Stabilization of freeze-dried lactic acid bacteria during long-term storage is challenging for the food industry. Water activity of the lyophilizates is clearly related to the water availability and ...maintaining a low a
during storage allows to increase bacteria viability. The aim of this study was to achieve a low water activity after freeze-drying and subsequently during long-term storage through the design of a lyoprotectant. Indeed, for the same water content as sucrose (commonly used lyoprotectant), water activity is lower for some components such as whey, micellar casein or inulin. We hypothesized that the addition of these components in a lyoprotectant, with a higher bound water content than sucrose would improve lactobacilli strains survival to long-term storage. Therefore, in this study, 5% whey (w/v), 5% micellar casein (w/v) or 5% inulin (w/v) were added to a 5% sucrose solution (w/v) and compared with a lyoprotectant only composed of 5% sucrose (w/v). Protective effect of the four lyoprotectants was assessed measuring Lactiplantibacillus plantarum CNCM I-4459 survival and water activity after freeze-drying and during 9 months storage at 25 °C.
The addition whey and inulin were not effective in increasing Lactiplantibacillus plantarum CNCM I-4459 survival to long-term-storage (4 log reduction at 9 months storage). However, the addition of micellar casein to sucrose increased drastically the protective effect of the lyoprotectant (3.6 log i.e. 0.4 log reduction at 9 months storage). Comparing to a lyoprotectant containing whey or inulin, a lyoprotectant containing micellar casein resulted in a lower water activity after freeze-drying and its maintenance during storage (0.13 ± 0.05).
The addition of micellar casein to a sucrose solution, contrary to the addition of whey and inulin, resulted in a higher bacterial viability to long-term storage. Indeed, for the same water content as the others lyoprotectants, a significant lower water activity was obtained with micellar casein during storage. Probably due to high bound water content of micellar casein, less water could be available for chemical degradation reactions, responsible for bacterial damages during long-term storage. Therefore, the addition of this component to a sucrose solution could be an effective strategy for dried bacteria stabilization during long-term storage.
Freeze-drying is the most widely used dehydration process in the food industry for the stabilization of bacteria. Studies have shown the effectiveness of an acid prestress in increasing the ...resistance of lactic acid bacteria to freeze-drying. Adaptation of bacteria to an acid stress is based on maintaining the properties of the plasma membrane. Indeed, the fatty acid composition of the membrane of lactic acid bacteria is often changed after an acid prestress. However, few studies have measured membrane fluidity after an acid stress during lactic acid bacterial strain cultivation.
In order to use two pH profiles, the strains
NCDO 712 and NZ9000 were cultivated in two media, without any pH control. The two pH profiles obtained were representative of the initial medium composition, medium buffering properties and strain metabolism. Absorbance at 600 nm and pH were measured during bacterial cultivation. Then, the two strains were freeze-dried and their survival rates determined. Membrane fluidity was evaluated by fluorescence anisotropy measurements using a spectrofluorometer.
Cultivation under more acidic conditions significantly increased the survival during freeze-drying (p<0.05, ANOVA) of both strains. Moreover, in both strains of
, a more acidic condition during cultivation significantly increased membrane fluidity (p<0.05, ANOVA). Our results revealed that cultivation under such conditions, fluidifies the membrane and allows a better survival during freeze-drying of the two
strains. A more fluid membrane can facilitate membrane deformation and lateral reorganization of membrane components, critical for the maintenance of cellular integrity during dehydration and rehydration.
A better understanding of the involvement of membrane properties, especially of membrane fluidity, in bacterial resistance to dehydration is provided in this study.
Background and objective: Presence of oxygen during production and rehydration of freeze-dried starters and probiotics can decrease viability of the bacteria. Indeed, removal of water from cells ...during freeze-drying can promote dysfunction in anti-oxidative mechanisms, resulting in oxidative stress by accumulation of reactive oxygen species. The aim of this study was to show how atmospheric or less oxidative gaseous conditions affect bacterial survival to freeze-drying and rehydration of two strains, including Lactobacillus casei, a widely used bacteria in biotechnology, and Escherichia coli, a laboratory model bacteria. Material and methods: Lactobacillus casei ATCC 334 and Escherichia coli K12 were freeze dried for 24h in 5% sucrose (m v-1). Two gaseous conditions (an oxygen-free gas and atmospheric air) were used during various steps of the process, including bacterial cultivation, mixing of the bacteria with the protectant and rehydration. Oxygen-free gas condition was obtained with an oxygen-free gas, composed of nitrogen, hydrogen and carbon dioxide (N2H2CO2)and an anaerobic chamber. Results and conclusion: Gaseous conditions included significant effects on bacterial survival rates (P<0.001 for Lactobacillus casei and Escherichia coli). Interestingly, for both bacteria, the optimal combination was atmospheric air during mixing of the bacteria with the lyoprotectant (P<0.001 for Lactobacillus casei and Escherichia coli) and N2H2CO2 during rehydration (P<0.001 for Lactobacillus casei and P<0.05 for Escherichia coli). Management of gaseous conditions during a freeze-drying process and rehydration (atmospheric air during mixing of the bacteria with lyoprotectant and oxygen-free gas during rehydration) enhances survival of the bacteria by preserving them from oxidative stress.
Research background. Freeze-drying is the most widely used dehydration process in the food industry for the stabilization of bacteria. Studies have shown the effectiveness of an acid prestress in ...increasing the resistance of lactic acid bacteria to freeze-drying. Adaptation of bacteria to an acid stress is based on maintaining the properties of the plasma membrane. Indeed, the fatty acid composition of the membrane of lactic acid bacteria is often changed after an acid prestress. However, few studies have measured membrane fluidity after an acid stress during lactic acid bacterial strain cultivation.
Experimental approach. In order to use two pH profiles, the strains Lactococcus lactis NCDO 712 and NZ9000 were cultivated in two media, without any pH control. The two pH profiles obtained were representative of the initial medium composition, medium buffering properties and strain metabolism. Absorbance at 600 nm and pH were measured during bacterial cultivation. Then, the two strains were freeze-dried and their survival rates determined. Membrane fluidity was evaluated by fluorescence anisotropy measurements using a spectrofluorometer.
Results and conclusions. Cultivation under more acidic conditions significantly increased the survival during freeze-drying (p<0.05, ANOVA) of both strains. Moreover, in both strains of L. lactis, a more acidic condition during cultivation significantly increased membrane fluidity (p<0.05, ANOVA). Our results revealed that cultivation under such conditions, fluidifies the membrane and allows a better survival during freeze-drying of the two L. lactis strains. A more fluid membrane can facilitate membrane deformation and lateral reorganization of membrane components, critical for the maintenance of cellular integrity during dehydration and rehydration.
Novelty and scientific contribution. A better understanding of the involvement of membrane properties, especially of membrane fluidity, in bacterial resistance to dehydration is provided in this study.
Pozadina istraživanja. Sušenje zamrzavanjem je često primjenjivani postupak dehidracije u svrhu stabilizacije bakterija koje se koriste u prehrambenoj industriji. Dosadašnja su istraživanja pokazala ...da se uzgojem u kiselom mediju uspješno povećava otpornost bakterija mliječno-kiselog vrenja na sušenje zamrzavanjem. Prilagodba bakterija na kiselinski stres ovisi o održavanju svojstava stanične membrane. Sastav masnih kiselina u membrani bakterija mliječno-kiselog vrenja često se mijenja nakon uzgoja u kiselom mediju. Međutim, u malom je broju istraživanja mjerena fluidnost membrane bakterija mliječno-kiselog vrenja nakon izlaganja kiselinskom stresu tijekom njihovog uzgoja.
Eksperimentalni pristup. Radi ispitivanja stope preživljavanja bakterija pri dvije pH-vrijednosti, sojevi bakterija Lactococcus lactis NCDO 712 i NZ9000 uzgojeni su na dvjema različitim hranjivim podlogama bez reguliranja pH-vrijednosti. Dva dobivena profila su odražavala razlike u početnom sastavu podloga, prilagodbi bakterija na promjenu pH-vrijednosti te metabolizmu sojeva. Tijekom uzgoja mjereni su apsorbancija pri 600 nm i pH-vrijednost podloga. Sojevi su zatim sušeni zamrzavanjem, te su praćene njihove stope preživljavanja. Fluidnost membrana je ispitana mjerenjem fluorescentne anizotropije pomoću spektrofluorometra.
Rezultati i zaključci. Uzgojem u kiseloj sredini bitno se povećala stopa preživljavanja obaju sojeva bakterije L. lactis tijekom sušenja zamrzavanjem (p<0,05; ANOVA). Osim toga, u oba se soja bakterije snižavanjem pH-vrijednosti tijekom uzgoja povećala fluidnost membrana (p<0,05; ANOVA). Dobiveni rezultati pokazuju da se uzgojem pri navedenim uvjetima fluidnost stanične membrane povećala, što je povećalo stopu preživljavanju ovih dvaju sojeva bakterija L. lactis tijekom sušenja zamrzavanjem. Veća fluidnost pospješuje deformaciju membrane i lateralnu reorganizaciju njezinih sastavnih elemenata, što je neophodno za održavanje integriteta stanice tijekom dehidracije i rehidracije.
Novina i znanstveni doprinos. Ovaj rad pridonosi boljem razumijevanju uloge stanične membrane, osobito njezine fluidnosti, u mehanizmu otpornosti bakterija na dehidraciju.
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