Determining Protein Structures Using X-Ray Crystallography.

X-ray crystallography is a robust and widely used technique that facilitates the three-dimensional structure determination of proteins at an atomic scale. This methodology entails the growth of protein crystals under controlled conditions followed by their exposure to X-ray beams and the subsequent analysis of the resulting diffraction patterns via computational tools to determine the three-dimensional architecture of the protein. However, achieving high-resolution structures through X-ray crystallography can be quite challenging due to complexities associated with protein purity, crystallization efficiency, and crystal quality.In this chapter, we provide a detailed overview of the gene to structure determination pipeline used in X-ray crystallography, a crucial tool for understanding protein structures. The chapter covers the steps in protein crystallization, along with the processes of data collection, processing, structure determination, and refinement. The most commonly faced challenges throughout this procedure are also addressed. Finally, the importance of standardized protocols for reproducibility and accuracy is emphasized, as they are crucial for advancing the understanding of protein structure and function.

Advancements in fertility preservation strategies for pediatric male cancer patients: a review of cryopreservation and transplantation of immature testicular tissue.

Recently, there has been increasing emphasis on the gonadotoxic effects of cancer therapy in prepubertal boys. As advances in oncology treatments continue to enhance survival rates for prepubertal boys, the need for preserving their functional testicular tissue for future reproduction becomes increasingly vital. Therefore, we explore cutting-edge strategies in fertility preservation, focusing on the cryopreservation and transplantation of immature testicular tissue as a promising avenue. The evolution of cryopreservation techniques, from controlled slow freezing to more recent advancements in vitrification, with an assessment of their strengths and limitations was exhibited. Detailed analysis of cryoprotectants, exposure times, and protocols underscores their impact on immature testicular tissue viability. In transplantation strategy, studies have revealed that the scrotal site may be the preferred location for immature testicular tissue grafting in both autotransplantation and xenotransplantation scenarios. Moreover, the use of biomaterial scaffolds during graft transplantation has shown promise in enhancing graft survival and stimulating spermatogenesis in immature testicular tissue over time. This comprehensive review provides a holistic approach to optimize the preservation strategy of human immature testicular tissue in the future.

Effects of exogenous glycine betaine on growth and development of tomato seedlings under cold stress.

Low temperature is a type of abiotic stress affecting the tomato (Solanum lycopersicum) growth. Understanding the mechanisms and utilization of exogenous substances underlying plant tolerance to cold stress would lay the foundation for improving temperature resilience in this important crop. Our study is aiming to investigate the effect of exogenous glycine betaine (GB) on tomato seedlings to increase tolerance to low temperatures. By treating tomato seedlings with exogenous GB under low temperature stress, we found that 30 mmol/L exogenous GB can significantly improve the cold tolerance of tomato seedlings. Exogenous GB can influence the enzyme activity of antioxidant defense system and ROS levels in tomato leaves. The seedlings with GB treatment presented higher Fv/Fm value and photochemical activity under cold stress compared with the control. Moreover, analysis of high-throughput plant phenotyping of tomato seedlings also supported that exogenous GB can protect the photosynthetic system of tomato seedlings under cold stress. In addition, we proved that exogenous GB significantly increased the content of endogenous abscisic acid (ABA) and decreased endogenous gibberellin (GA) levels, which protected tomatoes from low temperatures. Meanwhile, transcriptional analysis showed that GB regulated the expression of genes involved in antioxidant capacity, calcium signaling, photosynthesis activity, energy metabolism-related and low temperature pathway-related genes in tomato plants. In conclusion, our findings indicated that exogenous GB, as a cryoprotectant, can enhance plant tolerance to low temperature by improving the antioxidant system, photosynthetic system, hormone signaling, and cold response pathway and so on.

Cereus jamacaru DC. (mandacaru) fruit as a source of lactic acid bacteria with in vitro probiotic-related characteristics and its protective effects on Pediococcus pentosaceus during lyophilization and refrigeration storage.

This study isolated and identified autochthonous lactic acid bacteria (LAB) from mandacaru fruit and evaluated their potential probiotic and technological aptitudes in vitro, as well as the protective effects of freeze-dried mandacaru fruit on the most promising LAB isolate during lyophilization and refrigeration storage. Initially, 212 colonies were isolated from mandacaru fruit, and 34 were preliminarily identified as LAB. Thirteen isolates identified by 16S-rRNA sequencing as Pediococcus pentosaceus were negative for DNase, gelatinase, hemolytic, and biogenic amine production. The selected isolates showed proteolytic activity, diacetyl and exopolysaccharide production, and good tolerance to different NaCl concentrations while having low cellular hydrophobicity and antagonistic activity against pathogens. The survival of isolates sharply decreased after 3 h of exposure to pH 2 and had a good tolerance to 1 % bile salt. A principal component analysis selected P. pentosaceus 57 as the most promising isolate based on the examined technological and probiotic-related physiological properties. This isolate was lyophilized with mandacaru fruit and stored under refrigeration for 90 days. P. pentosaceus 57 lyophilized with mandacaru fruit had high viable cell counts (9.69 ± 0.03 log CFU/mL) and >50 % of physiologically active cells at 90 days of refrigeration storage. The results indicate that mandacaru fruit is a source of P. pentosaceus with aptitudes to be explored as potential probiotic and technological characteristics of interest for the food industry, besides being a good candidate for use in lyophilization processes and refrigeration storage of LAB due to its cryoprotective effects.

Development of sperm cryopreservation protocol for patin buah, Pangasius nasutus.

The development of sperm cryopreservation for Pangasius nasutus is necessary in order to serve the growing demand of this species through artificial fertilization and the preservation of valuable strains of male broodstocks. In the present study, the basic protocol of sperm cryopreservation for P. nasutus was established by identifying the optimal conditions for optimum cryoprotectant, toxicity of cryoprotectants, extenders, freezing condition and dilution ratio. Methanol (MeOH) at 10% concentration had the best post-thaw motility (26.3 ± 0.9%) and curvilinear velocity (VCL) compared to dimethyl acetamide and dimethyl sulfoxide. MeOH was the least toxic cryoprotectant; sperm suspended in 5 and 10% MeOH maintained motility up to 50 min. No significant differences were detected between the three types of extenders tested (0.9% sodium chloride, Calcium-free Hanks' Balance salt solution and ringer solution). P. nasutus sperm had a narrow range of optimal cooling rate. Significantly higher post-thaw motility was identified when cooling at 9.23 °C min-1, obtained by freezing at height of 14 cm above liquid nitrogen vapor for 7 min, showing lower cooling rate is suitable for this species. However, when cooling below and above the optimal cooling rate, post-thaw motility dropped drastically. There were no significant differences among the dilution ratios investigated, indicating the volume of cryodiluent at all tested ratios (1:9, 1:19 and 1:49) was sufficient for the protection of cells during the cryopreservation process. The development of the protocol for cryopreserved P. nasutus sperm will assist artificial seed production and provide an important tool for genetic and breeding research.

Effects of different cryopreservation parameters on the differences between trypan blue and fluorescent SYTO 13/GelRed assays.

Post-thaw cell viability assessment is very important in cryopreservation because it is the main assessment method used to optimize the cryopreservation protocols for each cell type; hence, having standardized accurate, quick, and reliable assays for post-thaw cell viability measurements is of utmost importance. The trypan blue exclusion assay and nucleic-acid-binding fluorescence-based assays are two different methods for cell viability assessment. Both assays identify cells with damaged membranes by whether they let a compound enter the cell. In this study, these two assays are compared in the context of cryopreservation and the impacts of important cryopreservation parameters on the differences in measurements are investigated. H9c2 myoblasts were cryopreserved with different freezing protocols. Cell membrane integrities were measured immediately after thaw as well as after cryoprotectant removal by a hemocytometer-based trypan blue dye exclusion assay and a dual fluorometric SYTO 13/GelRed assay; and the results were compared. This study quantifies how i) the absence or presence of different cryoprotectants, ii) different cell-cryoprotectant incubation conditions, and iii) the presence or removal of cryoprotectants after thaw affect the differences between these two viability assays.

Cryopreservation of the collector urchin embryo, Tripneustes gratilla.

The collector urchin, Tripneustes gratilla, is an ecologically important member of the grazing community of Hawai'i's coral reefs. Beyond its ability to maintain balance between native seaweeds and corals, T. gratilla has also been used as a food source and a biocontrol agent against alien invasive algae species. Due to overexploitation, habitat degradation, and other stressors, their populations face local extirpation. However, artificial reproductive techniques, such as cryopreservation, could provide more consistent seedstock throughout the year to supplement aquaculture efforts. Although the sperm and larvae of temperate urchins have been successfully cryopreserved, tropical urchins living on coral reefs have not. Here, we investigated the urchin embryos' tolerance to various cryoprotectants and cooling rates to develop a cryopreservation protocol for T. gratilla. We found that using 1 M Me2SO with a cooling rate of 9.7 °C/min on gastrula stage embryos produced the best results with survival rates of up to 85.5% and up to 50.8% maturation to the 4-arm echinopluteus stage, assessed three days after thawing. Continued research could see cryopreservation added to the repertoire of artificial reproductive techniques for T. gratilla, thereby assisting in the preservation of this ecologically important urchin, all while augmenting aquaculture efforts that contribute to coral reef restoration.

Novel Apoplastic Antifreeze Proteins of Deschampsia antarctica as Enhancer of Common Cell Freezing Media for Cryobanking of Genetic Resources, a Preliminary Study.

Antifreeze proteins (AFPs) are natural biomolecules found in cold-adapted organisms that lower the freezing point of water, allowing survival in icy conditions. These proteins have the potential to improve cryopreservation techniques by enhancing the quality of genetic material postthaw. Deschampsia antarctica, a freezing-tolerant plant, possesses AFPs and is a promising candidate for cryopreservation applications. In this study, we investigated the cryoprotective properties of AFPs from D. antarctica extracts on Atlantic salmon spermatozoa. Apoplastic extracts were used to determine ice recrystallization inhibition (IRI), thermal hysteresis (TH) activities and ice crystal morphology. Spermatozoa were cryopreserved using a standard cryoprotectant medium (C+) and three alternative media supplemented with apoplastic extracts. Flow cytometry was employed to measure plasma membrane integrity (PMI) and mitochondrial membrane potential (MMP) postthaw. Results showed that a low concentration of AFPs (0.05 mg/mL) provided significant IRI activity. Apoplastic extracts from D. antarctica demonstrated a cryoprotective effect on salmon spermatozoa, with PMI comparable to the standard medium. Moreover, samples treated with apoplastic extracts exhibited a higher percentage of cells with high MMP. These findings represent the first and preliminary report that suggests that AFPs derived from apoplastic extracts of D. antarctica have the potential to serve as cryoprotectants and could allow the development of novel freezing media.

Optimization of cryoprotectant formulation to enhance the viability of Lactiplantibacillus plantarum NBC99 isolated from human origin.

Freeze drying has been well applied in the preparation of high-efficiency viability probiotic powders. However, the process is generally accompanied by probiotic viability deficiency, which poses a problem for further application. In this study, various kinds of cryoprotectant formulations (skim milk, maltodextrin, and sucrose) were tested to enhance the survival of Lactiplantibacillus plantarum NBC99 after freezing and freeze-drying. An I-optimal experimental design-oriented optimization model was presented to optimize the cryoprotective medium, and the highest cell survival was observed with 25% skim milk, 8.71% maltodextrin, and 1.13% sucrose cryoprotectant as the optimum condition. L. plantarum NBC99 has been a good potential strain for the manufacture of an industrial probiotic, and this research has aimed to investigate the long-term protective effects of optimum cryoprotectant formulations on the viability of bacteria. The results showed the potential value of freeze-dried probiotic L. plantarum NBC99 culture for commercialization.

Investigating the cryoprotective efficacy of fructans in mammalian cell systems via a structure-functional perspective.

Fructans have long been known with their role in protecting organisms against various stress factors due to their ability to induce controlled dehydration and support membrane stability. Considering the vital importance of such features in cryo-technologies, this study aimed to explore the cryoprotective efficacy of fructans in mammalian cell systems where structurally different fructan polymers were examined on in vitro cell models derived from organs such as the liver, frequently used in transplantation, osteoblast, and cord cells, commonly employed in cell banking, as well as human seminal fluids that are of vital importance in assisted reproductive technology. To gain insights into the fructan/membrane interplay, structural differences were linked to rheological properties as well as to lipid membrane interactions where both fluorescein leakage from unilamellar liposomes and membrane integrity of osteoblast cells were monitored. High survival rates obtained with human endothelial, osteoblast and liver cells for up to two months clearly showed that fructans could be considered as effective non-permeating cryoprotectants, especially for extended periods of cryopreservation. In trials with human seminal fluid, short chained levan in combination with human serum albumin and glycerol proved very effective in preserving semen samples across multiple patients without any morphological abnormalities.

Molecular modelling studies reveal cryoprotective mechanism of L-Proline during the frozen storage of shrimp (Litopenaeus vannamei).

This study aimed to investigate the cryoprotective properties of proline (1% and 3% (w/v)) on shrimp. The cryoprotective mechanism was studied using physico-chemical experiments and molecular simulations. Proline had a notable positive impact on the thawing loss and texture of shrimp in comparison to the control. The denaturation of myosin in frozen shrimp was delayed by proline. Microscopy analysis demonstrated that proline effectively lowered the harm caused by ice crystals to shrimp muscle. Molecular simulations indicated that proline potentially exerted a cryoprotective effect primarily through the "water substitution" and "glassy state" hypotheses. Proline formed hydrogen bonds with myosin to replace the water molecules around myosin. Additionally, proline interacted with water molecules to form a glassy state, impeding the growth of ice crystals. Consequently, the stability of shrimp myosin was enhanced during freezing. In conclusion, proline demonstrated promise as an efficacious cryoprotectant for aquatic products.

Inulin Can Improve Red Blood Cell Cryopreservation by Promoting Vitrification, Stabilizing Cell Membranes, and Inhibiting Ice Recrystallization.

In transfusion medicine, the cryopreservation of red blood cells (RBCs) is of major importance. The organic solvent glycerol (Gly) is considered the current gold-standard cryoprotectant (CPA) for RBC cryopreservation, but the deglycerolization procedure is complex and time-consuming, resulting in severe hemolysis. Therefore, it remains a research hotspot to find biocompatible and effective novel CPAs. Herein, the natural and biocompatible inulin, a polysaccharide, was first employed as a CPA for RBC cryopreservation. The presence of inulin could improve the thawed RBC recovery from 11.83 ± 1.40 to 81.86 ± 0.37%. It was found that inulin could promote vitrification because of its relatively high viscosity and glass transition temperature (Tg'), thus reducing the damage during cryopreservation. Inulin possessed membrane stability, which also had beneficial effects on RBC recovery. Moreover, inulin could inhibit the mechanical damage induced by ice recrystallization during thawing. After cryopreservation, the RBC properties were maintained normally. Mathematical modeling analysis was adopted to compare the performance of inulin, Gly, and hydroxyethyl starch (HES) in cryopreservation, and inulin presented the best efficiency. This work provides a promising CPA for RBC cryopreservation and may be beneficial for transfusion therapy in the clinic.

Use of Powdered Milk in Semen Cryopreservation Protocols for Fish: A Systematic Review.

This systematic review provides an overview of the history and current status of cryopreservation of fish sperm and a detailed evaluation of cryoprotocols using powdered milk. A literature search was performed in PubMed, Scopus, Web of Science, and SciELO databases. Twenty-nine articles were selected after excluding duplicate articles or articles that did not meet the eligibility criteria. Rhamdia quelen and Danio rerio were the most studied species. Slow freezing method, dry-shipper, freezing rate of -35.6°C/min, thawing in water bath (35.93°C ± 10°C), and 0.25 and 0.5 mL plastic straws were the main approaches evaluated. Methanol was the most used permeable cryoprotectant in combination with powdered milk, yielding the best results at 10% concentration. Motility rate was the main analysis performed after cryopreservation in virtually all studies, being subjectively evaluated by most authors. Powdered milk at 15% promoted the best results in the analyzed studies. For motility rate, the gains with the addition of powdered milk were observed in the orders Perciformes (Oreochromis mossambicus), Siluriformes (Pangasius pangasius, Pseudoplatystoma corruscans, and Pseudoplatystoma mataense), and Cypriniformes (Tor soro and Barbonymus gonionotus). For fertilization, gains were observed in the order Siluriformes (P. mataense) and Cypriniformes (T. soro). Sperm viability gains were observed in the orders Siluriformes (P. pangasius), Characiformes (Piaractus brachypomus), and Cypriniformes (B. gonionotus). The scientific evidence we present in this study may contribute and serve as a starting point for new and more refined studies to be developed in the field.

Effect of freeze-dried protectants on the survival rate and fermentation performance of fermented milk's directed vat set starters.

A directed vat set (DVS) starter was proposed to improve the drawbacks of liquid starters in fermented production and enhance the survival rates of B. animalis subsp. lactis BZ11, S. thermophilus Q-1, and Lactiplantibacillus plantarum LB12. The protective agent formula was optimized using the response surface method (RSM), with the survival rate as the benchmark. The best combination of cryoprotectants was determined to be BZ11: 10 % skimmed milk powder, 3 % sodium glutamate, and 15 % trehalose; LB12: 10 % skim milk powder, 5 % glutamate sodium, and 10 % trehalose; Q-1: 10 % skimmed milk powder, 3 % sodium glutamate, and 10 % trehalose. The survival rate of BZ11 significantly increased to 92.87 ± 1.25 %. The DVS fermented milk did not differ significantly from the control group regarding cholesterol removal, live cell counts and pH (p > 0.05). All DVS can be stored for at least 2500 d at -20 °C-this DVS starter for fermented milk benefits from its large-scale and automated commercial production.

Development of heart organoid cryopreservation method through Fe3 O4 nanoparticles based nanowarming system.

Beyond single cell two-dimensional (2D) culture, research on organoids that can mimic human organs is rapidly developing. However, there are still problems in commercialization and joint research using organoids due to the lack of technology to safely store organoids. Since organoids are 3D complex structures with a certain size (0.1-5 mm) beyond the size of cells, the conventional cell-level cryopreservation method using cryoprotectant (CPA) cannot overcome the damage caused by volume change due to osmotic pressure difference and ice nucleation. Herein, we attempted to solve such limitations by applying a nanowarming system using CPA with high cell permeability and Fe3 O4 nanoparticles. By performing beat rate measurement, histological analysis, contractility analysis, and multi-electrode array, it was verified that the developed method could significantly improve functional recovery and survival of heart organoids after freezing and thawing. In this study, we demonstrated a successful organoid cryopreservation method based on a Fe3 O4 nanowarming system. The developed technology will provide clues to the field of tissue cryopreservation and spur the application of organoids.

Freeze-drying revolution: unleashing the potential of lyophilization in advancing drug delivery systems.

Lyophilization also known as freeze-drying is a technique that has been employed to enhance the long-term durability of nanoparticles (NPs) that are utilized for drug delivery applications. This method is used to prevent their instability in suspension. However, this dehydration process can cause stress to the NPs, which can be alleviated by the incorporation of excipients like cryoprotectants and lyoprotectants. Nevertheless, the freeze-drying of NPs is often based on empirical principles without considering the physical-chemical properties of the formulations and the engineering principles of freeze-drying. For this reason, it is crucial to optimize the formulations and the freeze-drying cycle to obtain a good lyophilizate and ensure the preservation of NPs stability. Moreover, proper characterization of the lyophilizate and NPs is of utmost importance in achieving these goals. This review aims to update the recent advancements, including innovative formulations and novel approaches, contributing to the progress in this field, to obtain the maximum stability of formulations. Additionally, we critically analyze the limitations of lyophilization and discuss potential future directions. It addresses the challenges faced by researchers and suggests avenues for further research to overcome these limitations. In conclusion, this review is a valuable contribution to the understanding of the parameters involved in the freeze-drying of NPs. It will definitely aid future studies in obtaining lyophilized NPs with good quality and enhanced drug delivery and therapeutic benefits.

Usage of ectoine as a cryoprotectant for cryopreservation of lactic acid bacteria.

Streptococcus thermophilus, the only Streptococcus species considered "Generally Recognized Safe", has been used widely in the food industry. This bacterium is one of the most valuable industrial lactic acid bacterial species. Due to the importance of this bacterium in industrial applications, it should be stored for a long time without losing its metabolic properties. The present study aimed to investigate the cryoprotectant effect of three compatible solutes (ectoine, trehalose, and sucrose) on bacterial cells stored at different temperatures (frozen at -80 °C or freeze-dried and subsequently stored at +4, -20, and -80 °C) for three months. The bacterial cells were tested for cell viability, bile salt tolerance, and lactic acid production before and after processing. The highest cell viability, bile salt tolerance, and lactic acid production were obtained with ectoine and under frozen (storage at -80 °C) conditions. In freeze-dried and subsequently stored at various temperatures, the best preservation was obtained at -80 °C, followed by -20 °C and +4 °C. Moreover, when ectoine's preservation potential was compared to other cryoprotectants, ectoine showed the highest preservation, followed by trehalose and sucrose. Although ectoine has a variety of qualities that have been proven, in the current work, we have shown for the first time that ectoine has cryoprotectant potential in yogurt starter cultures (S. thermophilus).

Poly(l-alanine-co-l-threonine succinate) as a Biomimetic Cryoprotectant.

We synthesized a series of [(l-Ala)x-co-(l-Thr succinate)y] (PATs), which are analogous to natural antifreezing glycoprotein with the structure of [l-Ala-l-Ala-l-Thr disaccharide]n, by varying the composition and degree of succinylation while fixing their molecular weight (Mn) and Ala/Thr ratio at approximately 10-12 kDa and 2:1, respectively. We investigated their ice recrystallization inhibition (IRI), ice nucleation inhibition (INI), dynamic ice shaping (DIS), thermal hysteresis (TH), and protein cryopreservation activities. Both IRI and INI activities were greater for PATs with higher l-Ala content (PATs-3 and PATs-4) than those with lower l-Ala content (PATs-1 and PATs-2). DIS activity with faceted crystal growth was clearly observed in PATs-2 and PATs-4 with a high degree of succinylation. TH was small with <0.1 °C for all PATs and slightly greater for PATs with a high l-Ala content. Except for PATs-1, the protein (lactate dehydrogenase, LDH) stabilization activity was excellent for all PATs studied, maintaining LDH activity as high as that of fresh LDH even after 15 freeze-thaw cycles. To conclude, the cryo-active biomimetic PATs were synthesized by controlling the l-Ala content and degree of succinylation. Our results showed that PATs with an l-Ala content of 65-70% and degree of succinylation of 12-19% exhibited the cryo-activities of IRI, INI, and DIS, and particularly promising properties for the cryoprotection of LDH protein.

Osmotic response during kidney perfusion with cryoprotectant in isotonic or hypotonic vehicle solution.

Organ cryopreservation would revolutionize transplantation by overcoming the shelf-life limitations of conventional organ storage. To prepare an organ for cryopreservation, it is first perfused with cryoprotectants (CPAs). These chemicals can enable vitrification during cooling, preventing ice damage. However, CPAs can also cause toxicity and osmotic damage. It is a major challenge to find the optimal balance between protecting the cells from ice and avoiding CPA-induced damage. In this study, we examined the organ perfusion process to shed light on phenomena relevant to cryopreservation protocol design, including changes in organ size and vascular resistance. In particular, we compared perfusion of kidneys (porcine and human) with CPA in either hypotonic or isotonic vehicle solution. Our results demonstrate that CPA perfusion causes kidney mass changes consistent with the shrink-swell response observed in cells. This response was observed when the kidneys were relatively fresh, but disappeared after prolonged warm and/or cold ischemia. Perfusion with CPA in a hypotonic vehicle solution led to a significant increase in vascular resistance, suggesting reduced capillary diameter due to cell swelling. This could be reversed by switching to perfusion with CPA in isotonic vehicle solution. Hypotonic vehicle solution did not cause notable osmotic damage, as evidenced by low levels of lactate dehydrogenase (LDH) in the effluent, and it did not have a statistically significant effect on the delivery of CPA into the kidney, as assessed by computed tomography (CT). Overall, our results show that CPA vehicle solution tonicity affects organ size and vascular resistance, which may have important implications for cryopreservation protocol design.

The protective effect of L-theanine addition to a cryoprotectant on the storage viability of lactic acid bacteria.

Lactic acid bacteria (LAB) that metabolize sugars to obtain energy and produce a large amount of lactate through the process are well known for their benefits. However, they can be used on a large scale only when good storage stability is guaranteed. The vitality and stability of several LAB strains were effectively protected in this investigation by L-theanine at 1% of the appropriate concentration (Lactiplantibacillus plantarum MG5023, Enterococcus faecium MG5232, Lactococcus lactis MG4668, Streptococcus thermophilus MG5140, and Bifidobacterium animalis subsp. lactis MG741). The inclusion of L-theanine as a protective agent significantly enhanced the viability of all strains throughout the freeze-drying process compared to that of the non-coated probiotics. The efficacy of L-theanine in improving bacterial stability and survivability was evaluated using accelerated stability tests, gastrointestinal (GI) tract survivability tests, and adhesion assays with intestinal epithelial cells. The cell surface was covered with substances including L-theanine, according to morphological findings, providing efficient defense against a variety of external stresses. Therefore, by exerting anti-freezing and anti-thawing properties, the adoption of L-theanine as a new and efficient protective agent may improve the stability and viability of a variety of probiotics.