Metabolic Engineering of Corynebacterium glutamicum for Highly Efficient Production of Ectoine.

Ectoine is a compatible solute that functions as a cell protector from various stresses, protecting cells and stabilizing biomolecules, and is widely used in medicine, cosmetics, and biotechnology. Microbial fermentation has been widely used for the large-scale production of ectoine, and a number of fermentation strategies have been developed to increase the ectoine yield, reduce production costs, and simplify the production process. Here, Corynebacterium glutamicum was engineered for ectoine production by heterologous expression of the ectoine biosynthesis operon ectBAC gene from Halomonas elongata, and a series of genetic modifications were implemented. This included introducing the de3 gene from Escherichia coli BL21 (DE3) to express the T7 promoter, eliminating the lysine transporter protein lysE to limit lysine production, and performing a targeted mutation lysCS301Y on aspartate kinase to alleviate feedback inhibition of lysine. The new engineered strain Ect10 obtained an ectoine titer of 115.87 g/L in an optimized fed-batch fermentation, representing the highest ectoine production level in C. glutamicum and achieving the efficient production of ectoine in a low-salt environment.

Global job satisfaction and fluctuation among community general practitioners: a systematic review and meta-analysis.

INTRODUCTION: Community General Practitioners (CGPs) are crucial to primary healthcare worldwide. Their job satisfaction significantly impacts the quality and accessibility of healthcare. However, a comprehensive global perspective on this issue remains absent, necessitating this systematic review and meta-analysis. METHODS: This systematic review and meta-analysis sourced literature from PubMed, Web of Science, CNKI, and Wanfang, up to June 14, 2023. Of the 2,742 identified studies, 100 articles were selected for meta-analysis to assess satisfaction levels, and 97 studies were chosen for comparative analysis of influential factors. We employed both meta-analytic and comparative analytic methodologies, focusing on varying geographical, economic, and temporal contexts. RESULTS: The pooled rate and corresponding 95% confidence interval (CI) for job satisfaction among CGPs was 70.82% (95%CI: 66.62-75.02%) globally. Studies utilizing 5-point score scale obtained a random effect size of 3.52 (95%CI: 3.43-3.61). Diverse factors influenced satisfaction, with remuneration and working conditions being predominant. A noticeable decline in job satisfaction has been observed since the coronavirus disease 2019 outbreak, with satisfaction rates dropping from an average of 72.39% before 2009 to 63.09% in those published after 2020. CONCLUSIONS: The downward trend in CGPs' job satisfaction is concerning and warrants urgent attention from policymakers, especially in regions with an acute shortage of CGPs. The findings from this comprehensive review and meta-analysis provide essential insights for informed healthcare policy-making. It highlights the urgency of implementing strategies to enhance CGP satisfaction, thereby improving the effectiveness of primary healthcare systems globally.

EGR1 suppresses HCC growth and aerobic glycolysis by transcriptionally downregulating PFKL.

BACKGROUND: Hepatocellular Carcinoma (HCC) is a matter of great global public health importance; however, its current therapeutic effectiveness is deemed inadequate, and the range of therapeutic targets is limited. The aim of this study was to identify early growth response 1 (EGR1) as a transcription factor target in HCC and to explore its role and assess the potential of gene therapy utilizing EGR1 for the management of HCC. METHODS: In this study, both in vitro and in vivo assays were employed to examine the impact of EGR1 on the growth of HCC. The mouse HCC model and human organoid assay were utilized to assess the potential of EGR1 as a gene therapy for HCC. Additionally, the molecular mechanism underlying the regulation of gene expression and the suppression of HCC growth by EGR1 was investigated. RESULTS: The results of our investigation revealed a notable decrease in the expression of EGR1 in HCC. The decrease in EGR1 expression promoted the multiplication of HCC cells and the growth of xenografted tumors. On the other hand, the excessive expression of EGR1 hindered the proliferation of HCC cells and repressed the development of xenografted tumors. Furthermore, the efficacy of EGR1 gene therapy was validated using in vivo mouse HCC models and in vitro human hepatoma organoid models, thereby providing additional substantiation for the anti-cancer role of EGR1 in HCC. The mechanistic analysis demonstrated that EGR1 interacted with the promoter region of phosphofructokinase-1, liver type (PFKL), leading to the repression of PFKL gene expression and consequent inhibition of PFKL-mediated aerobic glycolysis. Moreover, the sensitivity of HCC cells and xenografted tumors to sorafenib was found to be increased by EGR1. CONCLUSION: Our findings suggest that EGR1 possesses therapeutic potential as a tumor suppressor gene in HCC, and that EGR1 gene therapy may offer benefits for HCC patients.

A micro-nano interface integrated SERS-based microfluidic sensor for miRNA detection using DNAzyme walker amplification.

BACKGROUND: Precise and specific miRNA detection plays a vital role in exploring development mechanisms of cancer disease, thereby it can significantly improve relevant prevention and treatment strategies. RESULTS: In this work, a surface-enhanced Raman spectroscopy (SERS)-based microfluidic chip has been devised with a microcone array SERS substrate (MCASS) for the miR-141 detection. This substrate excels in unique SERS activity and large surface area for DNA oligonucleotide modification. As the presence of miR-141, the DNAzyme walker induced cleavage reaction took place on the finely designed and prepared dual DNA conjugated SERS nanoprobes. The SERS nanoprobes can anchor on MCASS by the DNA hybridization that achieved an impressive detection limit in the femtomolar level. SIGNIFICANCE: With this integrated SERS-based microfluidic chip, we provided a miRNA detection strategy using DNAzyme walker amplification technology. It is believed that this strategy could be a powerful tool for miRNA detection and related cancer screening test.

Flexible SERS Biosensor Based on Core-Shell Nanotags for Sensitive and Multiple Detection of T1DM Biomarkers.

Sensitive and multiple detection of the biomarkers of type 1 diabetes mellitus (T1DM) is vital to the early diagnosis and clinical treatment of T1DM. Herein, we developed a SERS-based biosensor using polyvinylidene fluoride (PVDF) membranes as a flexible support for the detection of glutamic acid decarboxylase antibodies (GADA) and insulin autoantibodies (IAA). Two kinds of silver-gold core-shell nanotags embedded with Raman probes and attached with GADA or IAA antibodies were synthesized to capture the targets, enabling highly sensitive and highly selective detection of GADA and IAA. The embedded Raman probes sandwiched between silver and gold layers guaranteed spectral stability and reliability. Moreover, the utilization of two Raman probes enables simultaneous and multiplexing detection of both GADA and IAA, improving the detection accuracy for T1DM. The proposed SERS-based method has been proven feasible for clinical sample detection, demonstrating its great potential in sensitive, reliable, and rapid diagnosis of T1DM.

Multifunctional protein-based self-assembled nanoplatform: overcoming hypoxic tumor microenvironment for enhanced imaging-guided photodynamic therapy.

Photodynamic therapy (PDT) has emerged as a promising modality for cancer treatment, but its efficacy is often limited by tumour hypoxia. Here, we report the development of a novel protein-based, self-assembled nanoplatform, CAT-I-BODIPY NPs (CIB NPs), to address this limitation. We first design and synthesize an I-BODIPY photosensitizer based on the heavy atom effect and modification of the electron-donating group, which exhibits excellent capabilities in generating reactive oxygen species and enabling near-infrared (NIR) fluorescence imaging. The incorporation of an oxygen-producing enzyme, catalase (CAT), within these nanoassemblies enables in situ oxygen generation to counteract hypoxic constraints. Controllable self-assembly by multiple supramolecular interactions into highly ordered architecture not only guarantees CAT's catalytic activity but also leads to excellent NIR fluorescence imaging ability and enhanced PDT efficacy. Notably, the visualization of optimal accumulation of CIB NPs within tumour sites 18 h post-injection offers precise PDT application guidance. Both in vitro and in vivo studies corroborate the remarkable anti-tumour efficacy of CIB NPs under NIR illumination, providing a significant advancement in PDT. The favourable biosafety profile of CIB NPs further emphasizes their potential for clinical application in hypoxic tumour therapy.

Fertilizer-induced manganese oxide formation enhances cadmium removal by paddy crusts from irrigation water.

Fertilization is a crucial agrological measure for agricultural production that can significantly impact the removal of Cd from irrigation water by paddy crusts (PC). In this study, laboratory and field experiments were conducted to investigate the impact of fertilization at low, medium, and high concentrations on the accumulation of Cadmium (Cd) in PC and the underlying mechanisms involved. The results showed that only low fertilizer concentration could promote the removal of Cd by PC, which reduced the Cd concentration in irrigation water from 19.52 µg/L to 5.35 µg/L. Conversely, medium and high fertilizer concentrations reduced the accumulation of Cd by PC. After fertilizer addition, the proportion of Fe-Mn oxidizable-Cd in PC reached 55 % (with low concentration of fertilizer treatment). The application of low concentration of fertilizer was found to stimulate the growth of filamentous green algae, leading to a significant increase in the relative abundance of sphingomonadaceae (by 1.39 %) and comamonadaceae (by 1.29 %). The XRD, SEM and correlation analysis show that a large amount of manganese oxide is formed on the surface of PC, which increases the fixation of Cd. These findings provide a new perspective for the remediation of heavy metal contamination in paddy fields.

The Promotion of Research Progress of Zinc Manganate Cathode Materials for Zinc-Ion Batteries by Characterization and Analysis Technology.

Zinc-ion batteries (ZIBs) have recently attracted great interest and are regarded as a promising energy storage device due to their low cost, environmental friendliness, and superior safety. However, the development of suitable Zn-ion intercalation cathode materials remains a great challenge, resulting in unsatisfactory ZIBs that cannot meet commercial demands. Considering that spinel-type LiMn2O4 has been shown to be a successful Li intercalation host, spinel-like ZnMn2O4 (ZMO) is expected to be a good candidate for ZIBs cathodes. This paper first introduces the zinc storage mechanism of ZMO and then reviews the promotion of research progress in improving the interlayer spacing, structural stability, and diffusivity of ZMO, including the introduction of different intercalated ions, introduction of defects, and design of different morphologies and in combination with other materials. The development status and future research directions of ZMO-based ZIBs characterization and analysis techniques are summarized.

Controllable Regulation of Ag2 S Quantum-Dot-Mediated Protein Nanoassemblies for Imaging-Guided Synergistic PDT/PTT/Chemotherapy against Hypoxic Tumor.

The combination of phototherapy and chemotherapy holds great potential for cancer treatment, while hypoxia in tumor as well as unexpected drug release largely restricts anticancer therapy. Inspired by the natural intelligence, herein, for the first time, a "bottom-up" protein self-assembly strategy mediated by near-infrared (NIR) quantum dots (QDs) with multicharged electrostatic interactions is presented to develop a tumor microenvironment (TME)-responsive theranostic nanoplatform for imaging-guided synergistic photodynamic therapy (PDT)/photothermal therapy (PTT)/chemotherapy. Catalase (CAT) possesses diverse surface charge distribution under different pH conditions. After modification by chlorin e6 (Ce6), the formulated CAT-Ce6 with patchy negative charges can be assembled with NIR Ag2 S QDs by regulating their electrostatic interactions, allowing for effective incorporation of specific anticancer drug oxaliplatin (Oxa). Such Ag2 S@CAT-Ce6@Oxa nanosystems are able to visualize nanoparticle (NP) accumulation to guide subsequent phototherapy, together with significant alleviation of tumor hypoxia to further enhance PDT. Moreover, the acidic TME triggers controllable disassembly through weakening the CAT surface charge to disrupt electrostatic interactions, allowing for sustained drug release. Both in vitro and in vivo results demonstrate remarkable inhibition of colorectal tumor growth with a synergistic effect. Overall, this multicharged electrostatic protein self-assembly strategy provides a versatile platform for realizing TME-specific theranostics with high efficiency and safety, promising for clinical translation.

Construction of HPQ-based activatable fluorescent probe for peroxynitrite and its application in ferroptosis and mice model of LPS-induced inflammation.

As one of the important members of reactive oxygen species, ONOO- plays a crucial role in signal transduction, immune response, and other physiological activities. Aberrant changes in ONOO- levels in the living organism are usually associated with many diseases. Therefore, it is important to establish a highly selective and sensitive method for the determination of ONOO- in vivo. Herein, we designed a novel ratio near-infrared fluorescent probe for ONOO- by directly conjugating dicyanoisophorone (DCI) to hydroxyphenyl-quinazolinone (HPQ). Surprisingly, HPQD was unaffected by environmental viscosity and responded rapidly to ONOO- within 40 s. The linear range of ONOO- detection was from 0 µM to 35 µM. Impressively, HPQD did not react with reactive oxygen species and was sensitive to exogenous/endogenous ONOO- in live cells. We also investigated the relationship between ONOO- and ferroptosis and achieved in vivo diagnosis and efficacy evaluation of mice model of LPS-induced inflammation, which showed promising prospects of HPQD in ONOO--related studies.

SERS-based biosensor for detection of f-PSA%: Implications for the diagnosis of prostate cancer.

In diagnosing prostate cancer and distinguishing it from other prostate diseases, the ratio of the concentration of free prostate-specific antigen (f-PSA) to total prostate-specific antigen (t-PSA), i.e., (f-PSA%) is more accurate than the concentration of t-PSA alone. Immunoassay based on surface-enhanced Raman scattering (SERS) frequency shift has been proven to be particularly suitable for detecting large biomolecules with high reproducibility. Along similar lines, the present study developed a SERS-based biosensor that simultaneously detects t-PSA and f-PSA. The 4-mercaptobenzoic acid (MBA) on the immunocapture substrate is coupled to the t-PSA antibody through the carboxyl group, and the combination of t-PSA induces the Raman frequency shifts of MBA. The immunocolloidal gold attached with f-PSA antibodies selectively capture the f-PSA that immobilized on the MBA-modified SERS substrates, allowing for f-PSA quantification according to the SERS intensities of the 5, 5'-Dithiobis (succinimidyl-2-nitrobenzoate) (DSNB) probe. The results show that f-PSA and t-PSA have good linear response in the concentration scale of 0.1-20 ng/mL, and 1-200 ng/mL, respectively. The biosensor combines Raman frequency shifts and intensities, which greatly simplifies traditional procedures for f-PSA% detection. All the results demonstrated the great potential of the proposed biosensor in highly reproducible and accurate diagnosis of prostate cancers.

Cadmium accumulation in rice grains is mitigated by duckweed-like hydrophyte through adsorption and increased ammonia nitrogen.

Although increasing attention has been paid to agronomic measures for reducing the heavy metal load in rice grain, the effects of duckweed-paddy co-cropping technology on the accumulation of cadmium (Cd) in rice grains remain unclear. To investigate its specific effects on Cd accumulation in paddy fields, three types of duckweed-like hydrophyte (DH), Azolla imbricata, Spirodela polyrrhiza, and Lemna minor were chosen for study. Their use resulted in a reduction of Cd content in rice grains from 0.40 mg/kg to <0.20 mg/kg, with A. imbricata yielding the best results (0.15 mg/kg). The three types of DH reduced the available Cd content in the soil by 10 % to 35 % after the paddy tillering stage. The reduction of available Cd content was attributed to the absorption, high pH, and increase of relative abundance of special bacteria of immobilizing Cd. In addition, DH could regulate soil nitrogen leading to ammonium nitrogen increased from 75 mg/kg to 100 mg/kg, while nitrate nitrogen decreased from 0.55 to 0.1-0.3 mg/kg. The increase of ammonium nitrogen content might induce the low Cd transfer ability in rice plant and then low Cd content in rice grain. This study demonstrated that DH has a good effect on the reduction of the Cd concentration in rice grains. Consequently, duckweed-paddy co-cropping technology offers a potential solution to heavy metal pollution and agricultural non-point source pollution, as it not only reduces Cd levels in rice plants, but also fixes nitrogen, reducing the need for nitrogen application.

Development of an activatable red emissive fluorescent probe for imaging hydrogen disulfide upregulation in living cells and zebrafish.

As a member of reactive sulfur molecules, hydrogen polysulfide (H2Sn) plays a vital role in cell protection, anti-oxidative stress and regulation of redox signaling. The highly selective and sensitive detection of H2Sn was still challenging due to its special nucleophilic and electrophilic reactivity. By incorporating phenyl 2-(benzoylthio) benzoate into semi-naphthofluorescein, we developed a novel red emissive fluorescent probe SNAFL-H2Sn for the detection of a representative H2Sn (e. g. H2S2). The addition of H2S2 would rapidly trigger SNAFL-H2Sn to produce significant turn-on fluorescence signal changes at 626 nm with a linear response over a range of 2-30 µM and a detection limit of 16 nM. SNAFL-H2Sn was capable of mapping exogenous and endogenous H2S2 in living cells and zebrafish. Moreover, SNAFL-H2Sn was applied to detect endogenous H2S2 under atorvastatin stimulation. The present study demonstrated that SNAFL-H2Sn potentially served as a promising tool for interrogating H2Sn functions in biological systems.

Designed symmetrical ß-hairpin peptides for treating multidrug-resistant salmonella typhimurium infections.

The rapid emergence and prevalence of multidrug-resistant salmonellosis lack effective therapies, which causes epidemic health problems and stimulates the development of antimicrobials with novel modes of action. In this research, 10 short symmetrical ß-hairpin peptides are synthesized by combining the ß-turn of Leucocin-A with recurring hydrophobic and cationic amino acid sequences. Those designed peptides exhibited potent antibacterial activities against drug-susceptible and drug-resistant Salmonella. One of the 10 peptides, WK2 ((WK)2CTKSGC(KW)2), displayed best cell selectivity towards Salmonella cells over macrophages and erythrocytes in a co-culture model. Fluorescent measurements and microscopic observations reflected that WK2 exerted its antimicrobial activity through a membrane-lytic mechanism. Moreover, the ß-hairpin peptides can bind to endotoxin (LPS) and suppress the production of LPS-induced proinflammatory cytokines in RAW264.7 cells, indicating as a potent anti-inflammatory activity. The preliminary in vivo studies can also demonstrate that WK2 decreased loads of Salmonella in the liver and spleen, mitigated Salmonella-caused inflammation and maintained the integrity of intestinal mucosal surfaces. Ultimately, the results highlight that WK2 is a promising therapeutic agent to prevent multidrug-resistant S. Typhimurium infections in humans and animals.

Evaluation of nitroreductase activity in nasopharyngeal carcinoma progression by an activatable two-photon fluorescent probe.

Nasopharyngeal carcinoma (NPC) originating from the epithelium cells is the most common malignant tumor of the head and neck. Small-molecule fluorescent probes for early diagnosis of NPC can effectively improve the 5-year survival rate of patients, which makes it become a research hotspot in recent years. Previous studies have suggested the expression levels of NTR in hypoxic tissues or cells and tumors increased relative to the normal state and were positively correlated with the degree of hypoxia. Regarding the mentioned above, we designed a two-photon fluorescent probe NaT-NTR for the detection of NTR in nasopharyngeal cell lines and tissues at different hypoxia levels. NaT-NTR showed high selectivity and sensitivity toward NTR in a complex physiological environment. Furthermore, imaging NTR in different cell lines revealed that the level of intracellular NTR might be positively correlated with the malignancy of nasopharyngeal carcinoma. More importantly, NaT-NTR was successfully applied to detect and image NTR in human nasopharyngeal carcinoma with a penetration depth of 100 µm. On this basis, NaT-NTR might be a powerful chemical tool for the early diagnosis of nasopharyngeal carcinoma.

SERS based Y-shaped aptasensor for early diagnosis of acute kidney injury.

Considering the pivotal role of biomarkers in plasma, the development of biomarker specific sensing platforms is of great significance to achieve accurate diagnosis and monitor the occurrence and progress in acute kidney injury (AKI). In this paper, we develop a promising surface-enhanced Raman scattering-based aptasensor for duplex detection of two protein biomarkers in AKI. Exploiting the base-pairing specificity of nucleic acids to form a Y-shaped self-assembled aptasensor, the MGITC labelled gold nanoparticles will be attached to the surface of magnetic beads. In the presence of specific AKI-related biomarkers, the gold nanoparticles will detach from magnetic beads into the supernatant, thus leading to a SERS signal increase, which can be used for the highly sensitive analysis of target biomarkers. In addition, the limit of detection calculated for each biomarker indicates that the SERS-based aptasensor can well meet the detection requirements in clinical applications. Finally, the generality of this sensor in the early diagnosis of AKI is confirmed by using a rat model and spiked plasma samples. This sensing platform provides a facile and general route for sensitive SERS detection of AKI-related biomarkers, which offers great promising utility for in vitro and accurate practical bioassay in AKI early diagnosis.

Biochemical and Metabolomic Responses of Antarctic Bacterium Planococcus sp. O5 Induced by Copper Ion.

Heavy metal pollution in the Antarctic has gone beyond our imagination. Copper toxicity is a selective pressure on Planococcus sp. O5. We observed relatively broad tolerance in the polar bacterium. The heavy metal resistance pattern is Pb2+ > Cu2+ > Cd2+ > Hg2+ > Zn2+. In the study, we combined biochemical and metabolomics approaches to investigate the Cu2+ adaptation mechanisms of the Antarctic bacterium. Biochemical analysis revealed that copper treatment elevated the activity of antioxidants and enzymes, maintaining the bacterial redox state balance and normal cell division and growth. Metabolomics analysis demonstrated that fatty acids, amino acids, and carbohydrates played dominant roles in copper stress adaptation. The findings suggested that the adaptive mechanisms of strain O5 to copper stress included protein synthesis and repair, accumulation of organic permeable substances, up-regulation of energy metabolism, and the formation of fatty acids.

An Optoelectronic thermometer based on microscale infrared-to-visible conversion devices.

Thermometric detectors are crucial in evaluating the condition of target objects spanning from environments to the human body. Optical-based thermal sensing tools have received extensive attention, in which the photon upconversion process with low autofluorescence and high tissue penetration depth is considered as a competent method for temperature monitoring, particularly in biomedical fields. Here, we present an optoelectronic thermometer via infrared-to-visible upconversion, accomplished by integrated light receiving and emission devices. Fully fabricated thin-film, microscale devices present temperature-dependent light emission with an intensity change of 1.5% °C-1 and a spectral shift of 0.18 nm °C-1. The sensing mechanism is systematically characterized and ascribed to temperature dependent optoelectronic properties of the semiconductor band structure and the circuit operation condition. Patterned device arrays showcase the capability for spatially resolved temperature mapping. Finally, in vitro and in vivo experiments implemented with integrated fiber-optic sensors demonstrate real-time thermal detection of dynamic human activity and in the deep brain of animals, respectively.

Construction of a Mitochondria-Endoplasmic Reticulum Dual-Targeted Red-Emitting Fluorescent Probe for Imaging Peroxynitrite in Living Cells and Zebrafish.

Peroxynitrite (ONOO- ) is one of the important reactive oxygen species, which plays a vital role in the physiological process of intracellular redox balance. Revealing the biological functions of ONOO- will contribute to further understanding of the oxidative process of organisms. In this work, we designed and synthesized a novel red-emitting fluorescent probe MCSA for the detection of ONOO- , which could rapidly respond to ONOO- within 250 s and exhibited high sensitivity to ONOO- with a low detection limit of 78 nM. Co-localization experiments demonstrated MCSA had the ability to localize into the mitochondria and endoplasmic reticulum. What's more, MCSA enabled monitoring ONOO- level changes during tunicamycin-induced endoplasmic reticulum stress. We have also successfully achieved the visual detection of exogenous and endogenous ONOO- in living cells and zebrafish. This work presented a chemical tool for imaging ONOO- in vitro and in vivo.

Improving the viability of powdered Lactobacillus fermentum Lf01 with complex lyoprotectants by maintaining cell membrane integrity and regulating related genes.

In this study, Lactobacillus fermentum Lf01, which was screened out in the early stage of the experiment, had better fermentation performance as the research objectives, and was prepared into powder by vacuum freeze-drying technology. We used response surface methodology to optimize the composition of the mixture used to protect powdered L. fermentum. Our data demonstrated that 10% skim milk, 12% sucrose, 0.767% tyrosine, and 2.033% sorbitol ensured the highest survival rate (92.7%) of L. fermentum. We have initially explored the potential mechanism of the complex protectants through the protection effect under the electron microscope, and the analysis methods of Fourier transform infrared spectroscopy and transcriptomics. The complex protectants could effectively maintain the permeability barrier and structural integrity of cell membrane and avoid the leakage of cell contents. Transcriptomic data have also indicated that the protective effect of the complex protectants on bacteria during freeze-drying was most likely achieved through the regulation of related genes. We identified 240 differential genes in the treatment group, including 231 up-regulated genes and 9 down-regulated genes. Gene ontology (GO) and Kyoto encyclopaedia of genes and genomes (KEGG) analyses of differential expression genes (DEGs) indicated that genes involved in amino acid metabolism, carbohydrate metabolism, membrane transport, fatty acid biosynthesis and cell growth were significantly up-regulated. These new results provided novel insights into the potential mechanism of lyoprotectants at the cellular level, morphological level, and gene level of the bacteria. PRACTICAL APPLICATIONS: In our study, a strain of Lactobacillus fermentum Lf01 with good fermentation performance was selected to be prepared into powder by freeze-drying technique. Bacterial cells were unavoidably damaged during the freeze-drying process. As a result, we investigated the protective effects on L. fermentum of ten distinct freeze-dried protectants and their mixtures. We were also attempting to explain the mechanism of action of the complex protectants at the cellular level, morphological level, and gene level of the bacteria. This presents very important theoretical and practical significance for the preservation of strains and the production of commercial direct-investment starter.