Progress 07/01/21 to 04/12/22

Outputs
Target Audience:1. Probiotic supplement industries (BioGaia, Probi AB, Winclove Probiotics B.V., Protexin, i-Health, UAS Labs, Arla Foods, Lallemand, Danone, Nestle, Danisco A/S, and Chr. Hansen): These companies lead the market space with their global presence through well-established manufacturing and distribution channels. Jun Innovations plan to develop partnerships with some of these companies by granting a licensing agreement to integrate the supercooling technology into their existing freeze-drying technology. 2. Human Microbiome industries 3. Freeze-dryer manufacturers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?1. Contact companies that Jun Innovations has connections with to give a product presentation 2. Begin licensing negotiations with interested parties 3. Attend tradeshows to generate demand and exposure of the supercooling technology 4. Continue to develop technology and commercially viable units for technology demonstrations 5. Publications, patents, and seminars What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. For proof-of-concept purposes, a freezing unit incorporated with the supercooling function was designed and fabricated. The supercooling system mainly consisted of Helmholtz coils, an in-house electrical control unit, and a freezing unit. Helmholtz coils were utilized in order to produce a uniform magnetic field almost throughout the entire storage space. The OMF intensity and frequency was determined as 10 mT and 30 Hz, respectively based upon the literature and our previous research outcomes. According to the literature, OMF with an intensity of 10 mT disturbed the energy balance of water-based aqueous systems and it would result in the enhancement of polarized feature and dipole moments of water molecules through the magnetic interaction, indicating that the applied OMF may be able to directly act upon water molecules and inhibited ice nucleation. Moreover, we were able to observe that the OMF treatment could potentially suppress ice nucleation within pure water and multiple food materials. The fabricated Helmholtz coils were placed in a 7.1 cubic feet commercial chest freezer and the freezer temperature was controlled by a PID controller. A cooling fan (air-flow rate of 0.03 m3/min) was employed to provide air-flow to the samples. A sample holder was created using a 3D printer and an Agilent DAQ with 16-channel multiplexer was used to collect the temperature data. The temperature at the surface of the sample container was measured using K-type thermocouples. 2. Frozen L. acidophilus cultures were obtained from the Food Microbiology Lab (University of Hawaii at Manoa, Honolulu, HI). All experiments were conducted in a certified biosafety level II laboratory. 100 μL of each stock was inoculated separately in 10 mL of MRS broth and incubated for 24 hrs at 35°C. Test samples were prepared from serial dilutions of the stock cultures using 0.1% peptone water. The initial concentrations of L. acidophilus stock cultures were obtained by plate counting methods on plate count agar. L. acidophilus was cultured in 50 mL of MRS broth at 37°C for 24 hrs and the supernatant was removed by centrifuging at 6,000 rpm for 15 min. The pellets were washed with 0.9% sterilized saline solution and centrifuged at 6,000 rpm for 5 min, and the washing step was repeated three times. The harvested cells were mixed with a cryoprotective solution (sterile solution of 10% skim milk powder) to obtain suspensions of approximately 10^9 to 10^10 CFU/mL. Prior to freeze-drying, four different pretreatment processes were performed for 12 hrs: (1) Supercooling at -7°C (SC -7°C), (2) Refrigeration at 7°C (R 7°C), (3) Freezing at -7°C (F -7°C), and (4) Freezing at -18°C (F -18°C). After the pretreatments, the samples were frozen at -40°C for 6 hrs and lyophilized with a 48 hrs freeze-drying protocol. The viability of freeze-dried L. acidophilus was determined after freeze-drying by the standard plate count method using MRS agar medium. The viable count of bacteria was conducted before and after freeze-drying separately. The collected cells or dried bacterial powder were randomly selected and added into the sterile saline solution for a series of dilutions. After that, the bacterial suspension was poured on the MRS agar plate, and the plates were held in the incubator at 37 °C for 48 h. The highest viability (78.83%) of L. acidophilus was achieved with supercooling pretreatment (SC -7°C) followed by refrigeration (R 7°C), freezing -18°C (F -18°C), and freezing -7°C (F -7°C). It is clearly demonstrated that supercooling pretreatment prior to the freezing step enhanced the viability of L. acidophilus by (1) inducing the formation of large numbers of small and homogeneous ice crystals and (2) promoting the expression of the surface layer protein (SLP). 3. The cell wall outer layer has various polymers such as polysaccharides and lipoteichoic acids and surface layer protein (SLP) that bounds to the rigid peptidoglycan containing layer via secondary cell wall polymers. The surface proteins form a close packing that leaves a significant fraction of the surface exposed to the outside. The SLP has a uniform structure and physicochemical properties, which can spontaneously couple with constituent subunits. It can be suggested that the SLP maintains the high viability of Lactobacillus during freeze-drying and storage. To evaluate the cold adaptation response of L. acidophilus, the formation of SLP on the cell surface was analyzed after freeze-drying. Freeze-dried L. acidophilus cells were fixed with 2.5 % glutaraldehyde in 0.1M sodium cacodylate buffer with 2mM calcium chloride, pH 7.4 for 1-2 hrs, washed in 0.1M cacodylate buffer, followed by post-fixation with 1% OsO4 in 0.1M cacodylate buffer for 1 hr. Tissue was dehydrated twice in an ethanol series of 30%, 50%, 70%, 85%, 95%, and three times in absolute ethanol, and then the cells were substituted with propylene oxide and embedded in LX112 epoxy resin. Ultrathin (60-80 nm) sections were obtained on an RMC PowerTome ultramicrotome, double-stained with uranyl acetate and lead citrate, viewed on a Hitachi HT7700 TEM (transmission electron microscopy) at 100 kV, and photographed with an AMT XR-41B 2k x 2k CCD camera. The average thickness was determined from the measurements for five bacteria at different regions of each cell. There was a significant difference in the surface layer thickness between various pretreatment groups. The thickest layer of 10.53 ± 1.32 nm was observed with the supercooling pretreatment at -7°C followed by F -7°C (8.04 ± 1.76 nm), R 7°C (7.24 ± 1.77 nm), and F -18°C (7.12 ± 1.16 nm). The SLP thickness values of L. acidophilus pretreated with supercooling @ -7°C and conventional refrigeration @ 4°C increased by 69.02% and 16.21%, respectively. This finding may support that a lower pretreatment temperature contributes to an increased rate of SLP synthesis. The enhanced production of SLP induced by the supercooling pretreatment can promote the survival rate of probiotic bacteria by protecting them against ice crystal damage during freeze-drying.

Publications

• Type: Theses/Dissertations Status: Published Year Published: 2022 Citation: Wang, Y. 2022. Enhanced viability of freeze-dried Lactobacillus acidophilus using supercooling pretreatment, MS Thesis, University of Hawaii