Title : Preservation methods for microorganisms in kefir grains
Abstract:
Introduction: Kefir is acidic, slightly alcoholic and a viscous fermented dairy beverage that has a long history in human nutrition due to its health benefits including anti-obesity, anti-oxidative, cholesterol-lowering, anti-allergenic, antiinflammatory, anti-tumour, and anti-microbial properties. Kefir is produced by two different methods as traditional and industrial methods. In traditional method, kefir grains are inoculated to milk and a milk is incubated at 20-25?C for an 18-24 h period. After incubation, kefir grains are sieved, washed and can be re-used for new kefir production. Kefir grains have a shape similar to small cauliflower or popcorn, with a diameter 0.3-2.0 cm and complex symbiotic microbial communities, including lactic acid bacteria, yeasts and acetic acid bacteria. The microorganisms in kefir grains should be kept viable by transferring kefir grains daily into milk. Besides, kefir grains can be stored in milk in refrigerator for 8-10 days. Different preservation methods such as freezing and freeze drying have been used to keep the microorganisms in kefir grains alive longer. The aim of this work was to investigate the effect of freeze drying on viability of microorganisms in kefir grains during storage.
Material and methods: The kefir grains used in this research were obtained from the Department of Food Engineering, Akdeniz University, Turkey. Skim milk powder, whey isolate powder and maltodextrin powder were suspended in distilled water at a concentration of 10% (w/w). Skim milk, whey isolate and maltodextrin solutions, which used as cryoprotective agents, were heat-treated at 62°C for 30 minutes and cooled to 25°C. After addition of kefir grains to cooled solutions (10%), mixtures were homogenized using a mechanical mixer. Freeze drying of the mixtures was performed with A FreeZone 2.5 Plus freeze dryer (Labconco Co., Kansas City, USA). Mixtures spread out at 1 mm layer on trays and frozen at -80°C for 24 h. Frozen mixtures were placed in the freeze dryer operated at -84°C and 0.02 mbar absolute pressure. After drying for 48 h, the dried mixtures were ground into fine powder using a grinder. All powders produced with different cryoprotective solutions were packaged under vacuum and atmospheric air conditions and stored at 25°C for 60 days. For kefir production, UHT skim milk was inoculated with the powders stored for 1, 30, and 60 days at a concentration of 0.5%. After inoculation, the milk samples were incubated at 25°C until the pH value decreased to 4.7. The pH values of the kefir samples were measured using a pH-meter (Orion 2 star, Thermo Scientific, Inc, USA) and titratable acidity was determined as % lactic acid according to Turkish Standards. The moisture content of the powders was determined using an infrared moisture balance (Precisa XM60, Intelligent Weighing Technology, Inc, Switzerland). Water activity measurements were performed at 25°C using an AquaLab water activity meter (4TE, Decagon Devices, Inc, Pullman, WA, USA). Microbiological analyses were run in duplicates for each mixture, powder and kefir sample. Ringer solution (1/4 strength) was used to prepare the dilutions for the microbiological analyses. Lactobacilli counts were performed on MRS medium at an incubation temperature of 30°C under anaerobic conditions for 3 days. Lactococci enumerations were carried out on M17 medium at an incubation temperature of 30°C under anaerobic conditions for 3 days. Yeast counts were performed on Yeast Extract Glucose Chloramphenicol (YGC) agar, with incubation at 25°C under aerobic conditions for 5 days. All analyses were performed on days 1, 30 and 60 of storage of the powders.
Results: The moisture content of the vacuum-packed powders ranged between 7.7 and 10.9%, which corresponded to water activity in the range of 0.32 and 0.48. The moisture content and water activity of the powders packed under atmospheric air were higher than those of the vacuum-packed powders. The initial counts of lactobacilli, lactococci and yeast in a mixture prepared from skim milk solution were 7.9, 6.5 and 5.5 log cfu/g, respectively. The highest counts of lactobacilli and lactococci were observed in the vacuum-packed powders obtained by skim milk solution. The reduction of lactobacilli, lactococci and yeast counts in all powders increased throughout the storage period. The pH values of milk samples inoculated with the 1-day stored powders decreased to 4.7 in between 24 and 30 hours of incubation, and the incubation time to reach a pH of 4.7 was extended for all samples inoculated with the 30-day stored powders. Kefir samples could not produced by using the 60-day stored powders because of the pH values of the milk samples inoculated with these powders did not decrease to pH 4.7 within 60 hours of incubation. The pH values of kefir samples obtained by using the 1- and 30-day stored powders were in the range of 4.41- 4.72 and 4.56- 4.73, respectively. The lowest pH value and the highest titratable acidity value were determined in kefir sample produced using by the vacuum-packed powder obtained by skim milk solution.
