Porcine Hemoglobin Research Articles

Biocontrol Strategy of Listeria monocytogenes in Ready-to-Eat Pork Cooked Ham Using Peptic Hydrolysates of Porcine Hemoglobin.

Listeria monocytogenes is a foodborne pathogen that represents a serious concern for ready-to-eat (RTE) meat products due to its persistence in production facilities. Among the different strategies for the control of this pathogen, the use of antimicrobial peptides derived from food by-products, such as slaughterhouse blood proteins, has emerged as a promising biocontrol strategy. This study evaluated for the first time the use of peptic hydrolysates of porcine hemoglobin as a biocontrol strategy of L. monocytogenes in RTE pork cooked ham. Pure porcine hemoglobin (Hb-P) and porcine cruor (P-Cru) were hydrolyzed using pepsin at different temperatures (37 °C for Hb-P and 23 °C for P-Cru) for 3 h. Then, the hydrolysates were characterized in terms of their degree of hydrolysis (DH), peptide population, color, and antimicrobial activity (in vitro and in situ) against three different serotypes of L. monocytogenes. Reducing the hydrolysis temperature of P-Cru by 14 °C resulted in a 2 percentage unit decrease in DH and some differences in the peptide composition. Nevertheless, the antimicrobial activity (in situ) was not significantly impacted, decreasing the viable count of L. monocytogenes by ~1-log and retarding their growth for 21 days at 4 °C. Although the color of the product was visibly altered, leading to more saturated reddish and yellowish tones and reduced brightness, the discoloration of the hydrolysates can be addressed. This biopreservation approach holds promise for other meat products and contributes to the circular economy concept of the meat industry by valorizing slaughterhouse blood and producing new antilisterial compounds.

Hemoglobin Hydrolyzate Promotes Iron Absorption in the Small Intestine through Iron Binding Peptides.

Hemoglobin is an excellent source of iron supplements, and its hydrolyzate spontaneously binds iron during digestion and promotes iron absorption in vivo. However, the underlying mechanisms of what peptides bind and how they bind iron ions remain unclear. This study prepared the porcine hemoglobin hydrolyzate through enzymatic hydrolysis and acid treatment and investigated the mechanisms of hemoglobin hydrolyzate on iron absorption through the determination of iron levels in dietary intervention mice, iron binding site analyses, peptide digestion analyses, molecular simulation docking, and INT407 cell validation. The results showed that ingestion of the hemoglobin hydrolyzate diets increased iron levels in the blood of mice, accompanied by the upregulation of duodenal iron circulation-related genes such as ferritin, PCBP1, and HP. Carboxyl, imidazole groups, and aromatic amino acid residues were iron binding sites of hemoglobin hydrolyzate during digestion. VDEVGGEA and VDEVGGE were found to involve the spontaneous and efficient binding of hemoglobin hydrolyzate to iron ions in the intestinal cavity. In particular, the DEVGGE peptide was the typical sequence for hemoglobin hydrolytic peptides to exert iron binding activity.

Candidate raw materials for a sustainable feeding of greater amberjack (Seriola dumerili) reared in recirculating aquaculture systems: Apparent digestibility, physical properties of diets and faeces, and postprandial nitrogen excretion

To evaluate the raw material digestibility, the physical characteristics of diet pellets and faeces, and the postprandial nitrogen excretion in greater amberjack (Seriola dumerili) reared in recirculating aquaculture systems (RAS), 10 diets were obtained by mixing a reference diet (CP: 51.8% DM; EE: 13.6% DM) with one of ten raw materials (7:3 ratio), i.e. fishmeal (FM), porcine haemoglobin (PH), fermented soybean (FS), soybean protein concentrate (SPC), hydrolysed wheat protein (HWP), potato protein (PP), beet pulp (BP), beer yeast without fat (BY), beer yeast with fat (BYF), and Lithothamnium seaweed (LS). Twenty fish (268.9 ± 94.3 g) were randomly distributed into 5 RAS tanks (4 fish per tank) and fed for 140 days the experimental diets. A Latin square experimental design was used based on 5 diets × 5 tanks for two rounds (10 ingredients, 2 weeks per diet per tank). The highest ADCs of dry matter and protein were recorded for PH (83.0% and 88.4%, respectively), FM (81.8% and 84.3%), SPC (77.4% and 82.5%), and BYF (73.0% and 80.9%) while the lowest ones for BY (64.4% and 70.8%), HWP (59.8% and 69.9%), PP (48.3% and 69.8%), and BP (53.7% and 55.8%) (p < 0.05). The ADCs of EAA were the highest for PH, FM and SPC compared to the other raw materials; ADCs of lipids was above 90% for all raw materials (p > 0.05). Regarding pellets properties, FM was associated with the highest value for oil leakage whereas BP to the lowest (0.18% vs. 0.11%; p < 0.05); FS, HWP, BYF, and PH produced less turbidity in water after 15 min than BY (3.68 mg L−1 on average vs. 6.01 mg L−1; p < 0.05). Regarding faeces size, fish fed FS, BYF, and FM produced the lowest volume of fresh faeces of fine-size class than those fed the other raw materials (62.1% vs. 69.2% on average; p < 0.05); the highest rate of dry faeces retained at a sieve mesh interval of 1.0 < mm < 1.2, was produced by fish fed BYF and the lowest rate by those fed BY (1.16% vs. 0.34%; p < 0.05). A maximum of postprandial nitrogen excretion (mg N-NH4+ × kgfish−1 × h) was reached between 2 and 4 h after feed ingestion without differences among diets (p > 0.05). Overall, the findings of this investigation will contribute to the sustainable formulation of RAS aquafeed optimized and tailored for this promising Mediterranean aquaculture species.

Efficient Secretory Expression for Mammalian Hemoglobins in Pichia pastoris

Mammalian hemoglobins (HB) are a kind of heme-binding proteins that play crucial physiological roles in various organisms. The traditional techniques employed for the extraction of HB are expensive and time-consuming, while the yields of mammalian HB in previous reports were quite low. The industrial Pichia pastoris is a highly effective platform for the secretory expression of heterologous proteins. To achieve efficient secretory expression of HB in P. pastoris, multiple strategies were applied, including the selection of a suitable host, the screening of optimal endogenous signal peptides, the knockout of VPS10, VTH1, and PEP5, and the co-expression of Alpha-Hemoglobin Stabilizing Protein (AHSP). In addition, the conditions for producing HB were optimized at shaking-flask level (BMMY medium with 100 mg/L of hemin, 2% methanol, and 24 °C). Based on these conditions, the higher titers of bovine hemoglobin (bHB, 376.9 ± 13.3 mg/L), porcine hemoglobin (pHB, 119.2 ± 7.3 mg/L), and human hemoglobin (hHB, 101.1 ± 6.7 mg/L) were achieved at fermenter level. The engineered P. pastoris strain and comprehensive strategies can also be applied to facilitate the synthesis of other high-value-added hemoproteins or hemoenzymes.

Effects of applied current modes on the migration selectivity of peptides from a porcine cruor hydrolysate during electrodialysis with ultrafiltration membrane

Pepsic hydrolysis of porcine hemoglobin produces various antimicrobial peptides, however, within such complex hydrolysates, certain peptide bioactivities remain unexpressed. Electrodialysis with ultrafiltration membrane (EDUF) addresses this by separating peptides from hydrolysates based on peptide charge and molecular mass, resulting in fractions with increased bioactivity. This study aims to assess the impacts of different current modes (continuous current (CC), pulsed electric field (PEF) and polarity reversal (PR)) on peptide migration selectivity, which has never been reported. Different combinations of pulse and pause or reversed pulse were tested: 1 s/1 s and 10 s/1 s. This investigation focused on the 31 most migrating peptides and their 6 main physicochemical characteristics. Three groups of current conditions with similar performances were observed: (1) CC, PEF 10 s/1 s and PR 10 s/1 s (Averaged final migration rate of 5.0 g/m2h and energy consumption of 48.7 Wh/g of peptide); (2) PEF 1 s/1 s (9.0 g/m2h and 61.0 Wh/g of peptide), and (3) PR 1 s/1 s (3.3 g/m2h and 95.6 Wh/g of peptide). CC, PEF, and PR 10 s/1 s facilitated the migration of mainly cationic peptides with low molecular masses and were the most selective conditions. PR 1 s/1 s allowed the migration of some anionic peptides with lower molecular masses, due to the short polarity reversal. This study demonstrated for the first time the effects of the electrical current mode and condition associated on migration selectivity of peptides during EDUF and their performances. Modifying the electrical current mode offers a new way to optimize separation selectivity and generate specific peptide populations in the recovery compartment with potentially different bioactivities.

Heme iron as potential iron fortifier for food application – characterization by material techniques

Abstract The modern food industry requires new analytical methods for high-demand food supplements, personalized diets, or bioactive foods development. One of the main goals of the food industry is to discover new ways of food fortification. This applies to food products or supplements for human and animal diets. In our research, we focused on the solid particles of AproTHEM (dried porcine hemoglobin), which is approved for animal feeding and as a meat product additive, and AproFER 1000 (heme iron polypeptides), which is still being investigated. The study showed the possible application of advanced techniques for the examination of iron-based food additives. We evaluated selected techniques for particle size and morphology examination such as laser diffraction, optical microscopy, as well as scanning electron microscopy, and briefly discussed their usefulness compared with other techniques. On the basis of our results, we proposed a path of microscopic analysis for the study of material homogeneity. The structure of heme iron was evaluated by X-ray diffraction, FT-IR, and Raman spectroscopy supported with thermal behavior analysis (differential scanning calorimeter). Furthermore, a portable colorimeter was applied for L*a*b* color analysis. Our study proved that for new food product development, particle size analysis as well as typically used advanced materials techniques can be successfully applied.

Decolorization of porcine hemoglobin hydrolysates: The role of peptide characteristics and pH values.

The unpleasant color caused by heme limits the utilization of hemoglobin as a food ingredient. Enzymatic hydrolysis has been used to decolorize hemoglobin, but the underlying mechanisms are poorly understood. The aim of this study was to investigate the decolorization efficiency of porcine hemoglobin using different enzymes and final pH values, and to elucidate their influence on decolorization. Based on higher yields and better decolorization, hemoglobin hydrolysates produced by papain, bromelain, savinase, and protease A were further studied. Compared to hydrolysates by savinase and protease A, a higher proportion of histidine-containing peptides was responsible for better decolorization by papain and bromelain. For all hydrolysates, a moderate reduction in pH to 4.0-5.0 facilitated decolorization of the hydrolysates. Similar peptide profiles of hydrolysates from the same enzyme treatment reflected that pH mainly affected the precipitation of the heme-containing fraction through heme-heme interaction rather than heme-peptide interaction. Overall, this study sheds light on the use of enzymatic hydrolysis to remove the heme group from hemoglobin. PRACTICAL APPLICATION: Slaughterhouses produce tons of protein-rich blood each year. Due to the presence of the heme group in hemoglobin, blood has a dark red color and metallic taste, making it generally unacceptable for consumers. This study provided information on the decolorization of porcine hemoglobin by removing the heme fraction, which should facilitate the utilization of decolored hemoglobin hydrolysates as nutritional food ingredients.

Effect of interaction between catechin and glycated porcine hemoglobin on its antioxidant functional and structural properties

This study aimed to investigate the interaction between catechins and glycated porcine hemoglobin (G-PHb). The effects of the interaction on the antioxidant function and structural characteristics of the complex (CG-PHb) of catechin and G-PHb were discussed. Porcine hemoglobin (PHb) and G-PHb were used as controls. The solubility, turbidity, emulsifying property, surface hydrophobicity, and oxidation resistance of CG-PHb were studied. The structural characteristics of the CG-PHb were analyzed via UV–visible spectroscopy, fluorescence spectrometry, fourier transform infrared spectroscopy, scanning electron microscope and molecular docking technology. Compared with the control group, these properties of the CG-PHb are significantly improved. The UV absorption peak of CG-PHb had a larger peak and a slight red shift. The fluorescence peak intensity was in the following order: CG-PHb > PHb > G-PHb. The β-sheet content of CG-PHb increased significantly (P < 0.05). Moreover, SEM showed that the structure of CG-PHb had changed. The surface pore structure increased, which facilitated its functional properties. Molecular docking showed that catechin likely interacted with PHb residues HEM-950, PHE-41, GLU-43, ARG-40 and LEU-96. Catechin and PHb formed a more stable complex because of hydrogen bonds. This study can provide new ideas for protein modification research and provide theoretical basis and reference for the property changes of the CG-PHb during food processing.

How Nanoparticles Modify Adsorbed Proteins: Impact of Silica Nanoparticles on the Hemoglobin Active Site.

The adsorption of proteins on surfaces has been studied for a long time, but the relationship between the structural and functional properties of the adsorbed protein and the adsorption mechanism remains unclear. Using hemoglobin adsorbed on silica nanoparticles, we have previously shown that hemoglobin's affinity towards oxygen increases with adsorption. Nevertheless, it was also shown that there were no significant changes in the quaternary and secondary structures. In order to understand the change in activity, we decided in this work to focus on the active sites of hemoglobin, the heme and its iron. After measuring adsorption isotherms of porcine hemoglobin on Ludox silica nanoparticles, we analyzed the structural modifications of adsorbed hemoglobin by X-ray absorption spectroscopy and circular dichroism spectra in the Soret region. It was found that upon adsorption, there were modifications in the heme pocket environment due to changes in the angles of the heme vinyl functions. These alterations can explain the greater affinity observed.

Increase of Kokumi γ-Glutamyl Peptides in Porcine Hemoglobin Hydrolysate Using Bacterial γ-Glutamyltransferase.

The kokumi sensation of protein hydrolysates could be enhanced by γ-glutamylation through forming a series of γ-glutamyl di- and tri-peptides. In this study, porcine hemoglobin hydrolysate was γ-glutamylated using enzymes from Bacillus amyloliquefaciens (Ba) or Bacillus licheniformis (Bl), which are sold as glutaminases but identified as γ-glutamyltransferases (GGTs). To yield more γ-glutamyl peptides, reaction conditions were optimized in terms of GGT source (BaGGT and BlGGT), substrate concentration (10, 20, and 40%), reaction time (3, 6, 12, and 24 h), and glutamine supplementation (20, 40, and 80 mM). Results showed that both the GGTs had the highest transpeptidase activity at similar pH values but different temperatures. In addition, BaGGT had stronger catalytic ability to form γ-glutamyl dipeptides, while BlGGT was more capable to generate γ-Glu-Val-Gly. Adding glutamine was more efficient to obtain more target peptides than adjusting the hydrolysate concentration and reaction time. This study contributes to the valorization of animal side streams.

Application of polymerized porcine hemoglobin in the ex vivo normothermic machine perfusion of rat livers.

Background: In contrast to traditional static cold preservation of donor livers, normothermic machine perfusion (NMP) may reduce preservation injury, improve graft viability and potentially allows ex vivo assessment of graft viability before transplantation. The polymerized porcine hemoglobin is a kind of hemoglobin oxygen carrier prepared by crosslinking porcine hemoglobin by glutaraldehyde to form a polymer. The pPolyHb has been proved to have the ability of transporting oxygen which could repair the organ ischemia-reperfusion injury in rats. Objective: In order to evaluate the effectiveness of rat liver perfusion in vitro based on pPolyHb, we established the NMP system, optimized the perfusate basic formula and explored the optimal proportion of pPolyHb and basal perfusate. Methods: The liver was removed and perfused for 6h at 37°C. We compared the efficacy of liver perfusion with different ratios of pPolyHb. Subsequently, compared the perfusion effect using Krebs Henseleit solution and pPolyHb perfusate of the optimal proportion, and compared with the liver preserved with UW solution. At 0h, 1h, 3h and 6h after perfusion, appropriate samples were collected for blood gas analysis and liver injury indexes detection. Some tissue samples were collected for H&E staining and TUNEL staining to observe the morphology and detect the apoptosis rate of liver cells. And we used Western Blot test to detect the expression of Bcl-2 and Bax in the tissues. Results: According to the final results, the optimal addition ratio of pPolyHb was 24%. By comparing the values of Bcl-2/Bax, the apoptosis rate of pPolyHb group was significantly reduced. Under this ratio, the results of H&E staining and TUNEL staining showed that the liver morphology was well preserved without additional signs of hepatocyte ischemia, biliary tract injury, or hepatic sinusoid injury, and hepatocyte apoptosis was relatively mild. Conclusion: Through the above-mentioned study we show that within 6h of perfusion based on pPolyHb, liver physiological and biochemical activities may essentially be maintained in vitro. This study demonstrates that a pPolyHb-based perfusate is feasible for NMP of rat livers. This opens up a prospect for further research on NMP.

Effect of Different Gas-Stunning Conditions on Heme Pigment Solutions and on the Color of Blood, Meat, and Small Intestine of Rabbits.

The objectives of this study were to elucidate the effect of different high-concentration levels of inert gases (Ar and N2) on heme-pigment solutions and on the color of the blood, meat, and small intestine of rabbits; and to figure out the proper concentration level of inert gas (Ar or N2) for the complete stunning of the rabbit. To observe the changing of color attributes, a research study was conducted in the abattoir of the National Institute of Animal Science (NIAS), RDA, Republic of Korea. This experiment had 2 parts, (i) A trial on heme-pigment solutions (hemoglobin and myoglobin solution) was conducted in a gas chamber with different high-concentration levels of carbon dioxide, argon, nitrogen, and normal air; and (ii) a rabbit experiment was conducted-stunning with different high-concentration levels of carbon dioxide, argon, nitrogen, and the Halal method (non-stunning). A small-size digital gas chamber was used for this experiment (size: width 850 mm × depth 1350 mm). Artificial hemoglobin (Hb) and myoglobin (Mb) solutions were created from "porcine hemoglobin lyophilized powder" and "equine skeletal muscle myoglobin lyophilized powder", respectively. In the heme-pigment solutions trial, 10 treatments were used: (i) 80% carbon dioxide (T1), (ii) 85% carbon dioxide (T2), (iii) 90% carbon dioxide (T3), (iv) 80% argon (T4), (v) 85% argon (T5), (vi) 90% argon (T6), (vii) 80% nitogen (T7), (viii) 85% nitogen (T8), (ix) 90% nitogen (T9), and (x) normal air (T10). Heme-pigment solutions (both Hb and Mb) were exposed with each treatment for four separate durations of time (30 s, 1 min, 2 min, and 4 min); and every sample (Hb and Mb) was exposed during each duration of time for 10 times (n = 10). In the rabbit experiment, seven treatments were used (i) 85% carbon dioxide (T1), (ii) 90% carbon dioxide (T2), (iii) 85% argon (T3), (iv) 90% argon (T4), (v) 96% nitrogen (T5), (vi) 98% nitogen (T6), and (vii) the Halal method (non-stunning) (T7). Forty-two rabbits (mixed-breed) were collected from the nearest commercial farm and randomly selected for a treatment group (n = 6). The average body weight was 2.3 kg. For stunning, each individual rabbit was separately kept in a gas chamber, after which each specific gas was used to fulfill the desired level in the pit. After reaching the desired level of concentration, rabbits were stunned within a very short time. It was observed that the time required for stunning in the T1, T2, T3, T4, T5 and T6 treatment was 79-82, 68-73, 97-103, 88-91, 164-173, and 108-115 s, respectively. In the case of the Halal method (non-stunning), as per the rules of Islam, rabbits were slaughtered without stunning. After slaughtering, in all treatments, the blood, meat, and small intestine of each animal were collected carefully and kept in a cool room in which the temperature was -2 °C, and after 24 h, the color attributes-lightness (L*), redness (a*), and yellowness (b*)-were measured via a Chroma Meter. It was found that in both experiments (trial on heme-pigment solutions and rabbit stunning), the color values (L*, a* and b*) showed a significant difference (p &lt; 0.05) among the treatment groups. The CO2-treated groups showed high redness (a*) and low lightness (L*) and yellowness (b*), which indicated a dark-red color, and N2-treated groups showed high lightness (L*) and yellowness (b*) and low redness (a*), which implied a bright-red color. The effect of the Ar was in between the CO2- and N2-treated groups. These phenomena were observed both in the heme-pigment solutions (Hb and Mb) and in the blood, meat, and small intestine of the rabbits. N2-stunned animals fulfill the fundamental desire of consumers to purchase bright red-colored fresh meat. Therefore, in view of color attributes, consumer satisfaction, and animal welfare, N2 gas can be thought of as a valuable alternative to stunning. Considering the time required for complete stunning and desirable color attributes, a 98% concentration of N2 is best for rabbit stunning. As such, it could be used as a better option for the gas stunning of animals.

Apparent digestibility of ten protein ingredients for largemouth bass ( Micropterus salmoides )

The study was conducted to investigate the apparent digestibility coefficients (ADC) of nutrients in commonly used protein sources for largemouth bass. A reference diet and test diets with 70% reference diet and 30% feedstuff were formulated with yttrium oxide as a digestibility indicator. Overall, the ADC of dry matter of tested protein ingredients was 69.5%–96.4%, and the ADC of dry matter in animal protein sources was significantly higher than in the other five plant protein sources. The ADC of crude proteins ranged from 89.1% to 97.3% for all tested feedstuffs. The ADC value of total amino acids in the tested ingredients is consistent with that of the protein. The ADC of crude lipid of tested ingredients ranged from 45.4% to 96.2%, and the lowest and highest values were observed in porcine haemoglobin powder and pet-grade poultry by-product meal, respectively. The ADC of total phosphorus of animal protein ingredients, except porcine haemoglobin powder, was significantly higher than that of plant protein ingredients, except fermented soybean meal. The results revealed the difference in availability of nutrients in plant and animal protein sources for largemouth bass, which could provide a theoretical basis for the selection of protein sources of commercial feed for largemouth bass.

Impact of Pulsed Electric Fields and pH on Enzyme Inactivation and Bioactivities of Peptic Hydrolysates Produced from Bovine and Porcine Hemoglobin.

The production of bioactive peptides from hemoglobin via peptic hydrolysis is a promising alternative to valorizing slaughterhouse blood proteins. Nevertheless, it has some limitations such as low yield, high cost of enzymes, and the use of chemical reagents. The latter is aggravated by the pH increase to inactivate the enzyme, which can affect the bioactivity of the peptides. Thus, this study aimed to evaluate the effect of pulsed electric fields (PEF) on the pepsin inactivation and biological activities (antimicrobial and antioxidant) of hemoglobin hydrolysates. Bovine (Hb-B) and porcine (Hb-P) hemoglobin were hydrolyzed with pepsin for 3 h and treated with PEFs to inactivate the enzyme. The degree of hydrolysis (DH) did not show significant changes after PEF inactivation, whereas peptide population analysis showed some changes in PEF-treated hydrolysates over time, suggesting residual pepsin activity. PEF treatments showed no significant positive or negative impact on antimicrobial and antioxidant activities. Additionally, the impact of pH (3, 7, and 10) on bioactivity was studied. Higher pH fostered stronger anti-yeast activity and DPPH-scavenging capacity, whereas pH 7 fostered antifungal activity. Thus, the use of hemoglobin from the meat industry combined with PEF treatments could fit the circular economy concept since bioactive peptides can be produced more eco-efficiently and recycled to reduce the spoilage of meat products. Nevertheless, further studies on PEF conditions must be carried out to achieve complete inactivation of pepsin and the potential enhancement of peptides' bioactivity.

Influence of growth medium and yeast species on the formation of γ-glutamyl peptides

γ-Glutamyl peptides are known to impart a kokumi sensation and increase the palatability of food. To compare the γ-glutamyl peptides produced by different yeasts, Saccharomyces cerevisiae and Debaryomyces hansenii were cultured in a conventional medium (YPG) and a medium composed of porcine hemoglobin hydrolysates (YHG) for six days. Quantification of γ-glutamyl peptides as well as analysis of yeast growth and γ-glutamyltransferase (GGT) activity were carried out. Results showed that both strains could form a range of γ-glutamyl dipeptides and glutathione in either medium. The total concentration of dipeptides and the concentration of glutathione in each medium showed an upward trend over fermentation time. Compared with yeast species, medium composition had a greater impact on the formation of γ-glutamyl dipeptides. During most of the fermentation period, D. hansenii produced more γ-glutamyl dipeptides (reaching a maximum of 2.395 μM on day 6 in YHG), while S. cerevisiae generated more glutathione (reaching a maximum of 13.465 μM on day 5 in YHG). In addition, D. hansenii showed higher GGT activity than S. cerevisiae in the latter half of fermentation. This study illustrates the influence of growth medium and the crucial choice of yeast species for obtaining target γ-glutamyl peptides for kokumi sensation.

Effects of γ-Glutamylated Hydrolysates from Porcine Hemoglobin and Meat on Kokumi Enhancement and Oxidative Stability of Emulsion-Type Sausages

Effects of γ-Glutamylated Hydrolysates from Porcine Hemoglobin and Meat on Kokumi Enhancement and Oxidative Stability of Emulsion-Type Sausages

Modulation of Functional Characteristics of Mesenchymal Stromal Cells by Acellular Preparation of Porcine Hemoglobin

Exploring the potential usage of the acellular preparation of porcine hemoglobin (PHb) isolated from slaughterhouse blood as a cell culture media component, we have tested its effects on the functional characteristics of stromal cells of mesodermal origin. Human peripheral blood mesenchymal stromal cells (PB-MSCs) were used in this study as a primary cell model system, along with three mouse cell lines (ATDC5, MC3T3-E1, and 3T3-L1), which represent more uniform model systems. We investigated the effect of PHb at concentrations of 0.1, 1, and 10 μM on these cells’ proliferation, cycle, and clonogenic and migratory potential, and found that PHb’s effect depended on both the cell type and its concentration. At the lowest concentration used (0.1 μM), PHb showed the least evident impact on the cell growth and migration; hence, we analyzed its effect on mesenchymal cell multilineage differentiation capacity at this concentration. Even under conditions that induce a specific type of MSC differentiation (cultivation in particular differentiation media), PHb modulated chondrogenic, osteogenic, and adipogenic differentiation, making it a potential candidate for a supplement of MSC culture. Through a model of porcine hemoglobin, these findings also contribute to improving the knowledge of extracellular hemoglobin’s influence on MSCs &gt;in vivo.

Flavor Characterization of Animal Hydrolysates and Potential of Glucosamine in Flavor Modulation.

Bovine (meat and heart) and porcine (hemoglobin and plasma) raw materials were hydrolyzed by Protease A (both endo- and exopeptidase activity), with or without glucosamine added during the enzyme inactivation step. Hydrolysates were characterized via peptide analysis (yield, UV- and fluorescence scanning spectroscopy, and peptide size distribution via size exclusion chromatography), sensory evaluation, and volatile compound analysis via gas chromatography mass-spectrometry (GC-MS) to determine if glucosamine-induced Maillard reaction improved taste and flavor. Porcine hemoglobin produced the most flavor-neutral hydrolysate, and could expectedly have the broadest application in food products. Both bovine meat and -heart hydrolysates were high in umami, and thereby good candidates for savory applications. Porcine plasma hydrolysate was high in liver flavor and would be suitable for addition to certain meat products where liver flavor is desirable. All hydrolysates had low perceived bitterness. Glucosamine-induced Maillard reaction had just a minor influence on the sensory profile via an increased perception of sweet taste (p = 0.038), umami taste (p = 0.042), and yolk flavor (p = 0.038) in the hydrolysates, irrespective of raw material. Glucosamine addition had a statistically significant effect on 13 of 69 volatiles detected in the hydrolysates, but the effect was minor and raw material-specific.

Effect of pH on the Antimicrobial Activity and Peptide Population of Pepsin Hydrolysates Derived from Bovine and Porcine Hemoglobins

Slaughterhouse blood is considered a valuable by-product of the meat industry that is mainly derived from the slaughter of cattle and pigs. Its cruor fraction is mainly constituted of hemoglobin, which has been widely described as an important source of several antimicrobial peptides that can be released by hydrolysis with pepsin. Despite the high sequence similarity between bovine (Hb-B) and porcine (Hb-P) hemoglobins, the release of antimicrobial peptides may be affected by varying hydrolysis conditions. This study presents, for the first time, the impact of pH (pH 2, 3, 4, and 5) on the antimicrobial peptide population of hydrolysates derived from the main hemoglobin sources by using pepsin (bovine and porcine). In the same way, their antibacterial, antifungal, and antiyeast activities are compared, as well as their type of mechanism of action (static or cidal effect). These characteristics have not been described previously in the literature. A total of 29 peptides from Hb-B and 35 peptides for Hb-P with potential antimicrobial peptides were identified by reversed-phase ultrahigh-performance liquid chromatography with tandem mass spectrometry (RP-UPLC–MS/MS) and bioinformatics tools. Similarities in the peptide population between Hb-B and Hb-P hydrolysates were observed by PCA, mainly in the enzymatic hydrolysis at pH 2 and pH 3. Both bovine and porcine hydrolysates produced at pH 2 and pH 3 showed antibacterial activity against L. ivanovii (MIC = 0.62–1.25 mg·mL–1) and antiyeast activity against R. mucilaginosa (0.31–1.25 mg·mL–1). Conversely, only antifungal activity against M. racemosus in the porcine hydrolysate at pH 3 was observed (1.25 mg·mL–1). This study confirms the similarities of Hb-B and Hb-P in terms of peptide profiles and antimicrobial activity, which indicates they are suitable sources of antimicrobial peptides with antibacterial, antifungal, and antiyeast activities that could be employed in the preservation of food products.

Globin Digest Improves Visceral Adiposity Through UCP1 Upregulation in Diet-Induced Obese Zebrafish and Mice.

Globin digest (GD), a bioactive oligopeptide derived from porcine hemoglobin proteins, has been demonstrated to have beneficial effects on improving postprandial hyperlipidemia, hyperglycemia, and liver injury. We previously reported the lipid-lowering effects of GD using a zebrafish obesogenic test. Here, we sought to evaluate the effect of GD on visceral adiposity and the underlying molecular mechanisms using zebrafish and mouse obesity models. GD ameliorated dyslipidemia and suppressed the accumulation of visceral adipose tissue (VAT) in adult obese zebrafish. Transcriptomic analysis by RNA sequencing of GD-treated adult zebrafish revealed that GD upregulated UCP1-related pathways. Further, we performed mouse experiments and found that GD intake (2 mg/g body weight/day) was associated with lowered plasma triglyceride and total cholesterol levels, decreased VAT accumulation, and improved adipocyte hypertrophy with the upregulation of Ucp1 expression in white adipose tissue at both the mRNA and protein levels. Taken together, these results indicate that GD improves visceral adiposity by upregulating UCP1 expression, providing a novel perspective on combating obesity.