Morphology-Based Prediction of Proliferation and Differentiation Potencies of Porcine Muscle Stem Cells for Cultured Meat Production.

Inconsistent efficiency of cell production caused by cellular quality variations has become a significant problem in the cultured meat industry. In our study, morphological information on passages 5-9 of porcine muscle stem cells (pMuSCs) from three lots was analyzed and used as input data in prediction models. Cell proliferation and differentiation potencies were measured by cell growth rate and average stained area of the myosin heavy chain. Analysis of PCA and heatmap showed that the morphological parameters could be used to discriminate the differences of passages and lots. Various morphological parameters were analyzed, which revealed that accumulating time-course information regarding morphological heterogeneity in cell populations is crucial to predicting the potencies. Based on the 36 and 60 h morphological profiles, the best proliferation potency prediction model (R2 = 0.95, RMSE = 1.1) and differentiation potency prediction model (R2 = 0.74, RMSE = 1.2) were explored. Correlation analysis demonstrated that morphological parameters selected in models are related to the quality of porcine muscle stem cells.

Tea polyphenols coated sodium alginate-gelatin 3D edible scaffold for cultured meat.

This study aimed to use edible scaffolds as a platform for animal stem cell expansion, thus constructing block-shaped cell culture meat. The tea polyphenols (TP)-coated 3D scaffolds were constructed of sodium alginate (SA) and gelatin (Gel) with good biocompatibility and mechanical support. Initially, the physicochemical properties and mechanical properties of SA-Gel-TP scaffolds were measured, and the biocompatibility of the scaffolds was evaluated by C2C12 cells. SEM results showed that the scaffold had a porous laminar structure with TP particles attached to the surface, while FT-IR results also demonstrated the encapsulation of TP coating on the scaffold. In addition, the porosity of all scaffolds was higher than 40% and the degradation rate during the incubation cycle was less than 40% and the S2-G1-TP0.1-3 h scaffold has excellent cell adhesion and extension. Subsequently, we inoculated rabbit skeletal muscle myoblasts (RbSkMC) on the scaffold and induced differentiation. The results showed good adhesion and extension behavior of RbSkMC on S2-G1-TP0.1-3 h scaffolds with high expression of myogenic differentiation proteins and genes, and SEM results confirmed the formation of myotubes. Additionally, the adhesion rate of cells on scaffolds with TP coating was 1.5 times higher than that on scaffolds without coating, which significantly improved the cell proliferation rate and the morphology of cells with extension on the scaffolds. Furthermore, rabbit-derived cultured meat had similar appearance and textural characteristics to fresh meat. These conclusions indicate the high potential of the scaffolds with TP coating as a platform for the production of cultured meat products.

Biotinylated caffeic acid covalent binding with myofibrillar proteins in alkaline conditions: Identification of protein-phenol adducts and alterations in protein properties.

In this study, the effects of covalent interactions between myofibrillar proteins (MP) and caffeic acid (CA) were investigated. Protein-phenol adducts were identified by biotinylated caffeic acid (BioC) used as a substitution of CA. The total sulfhydryls and free amines content were decreased (p < 0.05). The α-helix structure of MP increased (p < 0.05) and MP gel properties enhanced slightly at low dosages of CA (10 and 50 µM), and both were impaired significantly (p < 0.05) at high dosages of CA (250 and 1250 µM). Two prominent adducts of myosin heavy chain (MHC)-BioC and Actin-BioC were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which gradually increased at low concentrations of BioC (10 and 50 µM), and raised significantly at the concentration of 1250 µM. According to the correlation analysis, MHC-BioC and Actin-BioC adducts showed a significant negative correlation with gel properties, such as G', hardness, and water holding capacity (WHC) (p < 0.01), which indicated that the covalent interactions between MP and CA significantly affected the quality of meat products.

Preparation and Quality Evaluation of Cultured Fat.

Cultured meat is rapidly developing as an emerging meat production technology. Adipose tissue plays an essential role in the flavor of meat products. In this study, cultured fat was produced by cultured adipose-derived stem cells (ADSCs) based on collagen in vitro, with a 3D model. The research showed that ADSCs could attach to collagen hydrogels and differentiate into mature adipocytes. Texture analysis demonstrated that the springiness, cohesiveness, and resilience of cultured fat were consistent with porcine subcutaneous fat. Moreover, 28 volatile organic compounds (VOCs) were detected by headspace gas chromatography-ion mobility spectrometry. The relative contents of 17 VOCs in cultured fat were significantly higher than porcine subcutaneous fat and empty collagen hydrogels, and the relative contents of 5 VOCs in cultured fat were not significantly different from porcine subcutaneous fat. These findings assert the promising application of cultured fat in cultured meat production.

Identification of Protein-Phenol Adducts in Meat Proteins: A Molecular Probe Technology Study.

Plant polyphenols with a catechol structure can form covalent adducts with meat proteins, which affects the quality and processing of meat products. However, there is a lack of fast and effective methods of characterizing these adducts and understanding their mechanisms. This study aimed to investigate the covalent interaction between myofibrillar protein (MP) and caffeic acid (CA), a plant polyphenol with a catechol structure, using molecular probe technology. The CA-MP adducts were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected via Western blot and LC-MS/MS analyses. The Western blot analysis revealed that various specific adducts were successfully enriched and identified as bands around 220 kDa, 45 kDa, and two distinct bands between 95 and 130 kDa. Combined with the LC-MS/MS analysis, a total of 51 peptides were identified to be CA-adducted, corresponding to 31 proteins. More than 80% of the adducted peptides carried one adducted site, and the rest carried two adducted sites. The adducted sites were located on cysteine (C/Cys), histidine (H/His), arginine (R/Arg), lysine (K/Lys), proline (P/Pro), and N-terminal (N-Term) residues. Results showed that the covalent interaction of CA and MP was highly selective for the R side chain of amino acids. Moreover, the adducts were more likely to form via C-N bonding than C-S bonding. This study provides new insights into the covalent interaction of plant polyphenols and meat proteins, which has important implications for the rational use of plant polyphenols in the meat processing industry.

Effects of Cyclocarya paliurus (Batal.) Extracts on Oxidative Stability and Sensory Quality in Meat Products (Frankfurters).

Oxidation is one of the most common causes of the deterioration of meat and meat products. At the same time, synthetic antioxidants are becoming less accepted by consumers due to the potential health hazards they might cause. Therefore, a new trend to substitute these synthetic antioxidants with natural antioxidants has emerged. This study adds flavonoid extracts from Cyclocarya paliurus (C. paliurus) as a natural antioxidant for meat products (Frankfurters). The results showed that flavonoid extracts from C. paliurus had strong antioxidant and antibacterial activity. This is proportional to concentration, and the addition of extracts could significantly (p < 0.05) delay the lipid oxidation in the samples. In addition, we did not observe hazardous effects on the samples' pH and texture as a result of adding flavonoid extracts. We observed that flavonoid extracts from C. paliurus at concentrations of 0.06% and 0.12% did not affect the color and sensory evaluation of the samples. At a concentration of 0.18% and 0.24%, the flavonoid extracts had a negative impact on the color and sensory evaluation of the samples, likely due to the yellow-brown color of the extract itself. The findings showed that a low concentration of 0.12% flavonoid extracts from C. paliurus in meat products could effectively prevent lipid oxidation without affecting the sensory quality.

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.

Identification of Potential Peptide Marker(s) for Evaluating Pork Meat Freshness via Mass Spectrometry-Based Peptidomics during Storage under Different Temperatures.

This study applied peptidomics to investigate potential biomarkers for evaluating pork-meat freshness. The spoilage time points of pork meat stored at -2, 4, 10, and 25 °C were defined by evaluating meat freshness indicators (color, total viable count, pH, and total volatile basic nitrogen). Peptide MVHMASKE was identified as a potential peptide marker via multivariate analysis. Pearson correlation revealed a negative correlation between intensity of MVHMASKE and total viable count/total volatile basic nitrogen. In addition, the correlation between peptide content and the change in pork-meat freshness was verified using real-life samples, and the content of MVHMASKE showed a significant decline during storage under 4 and 25 °C, correspondingly reflecting the change of pork meat from fresh to spoiled. This study provides favorable evidence to evaluate pork-meat freshness by monitoring the change of peptide MVHMASKE in content based on mass spectrometry-based peptidomics.

Solidification/stabilization of heavy metals and its efficiency in lead-zinc tailings using different chemical agents.

Lead-zinc tailings are generated during the mining process which is considered as hazardous solid waste due to its high heavy metal content and leachability in the natural state. At present, the most effective technology for disposing heavy metals in solid wastes is the solidification/stabilization (S/S) technique. In terms of S/S technology, chemical stabilization is one of the most potential and practical method. This paper aims to investigate the S/S property of four typical chemical agents (Na2S, NaH2PO4, TMT and Na2EDTA) on the heavy metals in lead-zinc tailings. The results reveal that the heavy metals lead and zinc in tailings are stabilized more effectively by using chelating agents TMT than by using inorganic chemical agents Na2S and NaH2PO4. When the dosage of TMT reaches 4%, the leaching concentration of lead and zinc is 0.18 and 14.60 mg/L according to toxicity characteristic leaching procedure (TCLP), and the stabilization efficiency of lead and zinc is 99.31% and 80.92%, respectively, while the leaching concentration of lead and zinc just drops to 0.41 and 16.00 mg/L with addition of 10% NaH2PO4. Furthermore, the leaching concentration of heavy metal lead in tailings treated by 4% Na2EDTA increases to 53.44 mg/L which far exceeds the standard of pollution control. Therefore, considering stabilization efficiency and dosage, TMT is the preferred agent for solidifying heavy metals in lead-zinc tailings.

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.

Simultaneous Determination of Amphenicols and Metabolites in Animal-Derived Foods Using Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Amphenicols are widely used to prevent and treat animal diseases. However, amphenicol residues accumulate in livestock and poultry and harm consumers. We hypothesized that one can combine solid-phase extraction (SPE) with ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to simultaneously determine amphenicols and metabolites in pork, beef, lamb, chicken, and their products and meet government regulations for maximum residue limits. We extracted crude samples with ethyl acetate and ammonia water (98:2, v/v), purified the samples with a CNW Si SPE column, defatted the samples with acetonitrile-saturated n-hexane, and then determined the resulting analytes by UHPLC-MS/MS. The limit of detection of the analytes in livestock and poultry meat was 0.03-1.50 µg/kg, and the limit of quantification was 0.05-5.00 µg/kg. Measured chloramphenicol, thiamphenicol, and florfenicol concentrations were linear over the range 0.50-50 µg/kg; and the florfenicol amine concentration was linear over the range 5.00-200 µg/kg (all with correlation coefficients >0.9990). The recovery of the spiked samples was between 72% and 120%. The intraday relative standard deviation (RSD) ranged from 1% to 9%, and the interday RSD ranged from 1% to 12%. Based on the above results, the current method is sensitive, accurate, and reproducible with the detection limits being well below the maximum residue limits as per Chinese standard GB 31650-2019, and thus, our research hypothesis could be confirmed.

YAP Promotes Cell Proliferation and Stemness Maintenance of Porcine Muscle Stem Cells under High-Density Condition.

Muscle stem cells (MuSCs) isolated ex vivo are essential original cells to produce cultured meat. Currently, one of the main obstacles for cultured meat production derives from the limited capacity of large-scale amplification of MuSCs, especially under high-density culture condition. Here, we show that at higher cell densities, proliferation and differentiation capacities of porcine MuSCs are impaired. We investigate the roles of Hippo-YAP signaling, which is important regulators in response to cell contact inhibition. Interestingly, abundant but not functional YAP proteins are accumulated in MuSCs seeded at high density. When treated with lysophosphatidic acid (LPA), the activator of YAP, porcine MuSCs exhibit increased proliferation and elevated differentiation potential compared with control cells. Moreover, constitutively active YAP with deactivated phosphorylation sites, but not intact YAP, promotes cell proliferation and stemness maintenance of MuSCs. Together, we reveal a potential molecular target that enables massive MuSCs expansion for large-scale cultured meat production under high-density condition.

Combined application of high-throughput sequencing and UHPLC-Q/TOF-MS-based metabolomics in the evaluation of microorganisms and metabolites of dry-cured ham of different origins.

Ham fermentation relies on environmental and indigenous microorganisms forming a rich microbiome, which is pivotal to taste and flavor formation. Previous studies have focused on the appearance of differences of microorganisms and metabolites, this study aims to establish the relationship between microorganisms and metabolites over a period of two years in the fermentation of hams from Jinghua (JH2), Xuanwei (XW2), Rugao (RG2), Iberian (IB2) and Parma (PA2). We profiled bacterial communities by sequencing the V3-V4 region of the 16S rRNA genes and metabolites were analyzed using LC-Q-TOF-MS. LefSe analysis showed that different biomarkers in five ham groups. OPLS analysis showed that most differential metabolites are amino acids and were associated with four metabolic pathways. Correlation analysis implies a firm positive relationship between microorganisms and metabolites. This study provides novel insights into the taste and flavor quality of dry-cured hams of different origins due to fermentation.

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.

Label-free proteomics reveals the mechanism of bitterness and adhesiveness in Jinhua ham.

To obtain better understanding of the formation mechanisms of bitterness and adhesiveness, protease activities, proteolysis index and protein degradation were investigated among raw, normal and defective hams. Normal and defective hams both showed a decrease in cathepsin B and B + L activities compared with raw ham, while higher residual activities were observed in defective ham. Approximate 1.2-fold values of proteolysis index were observed in defective ham than in normal ham, indicating that cathepsin B and B + L activities were key contributors in degrading muscle proteins of dry-cured ham. 322 proteins were identified by label-free proteomics, and 49 down-regulated proteins were found in the comparison between normal and defective hams. Creatine kinase, myosin, α-actinin and troponin-T showed the most intense response to bitterness and adhesiveness of dry-cured ham, confirmed by partial least squares regression analysis. Myosin could be a suitable biomarker to monitor bitterness and adhesiveness of dry-cured ham.

Identification of Rosmarinic Acid-Adducted Sites in Meat Proteins in a Gel Model under Oxidative Stress by Triple TOF MS/MS.

Triple TOF MS/MS was used to identify adducts between rosmarinic acid (RosA)-derived quinones and meat proteins in a gel model under oxidative stress. Seventy-five RosA-modified peptides responded to 67 proteins with adduction of RosA. RosA conjugated with different amino acids in proteins, and His, Arg, and Lys adducts with RosA were identified for the first time in meat. A total of 8 peptides containing Cys, 14 peptides containing His, 48 peptides containing Arg, 64 peptides containing Lys, and 5 peptides containing N-termini that which participated in adduction reaction with RosA were identified, respectively. Seventy-seven adduction sites were subdivided into all adducted proteins including 2 N-terminal adduction sites, 3 Cys adduction sites, 4 His adduction sites, 29 Arg adduction sites, and 39 Lys adduction sites. Site occupancy analyses showed that approximately 80.597% of the proteins carried a single RosA-modified site, 14.925% retained two sites, 1.492% contained three sites, and the rest 2.985% had four or more sites. Large-scale triple TOF MS/MS mapping of RosA-adducted sites reveals the adduction regulations of quinone and different amino acids as well as the adduction ratios, which clarify phenol-protein adductions and pave the way for industrial meat processing and preservation.

Chemerin inhibition of myogenesis and induction of adipogenesis in C2C12 myoblasts.

Chemerin is an adipocyte-secreted adipokine that regulates the differentiation and metabolism of adipose through auto-/paracrine signaling. Its function in the differentiation of multipotent myoblast cells has thus far received little attention. In this study, C2C12 myoblast cells were cultured in the medium with Chemerin, and the differentiation potential of C2C12 myoblasts was analyzed. The results showed that Chemerin increased ROS levels and TG content of C2C12 cells. At the same time, the mRNA expressions and protein concentrations of the adipogenic factors PPARγ, C/EBPα and UCP1 were up-regulated, while the muscle specific transcription factors MyoD, Myogenin and MyHC were decreased in cultured C2C12 cells. In conclusion, the adipokine Chemerin promoted the adipogenic differentiation potential and altered the fate of myoblast cells from myogenesis to adipogenesis, which contributed in part to the up-regulated adipocyte genes. Our study reveals the importance of functional Chemerin signaling in adipogenesis and in directing the differentiation of multipotent myoblast cells.

Identification and quantification of adducts between oxidized rosmarinic acid and thiol compounds by UHPLC-LTQ-Orbitrap and MALDI-TOF/TOF tandem mass spectrometry.

LTQ Orbitrap MS/MS was used to identify the adducts between quinones derived from rosmarinic acid (RosA) and thiol compounds, including cysteine (Cys), glutathione (GSH), and peptides digested from myosin. Two adducts of quinone-RosA/Cys and quinone-RosA/2Cys, one quinone-RosA/GSH adduct, and three quinone-RosA/peptide adducts were identified by extracted ion and MS(2) fragment ion chromatograms. By using MALDI-TOF/TOF MS, the adduction reaction between RosA and myosin in myofibrillar protein isolates was determined, demonstrating that the accurate reaction site was at Cys949 of myosin. The effect of reaction conditions, including stirring time, temperature, and oxidative stress, on the formation of adducts was further investigated. The formation of quinone-RosA/Cys and quinone-RosA/GSH increased with stirring time. Both adducts increased with temperature, whereas the reactivity of the addition reaction of GSH was higher than that of Cys. With increasing oxidation stress, the formation of quinone-RosA/GSH adduct increased and that of quinone-RosA/Cys adduct decreased.