Submerged monoxenic culture medium development for Heterorhabditis bacteriophora and its symbiotic bacterium Photorhabdus luminescens: protein sources

Most medium formulations for improving culture of entomopathogenic nematodes (EPN) based on protein sources have used enriched media like animal feed such as dried egg yolk, lactalbumin, and liver extract, among other ingredients. Most results, however, showed unstable yields and longer production time. Many of the results do not show the detailed parameters of fermentation. Soy flour, cotton seed flour, corn gluten meal, casein powder, soytone, peptone, casein hydrolysates, and lactalbumin hydrolysate as protein sources were tested to determine the source to support optimal symbiotic bacteria and nematode growth. The protein hydrolysates selected did not improve bacterial cell mass compared with the yeast extract control, but soy flour was the best, showing 75.1% recovery and producing more bacterial cell number (1.4×10?/ml) than all other sources. The highest yield (1.85×10? IJs/ml), yield coefficient (1.67×10? IJs/g medium), and productivity (1.32×10? IJs/l/day) were also achieved at enriched medium with soybean protein.     

Scalable purification and characterization of the anticancer lunasin peptide from soybean

Lunasin is a peptide derived from the soybean 2S albumin seed protein that has both anticancer and anti-inflammatory activities. Large-scale animal studies and human clinical trials to determine the efficacy of lunasin in vivo have been hampered by the cost of synthetic lunasin and the lack of a method for obtaining gram quantities of highly purified lunasin from plant sources. The goal of this study was to develop a large-scale method to generate highly purified lunasin from defatted soy flour. A scalable method was developed that utilizes the sequential application of anion-exchange chromatography, ultrafiltration, and reversed-phase chromatography. This method generates lunasin preparations of >99% purity with a yield of 442 mg/kg defatted soy flour. Mass spectrometry of the purified lunasin revealed that the peptide is 44 amino acids in length and represents the original published sequence of lunasin with an additional C-terminal asparagine residue. Histone-binding assays demonstrated that the biological activity of the purified lunasin was similar to that of synthetic lunasin. This study provides a robust method for purifying commercial-scale quantities of biologically-active lunasin and clearly identifies the predominant form of lunasin in soy flour. This method will greatly facilitate the development of lunasin as a potential nutraceutical or therapeutic anticancer agent.     

Peptides and hydrolysates from casein and soy protein modulate the release of vasoactive substances from human aortic endothelial cells

Food proteins were shown to affect atherogenic risk factors, which is supposed to be related to specific peptide sequences encrypted within their primary sequence. The aim of this study was to evaluate the effects of peptides and hydrolysates from two food proteins, casein and soy protein, on endothelial cell functions (cell proliferation and release of vasoactive substances). Cell proliferation was not influenced by dipeptides and most of the tripeptides, whereas several total hydrolysates from casein and soy protein inhibited cell proliferation at higher concentrations (>0.25 mg/mL; P<0.05). The release of one or more of the vasoactive substances, thromboxan B2 (stable marker of thromboxan A2), 6-keto-prostaglandin F1alpha (stable marker of prostaglandin I2), endothelin-1, and nitric oxide, was significantly influenced by the incubation with various peptides compared with control cells (P<0.05). Various hydrolysate fractions from casein and soy protein influenced the release of 6-keto-prostaglandin F1alpha and nitric oxide (P<0.05) but did not influence the release of thromboxan B2 and endothelin-1. In conclusion, the present study demonstrates that peptides and hydrolysate fractions from casein and soy protein influence endothelial cell function as evidenced by the modulation of endothelial cell proliferation and alterations in the release of vasoactive substances.     

Usability of size-excluded fractions of soy protein hydrolysates for growth and viability of Chinese hamster ovary cells in protein-free suspension culture

To investigate the effect of size-excluded fraction of non-animal protein hydrolysate on growth, viability and longevity of Chinese hamster ovary (CHO) cells, several commercially available protein hydrolysates were evaluated as a feed supplement to chemically-defined protein-free suspension culture. Soy protein hydrolysates showed better supporting capability for cell growth and viability than the other types of hydrolysates. Maximal cell growth was not affected greatly by size exclusion of some soy hydrolysates such as bacto soytone and soy hydrolysates. CHO cells supplemented with size-excluded fractions of the two hydrolysates showed viable cell density and viability almost equal to those with their crude hydrolysates, although soy hydrolysates showed a little better performance. This suggested that the size-excluded hydrolysate fractions of some soy hydrolysate might be a potential culture medium additive to achieve better downstream operation in a large-scale production as well as enhanced productivity.     

Opposite Contributions of Glycinin- and β-Conglycinin-Derived Peptides to the Aggregation Behavior of Soy Protein Isolate Hydrolysates

The aggregation behavior as a function of pH was studied for hydrolysates obtained by hydrolysis of soy protein isolate (SPI) and glycinin- and β-conglycinin-rich protein fractions with subtilisin Carlsberg. The substrates were hydrolyzed up to degrees of hydrolysis (DH) of 2.2% and 6.5%. Compared with nonhydrolyzed SPI, a decrease in solubility was observed for the hydrolysates of SPI [0.8% (w/v) protein, I = 0.03 M] around neutral pH. At pH 8.0, glycinin hydrolysates had a much lower solubility (∼43% and 60%, respectively, for DH 2.2% and 6.5%) than SPI and β-conglycinin-derived hydrolysates, which were almost completely soluble. Peptides that aggregated were all larger than 5 kDa, and as estimated by size-exclusion chromatography their composition was almost independent of the aggregation pH. The solubility of hydrolysates of SPIs with a varying glycinin and β-conglycinin composition showed that glycinin-derived peptides are the driving force for the lower solubility of SPI hydrolysates. The solubility of SPI hydrolysates at pH 8.0 was shown not to be the sum of that of glycinin and β-conglycinin hydrolysates. Assuming that the separate hydrolysis of glycinin and β-conglycinin did not differ from that in the mixture (SPI), this indicates that β-conglycinin-derived peptides have the ability to inhibit glycinin-derived peptide aggregation.     

Use of soybean protein hydrolysates for promoting proliferation of human keratinocytes in serum-free medium

Human keratinocytes are generally cultured in media containing bovine pituitary extract (BPE), an animal product that can be a source of infectious contaminants. We investigated whether a safer plant product could replace BPE in the culture medium. Medium containing both BPE and soy protein hydrolysates (Bacto Soytone and Soy Hydrolysate) produced the largest number of viable cells, followed in descending order by medium supplemented only with BPE, only with the hydrolysates, and without supplementation (basal medium only). Soybean protein is thus an excellent source of nutrients for the growth of adherent keratinocytes, although they do not fully substitute for BPE.     

The bioactivity and fractionation of peptide hydrolysates in cultures of CHO cells

Peptide hydrolysate supplements in mammalian cell cultures provide enhanced growth and productivity. The objective of this study was to compare the bioactivity of ten different commercially available hydrolysates from plant, microbial, and animal sources. The peptide hydrolysates were tested as supplements to cultures of Chinese hamster ovary (CHO) cells that produce human beta interferon (β‐IFN). A soy hydrolysate was shown to support high cell growth but not protein productivity compared to an animal component hydrolysate (Primatone RL). On the other hand, a yeast hydrolysate showed lower cell growth, but comparable productivity of the recombinant protein. Glycosylation analysis showed that the glycan profile of β‐IFN produced in yeast hydrolysate supplemented cultures was equivalent to that from Primatone RL‐supplemented cultures. Fractionation of the yeast hydrolysate and Primatone RL produced a similar protein‐assayed pattern except for one extra peak at around 1 kDa in the Primatone RL profile. A fraction taken at a molecular weight range of 1.5–1.7 kDa showed the highest growth promoting activity in both samples. However, four other fractions in yeast hydrolysate and two in Primatone RL at lower molecular weights showed some growth promoting activity. In conclusion, the yeast hydrolysates provided a good alternative to the animal sourced Primatone RL for high productivity of β‐IFN from CHO cells with equivalent glycosylation. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:584–593, 2014     

Peptide and amino acid composition of different protein bases for culture media

The enzymatic hydrolysates under study, obtained from different raw materials, have been shown to contain a great variety of peptides with different molecular weight. The highest content of fractions with a molecular weight of 2000 D has been observed in enzymatic meat and casein hydrolysates manufactures in the GDR. Low-molecular fractions (100-200 D) prevail in amino peptide. A great variety of peptides with different molecular weight is observed in Hottinger's meat hydrolysate and in blood clot hydrolysate obtained from the blood of laboratory animals. All peptide fractions have been shown to contain a wide spectrum of free amino acids. These data on the peptide and amino acid composition of different protein bases facilitate their rational use of microbiological culture media.     

The high expression of Aspergillus pseudoglaucus protease in Escherichia coli for hydrolysis of soy protein and milk protein

Abstract

The hydrolysates of soy protein and milk protein are nutritional and functional food ingredients. Aspergillus pseudoglaucus aspergillopepsin I (App) is an acidic protease, including signal peptide, propeptide, and catalytic domain. Here, we cloned the catalytic domain App with or without propeptide in Escherichia coli. The results showed that the App without propeptide was not expressed or did not exhibit activity and App with propeptide (proApp) was highly expressed with a specific activity of 903?U/mg. Moreover, the denaturation temperature of proApp was 4.1?°C higher than App’s. The proApp showed 104?U/mg and 252?U/mg hydrolysis activities towards soy protein and milk protein under acidic conditions. By RP-HPLC analysis, the peptides obtained from the hydrolysates of soy protein and milk protein were hydrophilic peptides. This work first demonstrates efficient proteolysis of soy protein and milk protein through the functional expression of full-length proApp, which will likely have valuable industrial applications.     

Trypsin Hydrolysed Protein Fractions as Radical Scavengers and Anti-bacterial Agents from <Emphasis Type="Italic">Ficus deltoidea</Emphasis>

Different molecular sizes of protein hydrolysates were prepared from the crude protein extract of Ficus deltoidea using the technique of membrane ultrafiltration after trypsin hydrolysis. Gel electrophoretic images shows the presence of 12, 8, 7 and 7 protein bands for the protein fractions prepared from the molecular weight cut-off of 3, 10, 30 and 100 kDa, respectively. The protein hydrolysates were found to have higher radical scavenging activity than those unhydrolysed fractions at the similar molecular size. They exhibited significant differences in the radical scavenging activities based on one-way analysis of variance, except for the protein hydrolysates of 30 and 100 kDa. The smallest protein hydrolysates, 3 kDa appeared to have the comparable activity (30%) with bovine serum albumin as a positive control in this study. Similarly, the 3 kDa protein hydrolysates achieved the highest inhibitory activity (87.5%) against Pseudomonas aeruginosa at the concentration of 128 µg/mL. The protein hydrolysates were found to be more effective against gram negative bacteria (P. aeruginosa and Escherichia coli) because of lower minimum inhibitory concentration (MIC) and effective inhibitory concentration at 50% (EC₅₀) than gram positive bacterium (Staphylococcus aureus). Trypsin catalysed hydrolysis seemed to improve the anti-bacterial activity of protein hydrolysates in a bacterial strain dependent manner. The MIC could achieve 1–55 µg/mL at different molecular sizes of protein fractions. Mass spectra matching revealed that 26% of 226 identified proteins belonged to the category of plant defensive proteins in stress management and metal handling.     

Isolation and Characterization of Neutral Peptides in Soy Sauce

A soy sauce sample was fractionated by gel filtration on a Sephadex G–15 column, then the fractions were subfractionated on the basis of acidity by ion exchange chromatography on a QAE-Sephadex A–25 column. After preliminary fractionation, the components in the neutral subfractions were transformed to copper salts, and the salts were chromatographed on a DEAE-Sephadex A–25 column with a borate buffer and aqueous acetic acid to separate neutral peptide substances from a large amount of free amino acids. The peptide fractions were further fractionated on a preparative amino acid analyzer and by paper chromatography to separate the peptide substances.

Three glycodipeptides and eight dipeptides were isolated and characterized as the major neutral peptide components in soy sauce. However, it was difficult to anticipate any direct contribution of these peptides to the flavor construction in soy sauce on the basis of their taste intensities and contents.     

Physicochemical Properties and Specific Structural Features of Protein–Lipid Composites of Increased Nutritive Value

Fractional and component compositions of protein–lipid composites with increased nutritive value (compared to the protein preparations from which they were produced) were studied based on solubility and electrophoretic behavior. Differences in the fractional compositions of proteins and the amounts of hydrogen, ionic, and hydrophobic bonds were found. It was demonstrated that the water-, salt-, and alkali-soluble fractions of proteins changed during the manufacturing of the composites with soybean and wheat bran flour; the water- and alkali-soluble fractions, with protein concentrate from bran. Heterogeneity of the compositions and specific conformational features of composite proteins resulting from disulfide bonds were found. It was demonstrated that, during the manufacturing of composites, proteins of soybean flour aggregated (with the involvement of disulfide bonds), whereas protein products from wheat bran disaggregated. Breaks of interchain (wheat) or intrachain (concentrate) disulphide bonds accompanied the disaggregation. Overall the properties and specific structural features of the protein–lipid composites studied depended on the nature of the protein (soybean or wheat), type of initial preparations (flour or concentrate), and method of their production (emulsifying or drying).     

Ultrafiltration to fractionate wheat polypeptides

An ultrafiltration process allowing the fractionation of two kinds of polypeptides issued from limited chymotryptic hydrolysis of wheat gliadins was applied to wheat gluten hydrolysates. Hydrophilic and poorly charged polypeptides were well transmitted through an inorganic ZrO₂-based membrane at acidic pH, whereas hydrophobic and positively charged polypeptides were highly retained. By combining reversed-phase and cation-exchange chromatography (CEC), it was proved that the fractionation of the polypeptides was based on electrostatic repulsion of the charged polypeptides by the positively charged membrane. After a continuous diafiltration process, retentates containing 75 to 88% of hydrophobic polypeptide and permeates containing 84 to 90% of hydrophilic polypeptides were recovered, depending on the size of membrane used. Even if the ultrafiltration fractions were less purified than fractions issued from CEC, it was shown that they exhibited very different foaming properties: permeate did not produce nor stabilize foams, whereas retentate was more efficient than the whole hydrolysates and BSA.     

Physicochemical properties and specific structural features of protein-lipid composites of increased nutritive value

Fractional and component compositions of protein-lipid composites with increased nutritive value (compared to the protein preparations from which they were produced) were studied based on solubility and electrophoretic behavior. Differences in the fractional compositions of proteins and the amounts of hydrogen, ionic, and hydrophobic bonds were found. It was demonstrated that the water-, salt-, and alkali-soluble fractions of proteins changed during the manufacturing of the composites with soybean and wheat bran flour; the water- and alkali-soluble fractions, with protein concentrate from bran. Heterogeneity of the compositions and specific conformational features of composite proteins resulting from disulfide bonds were found. It was demonstrated that, during the manufacturing of composites, proteins of soybean flour aggregated (with the involvement of disulfide bonds), whereas protein products from wheat bran disaggregated. Breaks of interchain (wheat) or intrachain (concentrate) disulphide bonds accompanied the disaggregation. Overall the properties and specific structural features of the protein-lipid composites studied depended on the nature of the protein (soybean or wheat), type of initial preparations (flour or concentrate), and method of their production (emulsifying or drying).     

Purification and identification of novel angiotensin-I converting enzyme (ACE) inhibitory peptides from cultured marine microalgae (Nannochloropsis oculata) protein hydrolysate

Isolation of bioactive compounds and commercialization of marine microalgae sources are interesting targets in future marine biotechnology. Cultured biomass of the marine microalga, Nannochloropsis oculata, was used to purify angiotensin-I converting enzyme (ACE) inhibitory peptides using proteases including pepsin, trypsin, α-chymotrypsin, papain, alcalase, and neutrase. The pepsin hydrolysate exhibited the highest ACE inhibitory activity, compared to the other hydrolysates and then was separated into three fractions (F1, F2, and F3) using Sephadex G-25 gel filtration column chromatography. First fraction (F1) showed the highest ACE inhibitory activity and it was further purified into two fractions (F1-1 and F1-2) using reverse-phase high-performance liquid chromatography. The IC₅₀ value of purified ACE inhibitory peptides were 123 and 173 μM and identified as novel peptides, Gly-Met-Asn-Asn-Leu-Thr-Pro (GMNNLTP; MW, 728 Da) and Leu-Glu-Gln (LEQ; MW, 369 Da), respectively. In addition, nitric oxide production level (%) was significantly increased by the purified peptide (Gly-Met-Asn-Asn-Leu-Thr-Pro) compared to the purified peptide (Leu-Glu-Gln) and other treated pepsin hydrolysate fractions on human umbilical vein endothelial cells (HUVECs). Cell viability assay showed no cytotoxicity on HUVECs with the treated purified peptides and fractions. These results suggest that the isolated peptides from cultured marine microalga, N. oculata protein sources may have potentiality to use commercially as ACE inhibitory agents in functional food industry.     

α-amylase inhibitors in Triticum aestivum: Purification and physical-chemical properties

Two α-amylase inhibitors in aqueous extracts of wheat flour have been resolved by DEAE-Sephadex chromatography. α-Amylase inhibitor I, the major inhibitor, was homogeneous by disc gel electrophoresis. It had a MW of 20 000 daltons and an isoelectric point of 6·7. α-Amylase inhibitor II had two minor contaminants when analysed by electrophoresis. These inhibitors were classified as typical wheat albumin proteins. A third α-amylase inhibitor was discovered when it was observed that an albumin protein which is found only in Triticum aestivum varieties of wheat could also inhibit pancreatic α-amylase. All three of these inhibitors could be distinguished by their characteristic electrophoretic mobilities.     

Purification and identification of antioxidant peptides from walnut (Juglans regia L.) protein hydrolysates

Walnut proteins were hydrolyzed separately using three different proteases to obtain antioxidant peptides. The antioxidant activities of the hydrolysates were measured using 1,1-diphenyl-2-picryl hydrazyl (DPPH) assay. Among hydrolysates, pepsin hydrolysate obtained by 3 h exhibited the highest antioxidant activities, which could also quench the hydroxyl radical, chelate ferrous ion, exhibit reducing power and inhibit the lipid peroxidation. Then, 3-h pepsin hydrolysates were purified sequentially by ultrafiltration, gel filtration and RP-HPLC. The sequence of the peptide with the highest antioxidative activity was identified to be Ala-Asp-Ala-Phe (423.23 Da) using RP-HPLC-ESI-MS, which was identified for the first time from walnut protein hydrolysates. Last, the inhibition of the peptide on lipid peroxidation was similar with that of reduced glutathione (GSH). These results indicate that the protein hydrolysates and/or its isolated peptides may be effectively used as food additives.     

Sequence Determination of an Antioxidant Peptide Obtained by Enzymatic Hydrolysis of Oyster <Emphasis Type="Italic">Crassostrea madrasensis</Emphasis> (Preston)

Soft tissue from cultured farm fresh oysters (Crassostrea madrasensis) was subjected to two standard enzymatic peptide extraction procedures using pepsin and papain. The crude extracts obtained were partially purified by column chromatography and were freeze-dried. The hydrolysates obtained were compared with respect to their degree of hydrolysis (DH), antioxidant potential (AP) and total phenolic content (TPC). The hydrolysate showing better antioxidant property was further subjected to purification by high performance liquid chromatography and characterized by LC-MS/MS. Papain-digested oyster protein (OPHpap) hydrolysate showed higher DH, AP and TPC. OPHpap was further subjected to ultrafiltration and fractionated into 3 sizes namely, above 10, 3–10 and 1–3 kDa according to the molecular size. Antioxidant capacity of <3 kDa fraction OPHpap-3 evaluated by DPPH free radical scavenging assay, metal chelating activity, linoleic acid autoxidation assay showed maximum effectiveness. Of the seven fractions collected by purification of OPH-pap-3 on semi-preparative RP-HPLC, fraction 7 that showed the highest antioxidant activity was further characterized by LC-ESI-MS/MS and its sequence determined. An antioxidant peptide molecule with thirteen amino acids was identified in oyster protein hydrolysate obtained by papain digestion that may find application as a nutraceutical or may be utilized in food industry for prevention of rancidity in foods.     

Enzymatic preparation of immunomodulating hydrolysates from soy proteins

Soy protein hydrolysates with lower molecular weight were enzymatically prepared by several commercially available proteases (Alcalase 2.4L, Flavourzyme, Trypsin, Papain, Protease A and Peptidase R) with protein recovery varied from 42.59% to 79.87%. Relative content of positively charged peptides was determined on SP Sephadex C-25 using gradient sodium chloride solution as eluents. Immunomodulating properties were evaluated by measuring their effect on in vitro proliferation of murine spleen lymphocytes and phagocytic activity of peritoneal macrophages. The results showed that soy protein hydrolysates (SPHs) prepared with Alcalase and insoluble soy protein (InSP), preferable to other enzymes and soy proteins, have the highest immunomodulating activity and the optimum conditions were determined as follows: E/S=2% (Alcalase), 60 degrees C, pH 8.0, InSP concentration 6% and 225min. Positive correlations were obtained between the immunomodulating activity and content of positively charged peptides. The results suggested that lower molecular weight and positively charged peptides released from soy protein were effective in stimulating immunomodulating activity, thus provided insights into the preparation of potent immunomodulating products.     

Physicochemical properties of proteins from wheat grown under high-contrast climatic conditions

Studies using electrophoresis, gel chromatography, viscometry, and calorimetry revealed an interrelation of several physicochemical properties of proteins of soft wheat grown under conditions of cool and wet weather with rheological characteristics of gluten and dough and bread quality. The ratio of gliadin and albumin-globulin polypeptides in flour with short-tearing gluten was much lower compared to that in flour with normal gluten. Proteins from flour with short-tearing gluten, including the water-soluble and salt-soluble fraction, had a loose spatial structure. Gluten fractions of this gluten (gliadin and glutenin) were characterized by a more compact and elongated structure compared to normal gluten. As distinct from normal gluten, the conformation of protein particles in short-tearing gluten depended little on hydrophobic interactions. The results suggest that the main components of grain determine the rheological properties of short-tearing gluten.