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.
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.
Effect of high pressure (HP) treatment on the antimicrobial properties and the structure of nisin was evaluated. Nisin solutions at pH 2.8 or 6.1 were treated by HP at 500 MPa – 10 min – 20 °C and ...their antimicrobial potency was determined. It appeared that HP clearly impacted the antimicrobial activity of nisin, with respective activity loss of 22.5% and 49.9% at pH 2.8 and 6.1. Structural analysis of nisin by circular dichroism and Fourier transform-infrared spectroscopies revealed that the decrease of nisin antimicrobial activity was likely due to the unfolding of the protein induced by HP. A loss of nisin β-turns structure, particularly significant at neutral pH, was linked to the drastic drop in antimicrobial activity, as these structures are implicated in the nisin interaction with the bacterial membrane.
The combination of nisin and high pressure (HP) can be use at an industrial scale to inactivate bacteria. Nisin is allowed as a food additive (E234) and can be added at a final concentration ranging from 120 to 500 IU/g, depending on the product. In this work, we showed that HP can induce a significant reduction of nisin activity (-22.5% at pH 2.8 and -49.9% at pH 6.1). Therefore, this activity loss could be taken into account to manage the final nisin concentration in HP-treated food products.
•HP treatment (500 MPa – 10 min – 20 °C) reduced the antimicrobial activity of nisin of 22.5% at pH 2.8 and 49.9% at pH 6.1.•While nisin is randomly coiled at pH 2.8, the presence of β-turns at pH 6.1 induced a lower stability to HP.•The drop in antimicrobial activity after HP was due to the unfolding of nisin, resulting in a loss of the β-turns structure.
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•New additive-free yeast cells were dehydrated in a pilot fluidized bed dryer.•GSM and TSM medium promotes the synthesis of glutathione and trehalose, respectively.•High survival was ...found for glutathione-rich cells during dehydration at 45 °C–90 min.•Trehalose-rich yeast cells were more resistant to severe dehydration at 60 °C–60 min.
Fluidized bed drying is a dehydration process often applied to microorganisms and used for food particles stabilization. Despite its common use in yeast production, this process can lead to cell death depending on the strain and the growth conditions. During fermentation, yeasts undergo nutritional stressful environments according to media composition. In order to face these conditions, yeasts are able to synthesize protective molecules, e.g. glutathione (tripeptide, an antioxidant) and trehalose (disaccharide, a cell desiccation protector). However, the role of these two molecules during fluidized bed drying is still not well known. This work aimed to investigate if total glutathione (GSHt) and trehalose (TRE) accumulation leads to the creation of more resistant and stable yeast cells. The effect of GSHt accumulation after relatively nutrient richer cultivation on cell resistance to mild stress (45 °C–90 min) was obvious. This growing condition also improved the stabilization and performance of cells after rehydration. On the other hand, the highest amount of TRE stored by yeasts cultivated in nutrient poorer media led to cells being more resistant to severe stress (60 °C–60 min). This strategy could be used for the development of new bioprocesses focusing on improving the performance and resistance of cells after fluidized bed drying.
During slow freezing, spermatozoa undergo membrane alterations that compromise their ability of fertilizing. These alterations are cause either by cold shock or by the use of cryoprotectants known to ...be cytotoxic. However, little is known about the membrane changes that occurred during freezing. Here, we combined Generalized Polarization (GP), Time-resolved Fluorescence and laurdan fluorescence properties to investigate the changes in membrane fluidity and dynamics during slow freezing of bull sperm. We successfully demonstrated that laurdan may be distributed in three different local environments that correspond to different membrane lipid composition. These environments wont behave the same way when the cells will be subjected to either a chemical treatment (adding the cryoprotectants) or a physical treatment (freezing).
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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