ABSTRACT
Conjugated linoleic acid (CLA), a bioactive fatty acid that provides various physiological benefits, has gained increasing attention in the food industry, and various studies have focused on enhancing its content in dairy products. The factors influencing CLA content in dairy products vary significantly, including lactation stage, breed type, seasonality, feed, management methods of the animals, the manufacturing processes, storage, and ripening periods of the product. Additionally, the incorporation of CLA-producing probiotic bacteria, such as Lactobacillus, Lactococcus, Bifidobacterium, and Propionibacterium, is an emerging study in this field. Studies have revealed that factors affecting the CLA content in milk affect that in dairy products as well. Furthermore, the species and strains of CLA-producing bacteria, fermentation conditions, ripening period, and type of dairy product are also contributing factors. However, production of CLA-enhanced dairy products using CLA-producing bacteria while maintaining their optimal viability and maximizing exposure to free linoleic acid remains limited. The current review emphasized the factors affecting the CLA content and related mechanisms, challenges in the application of CLA-producing probiotic bacteria, and strategies to address these challenges and enhance CLA production in dairy products. Therefore, the development of functional dairy products with enhanced CLA levels is expected to be possible.
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Cultured meat is expected to become an important material for future food production; however, contrary to initial expectations, the full-scale industrialization of cultured meat is slow and the actual level and opened technology amount is very limited. This study reviews the publicly available technologies of cultured meat and suggests future developmental directions and research agenda. As a result of analyzing papers, patents, and press releases published over the past 10 years, it was found that cultured meat production technology is still at the prototype production level. This is because most papers published are about culture medium and scaffold development, culture conditions, and there is almost no research on finished cultured meat products. Worldwide, most of the filed patents are for producing cultured meat principles; most of them do not use food-grade materials and are not economically feasible for industrialization. Therefore, future research on the industrialization of cultured meat should focus on effective acquisition technologies for satellite cells; cell lineage and undifferentiated state maintenance technologies; the development of serum-free media and culture devices; the prevention of genetic modification, safety verification, and mass production. Furthermore, basic research on mechanisms and influencing factors related to cultured meat production is warranted.
ABSTRACT
Cell culture meat is based on the scaled-up expansion of seed cells. The biological differences between seed cells from large yellow croakers in the two-dimensional (2D) and three-dimensional (3D) culture systems have not been explored. Here, satellite cells (SCs) from large yellow croakers (Larimichthys crocea) were grown on cell climbing slices, hydrogels, and microcarriers for five days to analyze the biological differences of SCs on different cell scaffolds. The results exhibited that SCs had different cell morphologies in 2D and 3D cultures. Cell adhesion receptors (Itgb1andsdc4) and adhesion spot markervclof the 3D cultures were markedly expressed. Furthermore, myogenic decision markers (Pax7andmyod) were significantly enhanced. However, the expression of myogenic differentiation marker (desmin) was significantly increased in the microcarrier group. Combined with the transcriptome data, this suggests that cell adhesion of SCs in 3D culture was related to the integrin signaling pathway. In contrast, the slight spontaneous differentiation of SCs on microcarriers was associated with rapid cell proliferation. This study is the first to report the biological differences between SCs in 2D and 3D cultures, providing new perspectives for the rapid expansion of cell culture meat-seeded cells and the development of customized scaffolds.
Subject(s)
Cell Adhesion , Cell Culture Techniques , Cell Differentiation , Cell Proliferation , Hydrogels , Satellite Cells, Skeletal Muscle , Tissue Scaffolds , Animals , Satellite Cells, Skeletal Muscle/metabolism , Satellite Cells, Skeletal Muscle/cytology , Hydrogels/chemistry , Tissue Scaffolds/chemistry , Cell Culture Techniques, Three Dimensional/methods , Cells, Cultured , Desmin/metabolism , PAX7 Transcription Factor/metabolism , PAX7 Transcription Factor/genetics , Muscle DevelopmentABSTRACT
Fetal bovine serum (FBS) substitution remains one of the challenges to the realization of cultured meat production in the marketplace. In this study, three methods were developed to extract a substitute for FBS using egg white extract (EWE): using 25 mM CaCl2/2.5 % ammonium sulfate/citric acid (A); ethyl alcohol (B); and 5 % ammonium sulfate/citric acid (C). B EWE can effectively replace up to 50 % of FBS in growth media (10 % of the total). Ovalbumin in the extracts can promote cell proliferation, and components along the 12 kDa protein band have the potential to inhibit cell proliferation. Chick primary muscle cells applied with B EWE, an edible material that improved the cost and time efficiency of cultured meat production, effectively proliferated/differentiated. Therefore, EWE extracted using ethyl alcohol may be used as an FBS substitute to reduce animal sacrifices and should be considered a viable alternative to FBS for cultured meat.
ABSTRACT
Novel meat-inspired products, such as cell-cultivated meat and meat analogues, embrace environmental sustainability, food safety and security, animal welfare, and human health, but consumers are still hesitant to accept these products. The appearance of food is often the most persuasive determinant of purchasing decisions for food. Producing cultivated meat and meat analogues with similar characteristics to conventional meat could lead to increased acceptability, marketability, and profitability. Color is one of the sensorial characteristics that can be improved using color-inducing methods and colorants. Synthetic colorants are cheap and stable, but natural pigments are regarded as safer components for novel food production. The complexity of identifying specific colorants to imitate both raw and cooked meat color lies in the differences in ingredients and methods used to produce meat alternatives. Research devoted to improving the sensorial characteristics of meat analogues has noted various color-inducing methods (e.g., ohmic cooking and pasteurization) and additives (e.g., lactoferrin, laccase, xylose, and pectin). Additionally, considerations toward other meat components, such as fat, can aid in mimicking conventional meat appearance. For instance, the use of plant-based fat replacers and scaffolds can produce a marked sensory enhancement without compromising the sustainability of alternative meats. Moving forward, consumer-relevant sensorial characteristics, such as taste and texture, should be prioritized alongside improving the coloration of meat alternatives.
ABSTRACT
Expectations for the industrialization of cultured meat are growing due to the increasing support from various sectors, such as the food industry, animal welfare organizations, and consumers, particularly vegetarians, but the progress of industrialization is slower than initially reported. This review analyzes the main issues concerning the industrialization of cultured meat, examines research and media reports on the development of cultured meat to date, and presents the current technology, industrialization level, and prospects for cultured meat. Currently, over 30 countries have companies industrializing cultured meat, and around 200 companies that are developing or industrializing cultured meat have been surveyed globally. By country, the United States has over 50 companies, accounting for more than 20% of the total. Acquiring animal cells, developing cell lines, improving cell proliferation, improving the efficiency of cell differentiation and muscle production, or developing cell culture media, including serum-free media, are the major research themes related to the development of cultured meat. In contrast, the development of devices, such as bioreactors, which are crucial in enabling large-scale production, is relatively understudied, and few of the many companies invested in the development of cultured meat have presented products for sale other than prototypes. In addition, because most information on key technologies is not publicly available, it is not possible to determine the level of technology in the companies, and it is surmised that the technology of cultured meat-related startups is not high. Therefore, further research and development are needed to promote the full-scale industrialization of cultured meat.
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Peptides with bioactive effects are being researched for various purposes. However, there is a lack of overall research on pork-derived peptides. In this study, we reviewed the process of obtaining bioactive peptides, available analytical methods, and the study of bioactive peptides derived from pork. Pepsin and trypsin, two representative protein digestive enzymes in the body, are hydrolyzed by other cofactors to produce peptides. Bicinchoninic acid assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, chromatography, and in vitro digestion simulation systems are utilized to analyze bioactive peptides for protein digestibility and molecular weight distribution. Pork-derived peptides mainly exhibit antioxidant and antihypertensive activities. The antioxidant activity of bioactive peptides increases the accessibility of amino acid residues by disrupting the three-dimensional structure of proteins, affecting free radical scavenging, reactive oxygen species inactivation, and metal ion chelating. In addition, the antihypertensive activity decreases angiotensin II production by inhibiting angiotensin converting enzyme and suppresses blood pressure by blocking the AT1 receptor. Pork-derived bioactive peptides, primarily obtained using papain and pepsin, exhibit significant antioxidant and antihypertensive activities, with most having low molecular weights below 1 kDa. This study may aid in the future development of bioactive peptides and serve as a valuable reference for pork-derived peptides.
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Significance: Label-free quantitative phase imaging can potentially measure cellular dynamics with minimal perturbation, motivating efforts to develop faster and more sensitive instrumentation. We characterize fast, single-shot quantitative phase gradient microscopy (ss-QPGM) that simultaneously acquires multiple polarization components required to reconstruct phase images. We integrate a computationally efficient least squares algorithm to provide real-time, video-rate imaging (up to 75 frames / s ). The developed instrument was used to observe changes in cellular morphology and correlate these to molecular measures commonly obtained by staining. Aim: We aim to characterize a fast approach to ss-QPGM and record morphological changes in single-cell phase images. We also correlate these with biochemical changes indicating cell death using concurrently acquired fluorescence images. Approach: Here, we examine nutrient deprivation and anticancer drug-induced cell death in two different breast cell lines, viz., M2 and MCF7. Our approach involves in-line measurements of ss-QPGM and fluorescence imaging of the cells biochemically labeled for viability. Results: We validate the accuracy of the phase measurement using a USAF1951 pattern phase target. The ss-QPGM system resolves 912.3 lp / mm , and our analysis scheme accurately retrieves the phase with a high correlation coefficient ( â¼ 0.99 ), as measured by calibrated sample thicknesses. Analyzing the contrast in phase, we estimate the spatial resolution achievable to be 0.55 µ m for this microscope. ss-QPGM time-lapse live-cell imaging reveals multiple intracellular and morphological changes during biochemically induced cell death. Inferences from co-registered images of quantitative phase and fluorescence suggest the possibility of necrosis, which agrees with previous findings. Conclusions: Label-free ss-QPGM with high-temporal resolution and high spatial fidelity is demonstrated. Its application for monitoring dynamic changes in live cells offers promising prospects.
Subject(s)
Algorithms , Humans , Image Processing, Computer-Assisted/methods , Cell Line, Tumor , Microscopy, Phase-Contrast/methods , MCF-7 Cells , Microscopy, Fluorescence/methodsABSTRACT
Crusting has been developed as a non-chemical and non-machine intensive scaffold fabrication method. This method is based on the self-assembling ability of soy biomolecules, allowing the fabrication of a three-dimensional network for cell growth. Preliminary characterization revealed differences in pore size, water absorption, and degradation between pure soy-based scaffold (Y2R) and with added glycerol (Y2G). The Fourier-transform infrared spectrum absorbance peaks of functional groups related to proteins, carbohydrates, and lipids hinted the integration of soy biomolecules potentially via the Maillard reaction, as supported by the visible browning of the scaffold surface. Microscopic images revealed aligned myotubes in both scaffolds, with Y2G myotubes having greater proximity after 72 h of proliferation. Both spontaneous and electro-stimulated contractions were recorded as early as 72 h in proliferation medium. Crusting-fabricated soy-based scaffolds can further be explored for its application in cultured meat production.
Subject(s)
Glycine max , Tissue Scaffolds , Animals , Tissue Scaffolds/chemistry , Glycine max/chemistry , Meat/analysis , Soybean Proteins/chemistry , Cell Proliferation , Tissue Engineering , Mice , In Vitro MeatABSTRACT
Interest and investment in cultivated meat are increasing because of the realization that it can effectively supply sufficient food resources and reduce the use of livestock. Nevertheless, accurate information on the specific technologies used for cultivated meat production and the characteristics of cultivated meat is lacking. Authorization for the use of cultivated meat is already underway in the United States, Singapore, and Israel, and other major countries are also expected to approve cultivated meat as food once the details of the intricate process of producing cultivated meat, which encompasses stages such as cell proliferation, differentiation, maturation, and assembly, is thoroughly established. The development and standardization of mass production processes and safety evaluations must precede the industrialization and use of cultivated meat as food. However, the technology for the industrialization of cultivated meat is still in its nascent stage, and the mass production process has not yet been established. The mass production process of cultivated meat may not be easy to disclose because it is related to the interests of several companies or research teams. However, the overall research flow shows that equipment development for mass production and cell acquisition, proliferation, and differentiation, as well as for three-dimensional production supports and bioreactors have not yet been completed. Therefore, additional research on the mass production process and safety of cultivated meat is essential. The consumer's trust in the cultivated meat products and production technologies recently disclosed by some companies should also be analyzed and considered for guiding future developments in this industry. Furthermore, close monitoring by academia and the government will be necessary to identify fraud in the cultivated meat industry.
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Emerging technologies for cell-cultured fish meat as an environmentally friendly protein source for humans still have many obstacles, including large-scale production of high-quality cells, differentiation and bioassembly of cellular material, and improvement of the quality of meat products. Here, we used edible porous microcarriers as scaffolds to support scalable skeletal muscle cell expansion to prepare centimeter-scale cell-cultured fish (CCM) of Carassius auratus for the first time. The quality of CCM was assessed by analyzing the texture, nutrition, flavor, and safety. The results indicated that CCM demonstrated a softer texture than natural fish due to a high moisture content. CCM contained higher protein and lower fat contents, with no significant difference in energy from natural golden crucian carp meat (NGM). CCM had better digestible properties, and 17 volatile components were identified in CCM, ten cocontained compared to NGM. ELISA quantified penicillin, streptomycin, vitamin D, and insulin residues as risk factors in CCM. In conclusion, we utilized edible porous microcarriers to scale-up the expansion of Carassius auratus skeletal muscle cells and bioassembled high-quality CCM of Carassius auratus for the first time, which represents a state-of-the-art protocol applicable to different fish species and even to other economic animals and provides a theoretical basis for scaling up cell-cultured meat production.
Subject(s)
Goldfish , Muscle, Skeletal , Animals , Muscle, Skeletal/chemistry , Muscle, Skeletal/cytology , Porosity , Meat/analysis , Cell Culture Techniques , Fish Proteins/chemistry , Cells, Cultured , Seafood/analysisABSTRACT
BACKGROUND: Myostatin (MSTN) inhibition has demonstrated promise for the treatment of diseases associated with muscle loss. In a previous study, we discovered that Glycyrrhiza uralensis (G. uralensis) crude water extract (CWE) inhibits MSTN expression while promoting myogenesis. Furthermore, three specific compounds of G. uralensis, namely liquiritigenin, tetrahydroxymethoxychalcone, and Licochalcone B (Lic B), were found to promote myoblast proliferation and differentiation, as well as accelerate the regeneration of injured muscle tissue. PURPOSE: The purpose of this study was to build on our previous findings on G. uralensis and demonstrate the potential of its two components, Licochalcone A (Lic A) and Lic B, in muscle mass regulation (by inhibiting MSTN), aging and muscle formation. METHODS: G. uralensis, Lic A, and Lic B were evaluated thoroughly using in silico, in vitro and in vivo approaches. In silico analyses included molecular docking, and dynamics simulations of these compounds with MSTN. Protein-protein docking was carried out for MSTN, as well as for the docked complex of MSTN-Lic with its receptor, activin type IIB receptor (ACVRIIB). Subsequent in vitro studies used C2C12 cell lines and primary mouse muscle stem cells to acess the cell proliferation and differentiation of normal and aged cells, levels of MSTN, Atrogin 1, and MuRF1, and plasma MSTN concentrations, employing techniques such as western blotting, immunohistochemistry, immunocytochemistry, cell proliferation and differentiation assays, and real-time RT-PCR. Furthermore, in vivo experiments using mouse models focused on measuring muscle fiber diameters. RESULTS: CWE of G. uralensis and two of its components, namely Lic A and B, promote myoblast proliferation and differentiation by inhibiting MSTN and reducing Atrogin1 and MuRF1 expressions and MSTN protein concentration in serum. In silico interaction analysis revealed that Lic A (binding energy -6.9 Kcal/mol) and B (binding energy -5.9 Kcal/mol) bind to MSTN and reduce binding between it and ACVRIIB, thereby inhibiting downstream signaling. The experimental analysis, which involved both in vitro and in vivo studies, demonstrated that the levels of MSTN, Atrogin 1, and MuRF1 were decreased when G. uralensis CWE, Lic A, or Lic B were administered into mice or treated in the mouse primary muscle satellite cells (MSCs) and C2C12 myoblasts. The diameters of muscle fibers increased in orally treated mice, and the differentiation and proliferation of C2C12 cells were enhanced. G. uralensis CWE, Lic A, and Lic B also promoted cell proliferation in aged cells, suggesting that they may have anti-muslce aging properties. They also reduced the expression and phosphorylation of SMAD2 and SMAD3 (MSTN downstream effectors), adding to the evidence that MSTN is inhibited. CONCLUSION: These findings suggest that CWE and its active constituents Lic A and Lic B have anti-mauscle aging potential. They also have the potential to be used as natural inhibitors of MSTN and as therapeutic options for disorders associated with muscle atrophy.
Subject(s)
Chalcones , Muscle Fibers, Skeletal , Myostatin , Mice , Animals , Myostatin/metabolism , Molecular Docking Simulation , Cell Differentiation , Muscle Fibers, Skeletal/metabolism , Cell Proliferation , Muscle, Skeletal/metabolismABSTRACT
This study reviewed the current data presented in the literature on developing meat analogs using plant-, insect-, and protein-derived materials and presents a conclusion on future perspectives. As a result of this study, it was found that the current products developed using plant-, insect-, and mycoprotein-derived materials still did not provide the quality of traditional meat products. Plant-derived meat analogs have been shown to use soybean-derived materials and beta-glucan or gluten, while insect-derived materials have been studied by mixing them with plant-derived materials. It is reported that the development of meat analogs using mycoprotein is somewhat insufficient compared to other materials, and safety issues should also be considered. Growth in the meat analog market, which includes products made using plant-, insect-, and mycoprotein-derived materials is reliant upon further research being conducted, as well as increased efforts for it to coexist alongside the traditional livestock industry. Additionally, it will become necessary to clearly define legal standards for meat analogs, such as their classification, characteristics, and product-labeling methods.
ABSTRACT
Cultured meat (CM) is an alternative protein food and is produced by cultivating muscle satellite (stem) cells (MSCs) derived from livestock animals (bovine, chickens, and porcine) through myogenesis leading to generate muscle mass. Myostatin (MSTN) is well well-known negative regulator of myogenesis, and in the present study, in silico screening of natural compounds was performed to identify MSTN inhibitors. Interestingly, quercetin was found to inhibit MSTN (binding energy -7.40 kcal/mol), and this was further validated by a 100 ns molecular dynamics simulation. Quercetin was added to culture media to boost myogenesis, and its potent antioxidant property helped maintain media pH. Furthermore, quercetin increased the myotube thickness and length, increased MSC differentiation, and upregulated the gene and protein expressions of myoblast determination protein 1 (MYOD), Myogenin (MYOG), and Myosin heavy chains (MYH) in vitro. In addition, quercetin inhibited the activities of MSTN, activin receptor type-2B (ACVR2B), and SMAD2 and 3, and thus significantly enhanced MSC differentiation and myotube formation. Overall, this study shows that quercetin might be useful for enhancing large-scale CM production. It is hoped that this study provides a starting point for research in the CM area aimed to enhancing product quality, nutritional values, and the efficacy of large-scale production.
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This study was conducted to compare and analyze the changes in the biochemical characteristics and biological activity of peptide extracts derived from Chickso, Hanwoo, and Wagyu beef during digestion. The results of the in vitro digestion analysis revealed that the digestion rate, total free amino acid content, and antioxidant and antihypertensive activities of Chickso loin and shank myofibrillar proteins were significantly higher (p<0.05) than those of Hanwoo and Wagyu loin and shank myofibrillar proteins. Particularly, the peptide extracts of Chickso loin and shank had a high angiotensin-converting enzyme inhibitory activity. In mice in vivo digestion experiment, the blood serum of mice fed with Chickso loin peptide extract (<10 kDa) showed the highest antioxidant enzyme activity. Thus, Chickso peptide extracts were deemed to be similar or more bioactive than Hanwoo and Wagyu peptide extracts, and can be used as bioactive materials.
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This study aimed to analyze the leading research materials and research trends related to livestock food in Asia in recent years and propose future research agendas to ultimately contribute to the development of related livestock species. On analyzing more than 200 relevant articles, a high frequency of studies on livestock species and products with large breeding scales and vast markets was observed. Asia possesses the largest pig population and most extensive pork market, followed by that of beef, chicken, and milk; moreover, blood and egg markets have also been studied. Regarding research keywords, "meat quality" and "probiotics" were the most common, followed by "antioxidants", which have been extensively studied in the past, and "cultured meat", which has recently gained traction. The future research agenda for meat products is expected to be dominated by alternative livestock products, such as cultured and plant-derived meats; improved meat product functionality and safety; the environmental impacts of livestock farming; and animal welfare research. The future research agenda for dairy products is anticipated to include animal welfare, dairy production, probiotic-based development of high-quality functional dairy products, the development of alternative dairy products, and the advancement of lactose-free or personalized dairy products. However, determining the extent to which the various research articles' findings have been applied in real-world industry proved challenging, and research related to animal food laws and policies and consumer surveys was lacking. In addition, studies on alternatives for sustainable livestock development could not be identified. Therefore, future research may augment industrial application, and multidisciplinary research related to animal food laws and policies as well as eco-friendly livestock production should be strengthened.
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BACKGROUND: Doxycycline (DC) has been shown to possess non-antibiotic properties including Fas/Fas Ligand (FasL)-mediated apoptosis against several tumor types in the concentration range of 10-40 µg/mL. However, the effect of DC in apoptotic signaling at much low concentrations was not studied. METHODS: The present study investigated the attenuation effect of low dose of DC on FasL-induced apoptosis in HeLa cell by the methods of MTT assay, fluorescence microscopy, DNA fragmentation, flow cytometry analysis, and western blotting. RESULTS AND CONCLUSION: In the present findings we showed that low concentration of DC (<2.0 µg/mL) exhibited protective effects against FasL-induced apoptosis in HeLa cells. FasL treatment to HeLa cells resulted in a concentration-dependent induction of cell death, and treatment with low concentrations of DC (0.1-2 µg/mL) significantly (p < 0.001) attenuated the FasL-induced cell death as measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide (MTT) assay. Further, the FasL-induced apoptotic features in HeLa cells, such as morphological changes, DNA fragmentation and cell cycle arrest was also inhibited by DC (0.5 µg/mL). Tetracycline and minocycline also showed similar anti-apoptotic effects but were not significant when compared to DC, tested at same concentrations. Further, DC (0.01-16 µg/mL) did not influence the hydrogen peroxide- or cisplatin-induced intrinsic apoptotic pathway in HeLa cells. Protein analysis using Western blotting confirmed that FasL-induced cleavage/activation of cas-pase-8 and caspase-3, were inhibited by DC treatment at low concentration (0.5 µg/mL). Considering the overall data, we report for the first time that DC exhibited anti-apoptotic effects at low concentrations in HeLa cells by inhibition of caspase activation via FasL-induced extrinsic pathway.