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.

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.

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.

Morphophysiological responses of detached and adhered biofilms of Pseudomonas fluorescens to acidic electrolyzed water.

Pseudomonas spp. have emerged as the main spoilage bacteria, with many strains easily forming biofilms on food-contact surfaces and causing cross-contamination. The efficacy of disinfectants against bacteria is usually tested with planktonic cells; however, the disinfection tolerance of biofilms, especially detached biofilms, remains unknown. Here, we investigated the tolerance responses of detached and adhered biofilms of Pseudomonas fluorescens to acidic electrolyzed water (AEW) by determining tolerance responses by plate counting, comparing them using a Weibull model, and verifying changes in bacterial morphology by scanning electron microscopy. The experimental data and the responses calculated using Weibull a (scale) and b (shape) parameters agreed well (R2 values: 0.974-0.999), and we found that AEW exhibited effective antimicrobial activity against P. fluorescens, with adhered biofilms were more resistant than detached biofilms and planktonic cells. Additionally, AEW increased the bacterial membrane permeability and decreased the membrane potential, intracellular ATP concentrations, and intracellular pH while also triggering the disruption of extracellular polymeric substances. These results demonstrated that the morphophysiological responses of detached and adhered biofilms differed significantly and provided information on disinfectant-resistance strategies potentially beneficial to the development of novel disinfection approaches.

The effect of cooking temperature on the aggregation and digestion rate of myofibrillar proteins in Jinhua ham.

BACKGROUND: In order to evaluate the effect of cooking temperature on the nutrition quality of dry-cured hams, 60 biceps femoris samples from 16 Jinhua hams were divided into four groups (control, 70, 100 and 120 °C) and cooked for 30 min. Carbonyl content, sulfhydryl groups, surface hydrophobicity, microstructure, protein aggregation and digestibility of myofibrillar proteins were investigated. RESULTS: Cooking promoted carbonylation and decreased sulfhydryl groups in a temperature-dependent way. Scanning electron microscopy and Nile Red revealed that protein aggregation became a main phenomenon at 120 °C; it coincided with surface hydrophobicity. The increased carbonyl content and decreased sulfhydryl groups contributed to the formation of aggregates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles showed the initial difference in proteolysis rate among four groups. The in vitro digestibility of pepsin and of trypsin and α-chymotrypsin increased from the control to 100 °C and decreased from 100 to 120 °C. CONCLUSION: The increased digestibility could be attributed to the oxidation of proteins and exposing recognition sites of digestive enzymes, while the decreased digestibility was due to the formation of aggregates. Cooking was a main factor that affected the digestibility of Jinhua ham, and cooking at 100 °C could be an ideal way to gain the highest digestibility of Jinhua ham. © 2018 Society of Chemical Industry.

Evaluation of the spoilage potential of bacteria isolated from chilled chicken in vitro and in situ.

Microorganisms play an important role in the spoilage of chilled chicken. In this study, a total of 53 isolates, belonging to 7 species of 3 genera, were isolated using a selective medium based on the capacity to spoil chicken juice. Four isolates, namely Aeromonas salmonicida 35, Pseudomonas fluorescens H5, Pseudomonas fragi H8 and Serratia liquefaciens 17, were further characterized to assess their proteolytic activities in vitro using meat protein extracts and to evaluate their spoilage potential in situ. The in vitro studies showed that A. salmonicida 35 displayed the strongest proteolytic activity against both sarcoplasmic and myofibrillar proteins. However, the major spoilage isolate in situ was P. fragi H8, which exhibited a fast growth rate, slime formation and increased pH and total volatile basic nitrogen (TVBN) on chicken breast fillets. The relative amounts of volatile organic compounds (VOCs) originating from the microorganisms, including alcohols, aldehydes, ketones and several sulfur compounds, increased during storage. In sum, this study demonstrated the characteristics of 4 potential spoilage bacteria on chilled yellow-feather chicken and provides a simple and convenient method to assess spoilage bacteria during quality management.

Influence of modified atmosphere packaging on protein oxidation, calpain activation and desmin degradation of beef muscles.

BACKGROUND: Protein oxidation is widespread in biochemical systems. The objective of the study was to investigate the differences in protein oxidation, µ-calpain activity, desmin proteolysis and protein solubility of beef psoas major (PM) and semi-membranosus (SM) muscles under three packaging systems during postmortem ageing. At 24 h postmortem, beef muscles were packaged respectively in air-permeable film overwrap (AP), vacuum pack (VP) or modified atmosphere (MAP, 80% O2 + 20% CO2 ), and then displayed for 10 days at 4 °C. RESULTS: Carbonyl group values and thiol group content were significantly influenced by packaging type and storage time. The SM muscles from AP and MAP showed greater µ-calpain activity compared to VP. Desmin of PM and SM from AP and MAP samples showed decreased proteolysis compared with VP. CONCLUSION: The results suggested that the inhibition of µ-calpain activity of beef samples from AP and MAP could be closely associated with protein oxidation which further lowered the level of desmin degradation compared to VP. © 2017 Society of Chemical Industry.

The adipokine Chemerin induces lipolysis and adipogenesis in bovine intramuscular adipocytes.

The adipokine Chemerin is reported to regulate adipogenesis and glucose homeostasis in vivo and in 3T3-L1 cells. Our team is focused on the role of Chemerin in metabolism and intramuscular adipocyte differentiation because intramuscular fat is the basic material for the formation of marbling in livestock and poultry meat. In this study, bovine intramuscular mature adipocytes were cultured in medium with Chemerin, and the process of lipolysis of mature adipocytes and the adipogenesis of de-differentiated preadipocytes were investigated. The results showed that Chemerin induced significant lipolytic metabolism in intramuscular mature adipocytes, indicated by increased levels of glycerol, FFA, and up-regulated expression of the lipolysis critical factors HSL, LPL, and leptin. Meanwhile, the expressions of adipogenic key factors PPARγ, C/EBPα, and A-FABP were decreased by Chemerin during lipolysis or dedifferentiation in mature adipocytes. The de-differentiated preadipocytes could re-differentiate into mature adipocytes. Intriguingly, the formation of cells' lipid droplets was promoted by Chemerin during preadipocyte differentiation. In addition, mRNA and protein expressions of PPARγ, C/EBPα, and A-FABP were up-regulated by Chemerin during preadipocytes differentiation. These results suggest that Chemerin promotes lipolysis in mature adipocytes and induces adipogenesis during preadipocyte re-differentiation, further indicating a dual role for Chemerin in the deposition of intramuscular fat in ruminant animals.

Identification of antioxidant peptides of Jinhua ham generated in the products and through the simulated gastrointestinal digestion system.

BACKGROUND: The objective of this study was to purify and identify antioxidant peptides present in the extract of Chinese dry-cured Jinhua ham. Jinhua ham extracts were separated into five fractions (A-E) by size-exclusion chromatography. Each fraction was subjected to a simulated gastrointestinal (GI) digestion system and fractions showing strong antioxidant activities were collected and subjected to liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) for further purification and identification. RESULTS: Using MS/MS analysis, 33 peptides were identified in these fractions. Several key peptides were selected for synthesis and their antioxidant activity determined. The peptide showing strongest DPPH radical scavenging activity was GKFNV, which showed 92.7% antioxidant activity at a concentration of 1 mg mL(-1); the peptide LPGGGHGDL showed the highest hydroxyl radical scavenging activity; and LPGGGT and HA showed strong inhibition activity against erythrocyte hemolysis (about 45%) before digestion. On the other hand, KEER may contribute to Fe(2+) chelating ability in fraction C after GI digestion. CONCLUSION: Jinhua dry-cured ham seems to be a potential source of antioxidant peptides generated in the products and in GI digestion.

Emulsion stability, thermo-rheology and quality characteristics of ground pork patties prepared with soy protein isolate and carrageenan.

BACKGROUND: Plant proteins and polysaccharides are often utilised in ground meat products as meat binders, gelling agents, texture stabilisers or fat substitutes. The aim of this study was to investigate the effects of the incorporation of 57 g kg(-1) soy protein isolate (SPI), 7 g kg(-1) carrageenan (CAR) and SPI/CAR mixture on the quality of ground pork patties. RESULTS: Ground pork patties with individual SPI and CAR or SPI/CAR mixture showed either retained or improved emulsion stability, physicochemical properties and dynamic rheology compared with control samples. Although there were no significant colour differences among treatments, samples with SPI/CAR mixture presented higher texture profile values for hardness and chewiness compared with other treatments (P < 0.05). Patties with additives showed significantly lower cooking loss and better thermal emulsion stability than control samples owing to a lower release rate of water and fat (P < 0.05). Compared with control samples or those with individual SPI and CAR, patties with SPI/CAR mixture formed a smoother and more continuous structure with a more compact protein matrix. CONCLUSION: The results indicate that a mixture of SPI and CAR can be effectively used as a functional ingredient to improve the quality of ground pork patties.

Effect of ultrasound treatment on functional properties of reduced-salt chicken breast meat batter.

The effect of ultrasound treatments (40 kHz, 300 W) for different times (10, 20, 30 and 40 min) combined with different salt contents (1.0 %, 1.5 % and 2.0 %) on gel properties and water holding capacity (WHC) of chicken breast meat batter were investigated. Results showed salt level significantly (p < 0.05) affected the texture, storage modulus (G'), loss modulus (G″), cooking loss and WHC. Ultrasound treatments for 10 min and 20 min improved the texture and WHC, and had higher G' values. Compared with the controls (2 % salt), ultrasound treatment for 20 min with reduced-salt (1.5 %) had not significant effect (p > 0.05) on texture, cooking loss or WHC. However, longer ultrasound (40 min) treatment resulted in a decrease in hardness, G' value and WHC. Microstructural analysis revealed that gels treated with ultrasound for 20 min had a compact structure whereas those treated for 40 min contained more protein aggregations and more cavities. Low-field nuclear magnetic resonance (LF-NMR) indicated that ultrasound treatment for 20 min lowered the values of spin-spin relaxation time (T2) and increased the proportion of myofibillar water. Overall, high power ultrasound technology is a promising process which can improve the gelation properties and thereby allowing for a partial reduction in the salt levels in chicken meat gels.

The protective effect of rosmarinic acid on hyperthermia-induced C2C12 muscle cells damage.

High temperature will cause animal tissues or cells damage. Rosmarinic acid (RA) is a good antioxidant and health care product, but the roles of RA in muscle cells damage and the mechanisms which caused by high temperature is still unknown. In this study, the roles of RA on hyperthermia-induced apoptosis and damage of C2C12 muscle cells were investigated. C2C12 cells were cultured in medium with different concentration (0, 25, 50, 100 µM) RA and treated in 42 °C high temperature to induce cellular apoptosis and damage. Then, these cells were analyzed effect of different dose of RA on cells apoptosis and damage. The results indicated that RA has protective effect on heat-stress induced cellular damage, and the cells have the higher cell viability at the dose of 50 µM RA by MTT assay. Hochest33342/PI double staining showed that the cellular apoptosis of C2C12 cells were decreased in the presence of selected 50 µM RA. Malondialdehyde formation and reactive oxygen species levels were also decreased significantly, but cellular superoxide dismutase activity was increased significantly in the presence of RA even in the condition of 42 °C. Meanwhile, Caspase-3 mRNA expression, Caspase-3 activity, and Bax/Bcl-2 ratio were reduced significantly, but the mRNA expression of Hsp72 was increased significantly in those hyperthermia-induced C2C12 cells in the presence of 50 µM RA. Taken together, the results at least discovered that RA has protective effects on hyperthermia-induced cellular apoptosis and damage of muscle cells by change the expression of stress-genes and increasing intracellular antioxidant capability.

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.

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.

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.

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.

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.

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.

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.