Design of a new lyoprotectant increasing freeze-dried Lactobacillus strain survival to long-term storage

Abstract

Background 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 aw 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. Results 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). Conclusions 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.

Incorporating Cryopreservation Evaluations Into the Design of Cell-Based Drug Delivery Systems: An Opinion Paper

“…Cracking (microchanneling) of the PLGA polymer walls can also arise from overstretching of the pores during ice recrystallization thus further increasing the porosity of the microspheres which in turn is not favorable for sustained or prolonged drug release (14). The application of cryoprotectants in stabilizing lyophilized preparations has been explored in several studies (34)(35)(36)(37). Figueroa-Pizano et al utilized freezing-thawing in the fabrication of diflunisal loaded chitosan-polyvinyl alcohol (PVA) hydrogels.…”

Influence of Excipient Composition on Survival of Vaginal Lactobacilli in Electrospun Nanofibers

“…The OH groups in sucrose and alginate interact with the membrane proteins of bacteria. The addition of sucrose to the nanofibers loaded with bacteria resulted in hydrogen bonding with the membrane proteins and protecting the bacteria during electrospinning and long-term storage [26]. This explains the protective effect of sucrose on some species.…”

“…Specific excipients added to the nanofibers can act as lyoprotectants or prebiotics. Lyoprotectants prevent cell damage by protecting the protein structure of bacteria based on the "Water Replacement Hypothesis" [26], while prebiotics enhance bacterial growth. Excipients such as sucrose, trehalose, skim milk, lactose, and glycerol have been shown in previous studies to improve the survival of lactobacilli when added to nanofibers [20,25,27].…”

Novel Probiotic/Bacterial Cellulose Biocatalyst for the Development of Functional Dairy Beverage

“…Following Section 2.2 , cell mass was harvested by centrifugation at 7000 rpm for 10 min at 5 °C, and cells were collected after double-rinsing with Ringer’s solution (¼ strength) to remove substrate residues and fermentation by-products. Solutions of sucrose (2% w / v ) and whey protein (5% w / v ) were added to the cells as cryoprotectants [ 34 , 35 ]. Lyophilization process was carried out under vacuum in a freeze-drying system (BK-FD12P, Biobase Biodustry Co Ltd., Jinan, China) under the following conditions: cooling temperature −60 °C, pressure of 0.08 mbar, and lyophilization time of 24 h. The freeze-dried strains were placed in sealed vials and kept at 4 °C in a light-protected environment.…”