A review: analysis of technical challenges in cultured meat production and its commercialization.

The cultured meat technology has developed rapidly in recent years, but there are still many technical challenges that hinder the large-scale production and commercialization of cultured meat. Firstly, it is necessary to lay the foundation for cultured meat production by obtaining seed cells and maintaining stable cell functions. Next, technologies such as bioreactors are used to expand the scale of cell culture, and three-dimensional culture technologies such as scaffold culture or 3D printing are used to construct the three-dimensional structure of cultured meat. At the same time, it can reduce production costs by developing serum-free medium suitable for cultured meat. Finally, the edible quality of cultured meat is improved by evaluating food safety and sensory flavor, and combining ethical and consumer acceptability issues. Therefore, this review fully demonstrates the current development status and existing technical challenges of the cultured meat production technology with regard to the key points described above, in order to provide research ideas for the industrial production of cultured meat.

Maturity-onset diabetes of the young type 9 or latent autoimmune diabetes in adults: A case report and review of literature.

BACKGROUND: Maturity-onset diabetes of the young (MODY) is a monogenic genetic disease often clinically misdiagnosed as type 1 or type 2 diabetes. MODY type 9 (MODY9) is a rare subtype caused by mutations in the PAX4 gene. Currently, there are limited reports on PAX4-MODY, and its clinical characteristics and treatments are still unclear. In this report, we described a Chinese patient with high autoimmune antibodies, hyperglycemia and a site mutation in the PAX4 gene. CASE SUMMARY: A 42-year-old obese woman suffered diabetes ketoacidosis after consuming substantial amounts of beverages. She had never had diabetes before, and no one in her family had it. However, her autoantibody tested positive, and she managed her blood glucose within the normal range for 6 mo through lifestyle inter-ventions. Later, her blood glucose gradually increased. Next-generation sequencing and Sanger sequencing were performed on her family. The results revealed that she and her mother had a heterozygous mutation in the PAX4 gene (c.314G>A, p.R105H), but her daughter did not. The patient is currently taking liraglutide (1.8 mg/d), and her blood glucose levels are under control. Previous cases were retrieved from PubMed to investigate the relationship between PAX4 gene mutations and diabetes. CONCLUSION: We reported the first case of a PAX4 gene heterozygous mutation site (c.314G>A, p.R105H), which does not appear pathogenic to MODY9 but may facilitate the progression of latent autoimmune diabetes in adults.

The Effect of Long-Term Passage on Porcine SMCs' Function and the Improvement of TGF-ß1 on Porcine SMCs' Secretory Function in Late Passage.

Cultured meat is one of the meat substitutes produced through tissue engineering and other technologies. Large-scale cell culture is the key for cultured meat products to enter the market. Therefore, this study is aimed to explore the effect of long-term passage in vitro on smooth muscle cells (SMCs) and the effect of transforming growth factor-ß1 (TGF-ß1) on SMCs in the late passage. Multiple passages lead to the decline of the proliferation rate of SMCs in the proliferation stage and the differentiation ability in the differentiation stage. Transcriptome results showed that the ECM pathway and aging-related signaling pathways were significantly up-regulated in the late passage period. TGF-ß1 did not promote SMCs of late passage proliferation at the proliferation stage but promoted the gene and protein expression of collagen as the main protein of the extracellular matrix proteins at the differentiation stage. In addition, proteomic analysis revealed that TGF-ß1 promoted the expression of cell adhesion molecules which activate the Hippo signaling pathway and the HIF-1 signaling pathway and further promoted the production of collagen-containing extracellular matrix proteins. This could provide ideas for large-scale production of cultured meat products using SMCs.

Evaluation the food safety of cultured fat via detection of residues of adipogenic differentiation cocktail in cultured fat with high performance liquid chromatography and enzyme-linked immunosorbent assay.

Cultured fat is inducing adipose progenitor cells (APCs) to differentiate into mature adipocytes for consumption. The traditional adipogenic differentiation cocktail, including insulin, dexamethasone, indomethacin, isobutylmethylxanthine and rosiglitazone, has potential food safety problems in cultured fat. Therefore, the detection of these residues is necessary to ensure food safety. In this research, a method of high performance liquid chromatography (HPLC) was established to quantitatively analyze the potential residual content of dexamethasone, indomethacin, isobutylmethylxanthine and rosiglitazone in cultured fat and medium. Quantitative analysis showed that the content of four residues in cultured fat decreased to zero on Day 10. Subsequently, enzyme-linked immunosorbent assay (ELISA) was performed to detect the insulin content in the cultured fat and found that the insulin content in the cultured fat on Day 10 was 2.78 ± 0.21 µg/kg. After soaking with phosphate buffered saline (PBS), the insulin content decreased to 1.88 ± 0.54 µg/kg. In conclusion, this research provided an effective approach to clarify the content of potential residual components in cultured fat and it will provide reference for the safety of cultured fat in the future.

A Small RNA, SaaS, Promotes Salmonella Pathogenicity by Regulating Invasion, Intracellular Growth, and Virulence Factors.

Salmonella enterica serovar Enteritidis is a common foodborne pathogen that infects both humans and animals. The S. Enteritidis virulence regulation network remains largely incomplete, and knowledge regarding the specific virulence phenotype of small RNAs (sRNAs) is limited. Here, we investigated the role of a previously identified sRNA, Salmonella adhesive-associated sRNA (SaaS), in the virulence phenotype of S. Enteritidis by constructing mutant (ΔsaaS) and complemented (ΔsaaS/psaaS) strains. SaaS did not affect S. Enteritidis; it was activated in the simulated intestinal environment (SIE), regulating the expression of virulence target genes. We discovered that it directly binds ssaV mRNA. Caco-2 and RAW 264.7 cell assays revealed that SaaS promoted S. Enteritidis invasion and damage to epithelial cells while suppressing macrophage overgrowth and destruction. Furthermore, a BALB/c mouse model demonstrated that the deletion of SaaS significantly reduced mortality and attenuated the deterioration of pathophysiology, bacterial dissemination into systemic circulation, and systemic inflammation. Our findings indicate that SaaS is required for S. Enteritidis virulence and further highlight its biological role in bacterial pathogenesis. IMPORTANCE Salmonella is a zoonotic pathogen with high virulence worldwide, and sRNAs have recently been discovered to play important roles. We explored the biological characteristics of the sRNA SaaS and developed two cell infection models and a mouse infection model. SaaS is an SIE-responsive sRNA that regulates the expression of virulence-targeted genes. Additionally, it differentially mediates invasion and intracellular growth for survival and infection of the epithelium and macrophages. We further found that SaaS enhanced bacterial virulence by promoting lethality, colonization, and inflammatory response. These findings provide a better understanding of the critical role of sRNA in bacterial virulence.

Identification of porcine adipose progenitor cells by fluorescence-activated cell sorting for the preparation of cultured fat by 3D bioprinting.

Cultured meat is an emerging technology that is friendly for the environment and animal welfare. As a novel food ingredient, cultured fat is essential for the flavor and nutrition of cultured meat. In this study, we purified adipose progenitor cell (APC) from freshly isolated porcine stromal vessel fraction (SVF) by fluorescence-activated cell sorting (FACS) and identified the transcriptome characteristics of APC by RNA sequencing (RNA-seq). The results showed that APC had characteristics of high-efficiency proliferation and adipogenic differentiation and was distinct from SVF cell in transcriptome profiles. Subsequently, APC was used to prepare cultured fat by 3D bioprinting and to evaluate the differences in fatty acid composition between cultured fat and porcine subcutaneous adipose tissue (pSAT). The results indicated that the fatty acid composition and content of cultured fat had a certain similarity with pSAT; specifically, the content of key monounsaturated fatty acid (MUFA) that create pork flavor in cultured fat, such as C18:1(n-12), C18:1(n-9) and C19:1(n-9)T, were close to that of pSAT. Therefore, this research indicated that APC is a promising candidate cell type for the production of cultured fat.

Quality evaluation of cultured meat with plant protein scaffold.

Cultured meat technology is a promising new technology to solve the negative problems brought by traditional animal husbandry. Cultured meat should be further developed to appear on consumers' tables as alternative protein product. Therefore, this study used food grade peanut wire-drawing protein as scaffold to culture smooth muscle cells (SMCs) in vitro to obtain cultured meat productions containing both animal protein and plant protein. Multiple passages lead to the decline of the proliferation rate of SMCs in the proliferation stage and the differentiation ability in the differentiation stage, which means that the plasticity of cells decreased in the later stage of passage. SMCs can well adhere to the peanut wire-drawing protein scaffold and produce extracellular matrix protein and muscle protein, so as to form a cultured meat product with rich protein composition. This study provides a theoretical basis for the production of nutrient-rich cultured meat products.

Effects of selected flavonoids oncellproliferation and differentiation of porcine muscle stem cells for cultured meat production.

Stemness decline of muscle stem cells (MuSCs) is a significant problem in cultured meat processing. In the present study, three flavonoids (quercetin, icariin, and 3,2'-dihydroxyflavone) with multi concentrations were evaluated to promote the proliferation and differentiation of porcine muscle stem cells. In the proliferation phase, 3,2'-dihydroxyflavone (10 µM) significantly amplified the cells by 34% and up-regulated the expression of paired box transcription factor 7 (PAX7) by 60%, which was higher than quercetin (75 nM) and icariin (7.5 nM). In the differentiation phase, quercetin (50 nM) showed the best pro-differentiation effect and up-regulated the expression of myosin heavy chain (MYHC) by 4.73-fold compared with the control group. These results indicated that flavonoids had a significant impact on promoting the proliferation and differentiation of porcine MuSCs, and 3,2'-dihydroxyflavone (10 µM) for proliferation and quercetin (50 nM) for differentiation were the optimal combinations.

Production of cultured fat with peanut wire-drawing protein scaffold and quality evaluation based on texture and volatile compounds analysis.

Cultured meat is an emergent technology that cultivates cells in three-dimensional scaffolds to generate tissue for consumption. Fat makes an important contribution to the flavor and texture of traditional meat, but there are few reports on cultured fat. Here, we demonstrated the construction of cultured fat by inoculating porcine adipose-derived mesenchymal stem cell (ADSC) on peanut wire-drawing protein (PWP) scaffolds. First, we demonstrated that basic fibroblast growth factor (bFGF) promoted cell proliferation and maintained adipogenic differentiation ability. Then, we generated cultured fat and found that cultured fat decreased the texture of PWP scaffolds. Moreover, 43 volatile compounds were detected by headspace gas chromatography-ion mobility spectrometry (GC-IMS), of which 17 volatile compounds showed no significant differences between cultured fat and porcine subcutaneous adipose tissue (pSAT), which indicated that cultured fat and pSAT had certain similarities. Collectively, this research has great promise for improving the quality of cultured meat.

Production of cultured meat by culturing porcine smooth muscle cells in vitro with food grade peanut wire-drawing protein scaffold.

Cultivating meat is a promising solution to the negative problems brought by traditional animal husbandry. To make cultured meat have the sensory and nutritional characteristics of conventional meat as much as possible, many studies have been conducted on various cell types and scaffold characteristics. Therefore, this study aims to produce a low-cost cultured meat with a quality closer to that of conventional meat. Tissue generation requires three-dimensional (3D) scaffolds to support cells and simulate extracellular matrix (ECM). Here, we used peanut wire-drawing protein (a biomaterial based on edible porous protein) as a new culture meat scaffold to culture cells. The scaffold can support cell attachment and proliferation to create 3D engineered porcine muscle tissue. The differentiation of smooth muscle cells (SMCs) was induced by a low serum medium to produce more extracellular matrix proteins. After differentiation, it was found that peanut wire-drawing protein scaffolds could be used for porcine smooth muscle cell adhesion and growth. The ECM protein and muscle protein produced by SMCs can endow cultured meat with better quality. This technology provides an innovative pathway for the industrialized production of cultured meat.

Insights into ultrasonic treatment on the mechanism of proteolysis and taste improvement of defective dry-cured ham.

To investigate the effects of ultrasonic treatment on proteolysis and taste development of defective dry-cured ham, sensory attributes, enzyme activities, protein degradation and free amino acids were evaluated after different ultrasonic treatments. The ultrasonic treatment of 1000 W & 50 °C significantly increased the intensities of overall taste, umami, sweetness and richness, and decreased bitterness values compared with other groups. The residual activities of DPP I and cathepsin B + L in 1000 W & 50 °C maintained 48.71% and 24.94% of control group, respectively; the intense degradation of structural proteins was observed by label-free proteomics, accordingly. The contents of total free amino acids from 4522.64 mg/100 g muscles in control group increased to 5838.75 mg/100 g muscles in 1000 W & 50 °C; the largest increase of sweet and umami amino acids observed in 1000 W & 50 °C was responsible for the improvement of taste quality of defective dry-cured ham.

Evaluation of the effect of smooth muscle cells on the quality of cultured meat in a model for cultured meat.

While the research on improving the meat quality of cultured meat is in full swing, few studies have focused on the effect of smooth muscle cells (SMCs) on the meat quality of cultured meat. Therefore, this study aimed at building a cultured meat model containing smooth muscle cells, and further evaluating the effect of smooth muscle cells on the quality of cultured meat, so as to reveal the contribution of smooth muscle cells in the production of cultured meat. In this study, we isolated high purity of smooth muscle cells from vascular tissues. The addition of basic fibroblast growth factor (bFGF) to the medium significantly increased the growth rate of smooth muscle cells and the expression of extracellular matrix related genes, especially collagen and elastin. Smooth muscle cells were seeded in a collagen gel to construct a culture meat model. It was found that the pressure loss of the model meat significantly decreased from 98.5 % in control group to 54 % with the extension of culture time for 9 days, while the total collagen content of model meat increased significantly (P < 0.05). In addition, the hydrogel tissue with smooth muscle cells compacted more dramatically and were more tightly, accompanied by significantly increased hardness, springiness and chewiness compared to the control one (P < 0.05). These results indicate that smooth muscle cells can secrete extracellular matrix proteins such as collagen, which can significantly enhance the texture of cultured meat models prepared by hydrogel.

A comprehensive review on molecular mechanism of defective dry-cured ham with excessive pastiness, adhesiveness, and bitterness by proteomics insights.

Excessive bitterness, pastiness, and adhesiveness are the main organoleptic and textural defects of dry-cured ham, which often cause a lot of financial losses to manufacturers and seriously damage the quality of the product. These sensory and textural defects are related to the protein degradation of dry-cured ham. Proteomics shows great potential to improve our understanding of the molecular mechanism of sensory and textural defects and identify biomarkers for monitoring their quality traits. This review presents some of the major achievements and considerations in organoleptic and textural defects of dry-cured ham by proteomics analysis in the recent decades and gives an overview about how to correct sensory and textural defects of dry-cured ham. Proteomics reveals that muscle proteins derived from myofibril and cytoskeleton and involved in metabolic enzymes and oxygen transport have been identified as potential biomarkers in defective dry-cured ham. Relatively high residual activities of cathepsin B and L are responsible for the excessive degradation of these protein biomarkers in defective dry-cured ham. Ultrasound-assisted mild thermal or high-pressure treatment shows a good correction for the organoleptic and textural defects of dry-cured ham by changing microstructure and conformation of muscle proteins by accelerating degradation of proteins and polypeptides into free amino acids.

Enhanced flavor strength of broth prepared from chicken following short-term frozen storage.

This study investigated the effect of frozen storage periods (0, 2, 4, 6, or 8 weeks) of raw meat and stewing on the flavor of chicken broth. With the increased storage duration of frozen raw material, the contents of the free amino acids, nucleotides and mineral elements in the broth decreased significantly, especially within the first 4 weeks, and then increased significantly. Meanwhile, the volatile compounds showed the reverse trend. The results from the E-nose, E-tongue and sensory evaluation indicated a progressive difference in overall flavor profiles between the samples. The sensory scores of the meaty and fatty traits reached a maximum as raw chicken meat was stored for 4 weeks at -18 °C, which should be related to the increased contents of aldehydes and 2-pentyl furan. Overall, the limited storage duration of frozen raw meat can enhance the flavor of chicken broth.

Structural basis for high-pressure improvement in depolymerization of interfacial protein from RFRS meat batters in relation to their solubility.

High-pressure processing (HPP) can modify the construction of interfacial proteins (IPs) to improve the properties of reduced-fat and reduced-salt (RFRS) meat batters. In this study, the relationship between the construction of IPs and their solubility at fat droplet/water interface in RFRS meat batters with HPP treatments was investigated. When 200 MPa for 2 min was applied, the IPs exhibited the highest solubility due to a high concentration of absorbed myosin with the content of random coil 65.62%, but the particle diameter was in reverse. The microscopy revealed the depolymerization of IPs occurred at low pressure, while macromolecular aggregates were produced as the cross-linking of IPs to some degree at pressure ≥ 200 MPa. This phenomenon was supported by the result of SDS-PAGE and the sulfhydryl of IPs. In conclusion, the HPP induced solubility alteration of IPs was achieved by modifying their construction through adjusting the secondary structures and regulating bond interactions.

Evaluating the effect of cooking temperature and time on collagen characteristics and the texture of hog maw.

This study evaluated the texture of hog maw and the degradation of Types I and III collagen in the intramuscular connective tissue (IMCT) of hog maw at different cooking temperatures (75-95°C) and times (50-130 min). The cooking loss, shear force, collagen content, collagen solubility, and IMCT strength of hog maw cooked in water baths were measured. The instrumental texture profile analysis showed that the brittleness, springiness, chewiness and hardness of the cooked hog maw significantly increased with the increase of cooking temperature, while the hardness, springiness and chewiness increased first and then decreased with increasing cooking time. Cooking loss exhibited a 38% increase between the raw meat and meat cooked at 95°C. The collagen solubility significantly increased from 5.5 mg/g for raw meat to 8.6 mg/g for meat cooked at 95°C, accompanied by decreases in the shear force and IMCT strength associated with the increase in cooking temperature and time. These results show that the texture and collagen characteristics of hog maw are dramatically affected by the cooking temperature and time. Sodium dodecyl sulfate electrophoresis and immunofluorescence staining further showed that collagen degradation occurred after cooking, and the degradation of Type I collagen was higher than that of Type III collagen. These results indicated that the degradation of Type I collagen was mainly responsible for the sensory and textural improvements of the cooked hog maw.

Highly linearly polarized light emission from flexible organic light-emitting devices capitalized on integrated ultrathin metal-dielectric nanograting.

Although there have been tremendous achievements ever since the first work on an organic electroluminescent (EL) device that emitted polarized light, the development of flexible polarized emission organic light-emitting devices (OLEDs) is not without hurdles, and the challenge towards real-world applications still requires tremendous effort. In this paper, we proposed highly linearly polarized light-emission from flexible green OLEDs capitalized on integrated ultrathin metal-dielectric nanograting. The acquired polarized device with meticulously optimized geometric parameters yields an angle-invariant average extinction ratio beyond 20.0 dB within a viewing angle range of ± 60°. The detailed analysis illustrates that surface plasmons and cavity modes are simultaneously contributed to the TM-polarized light selection. We hope that the presented approach will open new opportunities for designing flexible polarized light sources.

Insights into the evolution of myosin light chain isoforms and its effect on sensory defects of dry-cured ham.

To better understand the contribution of myosin light chain (MLC) isoforms to sensory defects in Jinhua ham, dipeptidyl peptidase (DPP) activities, peptide fragments, cleavage sites and the potential of DPP to develop sensory defects of dry-cured ham were evaluated and discussed in normal and defective hams. Higher residual activities of DPP I were found in defective ham compared with normal ham; approximate 3-fold peptide fragments were identified in defective ham than in normal ham. These regions of positions 11-35 and 116-141 in MLC 1, 13-53 and 139-156 in MLC 2, and 18-50 in MLC 3 contributed to the intense generation of peptide fragments in defective ham. PLS-DA further revealed DPP I showing intense response to degrade peptides. Cleavage sites including Glu-128, Tyr-132 and Glu-133 were responsible for the intense release of dipeptides in defective ham. These cleavages could play key role in discriminating taste attributes between defective and normal hams.

Role of protein S-nitrosylation in regulating beef tenderness.

This research aimed to explore the role of protein S-nitrosylation in regulating the tenderness of postmortem beef, from the perspective of µ-calpain autolysis and protein proteolysis. Five bovine semimembranosus muscles were incubated with three treatments including S-nitrosoglutathione (GSNO, nitric oxide donor), normal saline and Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME, nitric oxide synthase inhibitor). The results showed that the level of protein S-nitrosylation was improved by GSNO treatment and reduced by L-NAME treatment (p < 0.05). Compared to the control, GSNO treatment had higher shear force while L-NAME treatment presented lower shear force at 7 d postmortem (p < 0.05). In addition, µ-calpain autolysis, myofibrillar protein and desmin degradation were reduced by GSNO treatment and accelerated by L-NAME treatment (p < 0.05). Therefore, it can be speculated that protein S-nitrosylation could affect beef tenderization by regulating the autolysis of µ-calpain and the degradation of myofibrillar proteins.

Protein degradation and peptide formation with antioxidant activity in pork protein extracts inoculated with Lactobacillus plantarum and Staphylococcus simulans.

This study focused on sarcoplasmic and myofibrillar protein degradation and the formation of peptides with antioxidant activity by mixed starters (Lactobacillus plantarum CD101 and Staphylococcus simulans NJ201). Gel electrophoresis indicated that the mixed starters can hydrolyze both sarcoplasmic and myofibrillar proteins, and the concentration of peptides increased (P < .05). Compared with the control group, using mixed starters led to a significant increase (P < .05) in the DPPH radical scavenging activity, Fe2+ chelating activity, and ABTS radical scavenging activity of sarcoplasmic proteins, but demonstrated no significant difference in myofibrillar proteins. Two hydrophobic fractions (C2, C5) separated by RP-HPLC in the inoculation groups with sarcoplasmic proteins showed high DPPH radical scavenging activity (66.60%, 60.50%). Eighteen peptides were identified by LC-MS/MS, which mainly arose from triosephosphate isomerase, creatine kinase M-type, and glyceraldehyde-3-phosphate dehydrogenase. Hydrophobic amino acids accounted for a large proportion. Our results indicate that mixed starters affect proteolytic characterization and contribute to the formation of peptides with antioxidant capacity in sarcoplasmic proteins.