control sample had lowest serum separation (52.6%). Depending on the amount of electric field strength and the number of pulses applied, the amount of serum separation in Doogh samples were changed.  In Doogh sample subjected to 1 kv / cm electric field strength and pulses number= 50, serum separation was equal to 53.2 ± 0.5% and in sample subjected to electric field strength= 4 kv / cm and 250 pulses number, the highest serum separation was observed (72±0.8%), and results might be due to the coagulation of proteins during application of pulsed electric field resulting in an increase in serum separation level. Applying pulsed electric field operation did not significantly change the rheological behavior of Doogh samples and it can be concluded that the conditions of pulsed electric field operation studied in this study did not have a significant effect on the intermolecular bonds of the sample proteins. Investigation of the apparent viscosity of Doogh samples under pulsed electric field operation showed that the viscosity values ​​did not change significantly in samples that were exposed to 1 kV/cm and 50 and 100 pulses number, but in other treatments, it caused changes in viscosity compared to the control sample. The highest decrease in viscosity was observed in the treated sample under conditions of 4 kV /cm and 250 pulse number. One of the possible reasons for the decrease in apparent viscosity is that the pulsed electric field results in the coagulation of proteins and the size of distribution of fat globules (Xiang et al., 2011). The results of morphological examination of yeast cells, showed that the pulsed electric field treatmentcaused a change in the morphology of the yeast cell.
Conclusion: The results of this study showed that application of non-thermal operation of pulsed electric field reduces the population of Kluyveromyces marxianus, a major causative of swelling of this product. At electric field strength of 4 kv/cm and pulses numbers of 100, 150, 200 and 250, the log of yeast count reaches to the lowest level. Applying pulsed electric field operation caused some changes in the rheological properties of the Doogh and reduced its apparent viscosity compared to the control sample. In addition, the percentage of serum separation, increased as a result of applying this operation.