Injury Mechanism and Protection Methods of Spray-dried Lactobacillus Bulgaricus

Author:Gong Zuo Min

Supervisor:zhang lan wei


Degree Year:2019





Spray drying has the advantages of low cost,high efficiency,rapidity and continuousness.It is a potential drying technology which can replace freeze-drying to prepare lactic acid bacteria(LAB)powder.However,the lethal effect of hot air on LAB during spray drying leads to the low survival rate.At present,the lack of indepth research on the damage mechanism of LAB during the spray drying process has limited the application of spray drying in the preparation of LAB powder.At first,a novel sampling method was established to study changes in the activity of LAB during spray drying.A sampling device with a refrigerant was designed to maintain the low temperature environment in the sampling cup and was pre-cooled before spray dying.Carbonyl iron powder(CIP)was added to the feed suspensions to capture samples with different water content at different sites by the magnetic effect between CIP and magnet on the bottom of the sampling cup.During drying,a temperature sensor measured the particle contact temperature.The height of the sampling cup should be increased with the increase of contact temperature to maintain the moisture content of the captured samples.A sampling cup with a depth of 3.5 cm(for contact temperatures below 79.1 °C)or 5 cm(for contact temperatures below 89.1 °C).The ratio of CIP to skim milk powder was 1:10,and reconstituted skim milk(RSM)concentration was not lower than 20%(w/w)to get enough mass of samples.Sterile water placed in the sampling cup prior to drying resulted in a more accurate measurement of cell viability.The samples of LAB at different stages of spray drying can be obtained accurately and reliably by this method.The relationship between survival rates of Lactobacillus bulgaricus sp1.1 and water content/activities was established to identify the injury mechanism in cellular structures and constituents during 120/70 ℃ spray drying.The results revealed that 90% of bacterium were killed when the water content was <23.52%(aw?< 0.82).Results of Fourier-transform infrared measurement and changes in viable cells on differential culture mediums showed that cell wall was the first site to be injured when the water content reached <61.50%,and the α-helices in the overall protein changed at the beginning stage of drying.Permeability of the cell membrane was damaged at < 23.52%,and leading most bacterium to death,which was the critical injury site.After drying,the membrane changes and the repair function of cell wall is inhibited.The structural damage sequence of LAB was characterized from outside to inside.Study on the effects of different inlet and outlet temperatures on the activity of spray-dried LAB showed the survival rate increased with the decrease of the outlet temperature.When the outlet temperature was lower than 65 °C,the activity of LAB no longer changed,and the survival rate was about 28%.Optimized temperature condition of spray-dried LAB was 120/60 °C,which led to high survival rate and water content < 7%.From the relationship between LAB,water and temperature during spray drying at inlet and outlet temperatures of 120/70 °C,120/60 °C and 110/60 °C,we found that lowering the outlet temperature would cause the prolong the movement of droplets in the main circulation area to extend the drying time.When the outlet temperature was 70 °C,cells were simultaneously damaged by heat damage and dehydration,number of cells decreased with the decreasing moisture content.When the outlet temperature was 60 °C,cells were only inactivated by dehydration,the number of viable cells was unchanged when the moisture content was reduced to a certain value.Reduce in outlet temperature effectively increased the survival rate by decreasing the fatal cell membrane damage.The higher outlet temperature lower the key moisture contents for spray– dried LAB.When the outlet temperature was 60 °C,the key moisture content of most LAB death was 27.50%.When the outlet temperature was 70 °C,the key moisture content of most cells’ death decreased to 21.99%.According to the characteristics of lactobacillus from outside to inside in the process of spray drying,the electrostatic attraction between basic proteins and lactobacillus and acidic proteins and the cross-linking effect of Transglutaminase(TGase)on protein molecules are utilized to shorten the distance between proteins and bacteria so as to improve the extracellular protection.The results showed that the mixing of basic proteins with acidic proteins and TGase treatment increased the survival rates.84.78% of cells remained after spray drying because TGase-treated milk basic protein(MBP)-soy protein isolate(SPI)protected cell membrane significantly.Positively charged MBP formed tight closed loop-shaped complexes with SPI by electrostatic attraction,and adhered to more bacteria.TGase did not changes the charge of proteins,but can decrease the volumes of protein-cell complex by strengthening the hydrogen,C=O and C=N bonds,and expose the hydrophobic group of inner α-helix to adhere to bacteria.Cell membrane is the key damage site of LAB spray drying.Trehalose can be loaded into cells by electroporation to enhance the protection of cell membrane.The results showed that the survival rates of spray-dried bacteria increased at an intracellular trehalose content of > 3.47 μg/107 CFU and reached 99.59% when intracellular trehalose content was 10.09 μg/107 CFU.Electroporation damaged the cell membrane,but intracellular trehalose protected cell membrane from further damage by electroporation and spray drying,so moderate electroporation did not decrease the overall survival rate of lactobacillus after spray drying.The loading of trehalose by 2.5 kV/cm for 2 pulses can increase the total survival rate to 61.30%.Finally,the survival rate of spray-dried LAB was as high as 93.5% when the twotreatment mentioned above were combined.In this study,sampling during spray drying process was achieved,and the injury mechanism of LAB during the spray drying was determined.Transglutaminase treatment of acid-basic protein and electroporation-loading trehalose technology can be applied to improve the survival rate of spray-dried LAB.