The production of polyhydroxybutyrate by Methylobacterium rhodesianum and Ralstonia eutropha in media containing glycerol and casein hydrolysates

Methylobacterium rhodesianum and Ralstonia eutropha were cultivated to produce polyhydroxybutyrate (PHB) using media which contained glycerol and casein hydrolysates as C/N-substrates. In these media the pH had not to be regulated during the fermentations. The first strain accumulated an average of 39% PHB during 92 h of cultivation in flasks and 50% PHB during 45 h of cultivation in fermenters. The second one yielded an average of 47% PHB during 67 h of cultivation using casein peptone and 65% PHB during 45 h of cultivation using Casamino acids in the medium. Calculated N-balances showed that about 65% of the supplied nitrogen was used for growth of non-PHB cell dry mass. The conversion of glycerol to PHB was 17% (w/w).     

Effect of the peptide and vitamin composition of casein hydrolysates on the β-galactosidase activity of Kluyveromyces bulgaricus cells

Instead of yeast extracts, casein hydrolysates from several suppliers were added to culture media and analyzed with respect to their ability to stimulate β-galactosidase activity in Kluyveromyces bulgaricus cells. Four enzymatic casein hydrolysates caused a significantly higher stimulation of enzyme activity, while acid casein hydrolysates clearly reduced the enzyme activity. Enzymatic casein hydrolysates, inducing high and low lactase activity, were analyzed with respect to average peptide length (APL), vitamins (niacin, pathothenate) as well as free and bound amino acids. The molecular weights of these casein hydrolysates were estimated by gel filtration. No correlation was found between the degree of enzyme stimulation and the vitamin contents, the APL values and the free amino acid contents of the casein hydrolysates. Casein hydrolysates stimulating the lactase activity were less soluble in water and, in a gel filtration column, they showed three peaks with slightly lower molecular weights than the three peaks seen in hydrolysates which had no effect on activity. APL values of alcoholic precipitates of casein hydrolysates showed an inverse correlation to lactase activity. The molecular weights of alcoholic precipitates of lactase stimulating digests were also lower compared to non-stimulating ones. Alcoholic precipitates with lactase-stimulating activity were more hydrophilic, as was shown by a smaller proportion absorbed in a C18 column and by amino acid analysis. Our results sugest that alcoholic precipitates could probably be important in the lactase stimulation and their composition should be further investigated. The mechanism is nevertheless complex and may be caused by various simultaneous factors.     

Continuous production of high degree casein hydrolysates by immobilized proteases in column reactor

The enzymatic complete hydrolysis of casein was investigated by using immobilized endopeptidase and exopepti dase packed in the jacketed column reactors. The mass transfer efficiency of proteins was improved by using sliced shrimp chitin hull as enzyme support, which formed a network structure inside the column reactor that prevented the formation of protein precipitate and increased the line flow rate of protein solution. The specificity of the protease was of crucial importance for both the hydrolysis degree and the free amino acid content of the hydrolysates. Of the enzymes tested, the immobilized A. oryzae protease was the most effective enzyme in breaking down the casein molecules and releasing the free amino acid from casein hydrolysates. The immobilized pancreatic and kidney exopeptidase could lead to a 20% increase of free amino acids. The free amino acid content of casein hydrolysates was 34.81% after processing and could reach to 64% if the column length was doubled, but 100% hydrolysis was impossible as the reverse reaction was also taking place. The casein hydrolysates was characterized by its high degree of hydrolysis and high content of free amino acids. It can be applied in infant formula, element diet, and as a protein ingredient for food industry.     

Ethanol production from concentrated food waste hydrolysates with yeast cells immobilized on corn stalk

The aim of the present study was to examine ethanol production from concentrated food waste hydrolysates using whole cells of S. cerevisiae immobilized on corn stalks. In order to improve cell immobilization efficiency, biological modification of the carrier was carried out by cellulase hydrolysis. The results show that proper modification of the carrier with cellulase hydrolysis was suitable for cell immobilization. The mechanism proposed, cellulase hydrolysis, not only increased the immobilized cell concentration, but also disrupted the sleek surface to become rough and porous, which enhanced ethanol production. In batch fermentation with an initial reducing sugar concentration of 202.64 ± 1.86 g/l, an optimal ethanol concentration of 87.91 ± 1.98 g/l was obtained using a modified corn stalk-immobilized cell system. The ethanol concentration produced by the immobilized cells was 6.9% higher than that produced by the free cells. Ethanol production in the 14th cycle repeated batch fermentation demonstrated the enhanced stability of the immobilized yeast cells. Under continuous fermentation in an immobilized cell reactor, the maximum ethanol concentration of 84.85 g/l, and the highest ethanol yield of 0.43 g/g (of reducing sugar) were achieved at hydraulic retention time (HRT) of 3.10 h, whereas the maximum volumetric ethanol productivity of 43.54 g/l/h was observed at a HRT of 1.55 h.     

Adsorption and recovery of cellulases during hydrolysis of newspaper

The adsorption of cellulases from Trichoderma viride was studied during the hydrolysis of newspaper. By measuring individual enzyme activities it was found that in the early stage of hydrolysis enzyme components showing C_xA; were adsorbed preferentially to those showing C₁A; afterwards ths situation was inverted. Electrophoretic resolution of proteins in hydrolysates showed a continuous decrease of enzyme proteins in solution, and furthermore suggested that the enzymes once adsorbed remained immobilized on the substrate (even after extensive digestion). Experiments to recover the enzyme that had remained in solution after typical hydrolysis showed a potential saving of up to 40%.     

Optimization of Penicillium aurantiogriseum protease immobilization on magnetic nanoparticles for antioxidant peptides’ obtainment

This work reports an optimization of protease from Penicillium aurantiogriseum immobilization on polyaniline-coated magnetic nanoparticles for antioxidant peptides’ obtainment derived from bovine casein. Immobilization process was optimized using a full two-level factorial design (2⁴) followed by a response surface methodology. Using the derivative, casein was hydrolyzed uncovering its peptides that were sequenced and had antioxidant properties tested through (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) (ABTS) radical scavenging and hydrogen peroxide scavenging assays. Optimal conditions for immobilization were 2?hr of immobilization, offered protein amount of 200?µg/mL, immobilization pH of 6.3 and 7.3?hr of activation. Derivative keeps over 74% of its original activity after reused five times. Free and immobilized enzyme casein hydrolysates presented similar peptide mass fingerprints, and prevalent peptides could be sequenced. Hydrolysates presented more than 2.5× higher ROS scavenging activity than nonhydrolyzed casein, which validates the immobilized protease capacity to develop casein-derived natural ingredients with potential for functional foods.     

Production of Candida utilis protein from peat extracts

Sphagnum peat extracts or hydrolysates have been obtained and used as a culture medium for the production of Candida utilis biomass as single cell proteins. Acid hydrolysis of ground peat (4–60 mesh) in an autoclave operated under a set of conditions for acid strength (0.3-1.5 (v/v) H₂SO₄), holding time (1–4 hr), temperature (100–165°C), and weight ratio of dry peat to solution (3.3–16.7 g dry peat/100 g solution) yielded carbohydrate-rich extracts of different concentrations (1–34g/liter). The best yield (mg total carbohydrate/g dry peat) was obtained for a holding time of I hr and a temperature of 152°C. Low peat concentratio (4.1 g dry peat/100 g solution)resulted in high yield(280mg total carbohydrate/gdry peat) with a corresponding low carbohydrate content in hydrolysate (13 g/liter), while a lower yield with a higher carbohydrate content (34 g/liter)in hydrolysate were found when increasing peat concentration (16.7 g dry peat/100 g solution). Shake-fladk experiments using peat hydrolysates as the culture medium together with NH₄OH (～4.8 g/liter) and K₂HPO₄(5 g/liter) as nitrogen and phosphate supplement, respectively, gave a maximum biomass concentration of 7.5 g/liter after 60 hr at 30°C and 200rpm. Batch cultivation in a fermentor under controlled conditions for aeration (4.2 liter/min), agitation (500rpm), temperature (30°C), and pH (5.0) produced a maximum biomass of 10 g/liter after 20 hr with a specific growth rate of 0.13 hr^?1. For the continuous cultivation, a maximal biomass productivity of 1.24 g/gliter-he was obtained at a dilution rate of 0.125 hr ^?1. Monod's equation's equation has been used for the estimation of the coefficients μ_Max, K_s, and Y. It was found that the yield coefficient Y is not constant during the progress of batch cultivation.     

Peptides with antioxidant properties identified from casein,whey, and egg albumin hydrolysates generated by two novel fungal proteases

Abstract

There are many diseases linked to oxidative stress, including cancer. Importantly, endogenous antioxidants are insufficient to protect against this process. Peptides derived from food proteins produced by hydrolysis have been investigated as exogenous antioxidants. The present study aimed to identify novel peptides with antioxidant potential produced from egg and milk proteins hydrolysis with two new fungal proteases isolated from Eupenicillium javanicum and Myceliophthora thermophila. The degree of hydrolysis at several time points was calculated and correlated to DPPH scavenging and metal chelating assays, all hydrolysates presented antioxidant activity. Casein hydrolyzed by the M. thermophila protease showed the best antioxidant activity. The identified sequences showed that the proportions of amino acids that influence antioxidant activity support the antioxidant assay. Our data reveal the conditions necessary for the successful generation of antioxidant peptides using two novel fungal proteases. This opens a potential new avenue for the design and manufacture of antioxidant molecules.     

PVA-biocatalyst with entrapped viable Bacillus subtilis cells

Bacillus subtilis cells were entrapped in polyvinyl alcohol (PVA)-cryogel beads without decay in their viability and capability of secretion of proteolytic enzymes (metalloproteinase and subtilisin). Conditions for preparation of the PVA-biocatalyst with suitable stability and viability of B. subtilis cells were optimized. Diffusion of various compounds into the cryogel (sliced beads) has been monitored on-line using image analysis system. Optimal working conditions and kinetic constants for hydrolysis of proteins catalyzed by the PVA-biocatalyst containing whole B. subtilis cells were estimated. The PVA-biocatalyst was applied in the hydrolysis of casein. The productivity of the biocatalyst (expressed as an amount of liberated aromatic amino acids) reached a maximal level of 12 mg g⁻¹ h⁻¹. Composition of mixture of peptides was dependent on pH, concentrations of Na⁺ and glucose, and in the reaction milieu. Protein hydrolysates of desired composition can be obtained using B. subtilis viable cells immobilized in PVA-gel. Incubation of the immobilized cells in a nutrient medium with casein successfully regenerated proteolytic activity of the biocatalyst.     

Cellulose hydrolysis by immobilized <Emphasis Type="Italic">Trichoderma reesei</Emphasis> cellulase

Cellulose hydrolysis by immobilized Trichoderma reesei cellulase in the presence of a low viscosity ionic liquid, 1-ethyl-3-methylimidazolium diethyl phosphate (EMIM-DEP), was investigated. Preparation of the carrier-free immobilized cellulase was optimized with respect to concentration of the cross-linker and the type of precipitant. The addition of 2% (v/v) EMIM-DEP during hydrolysis gave an initial reaction rate 2.7 times higher than the hydrolysis rate with no ionic liquid. The initial yield after 2 h was 0.7 g glucose/g cellulose, and the carrier-free immobilized cellulase (CFIC) was effectively re-used five times.     

Control of the degree of hydrolysis of a protein modification with immobilized protease by the pH-drop method

A mathematical model, DH = 1/k(10^?2ΔpH ? 1), between the pH-drop (ΔpH) and degree of hydrolysis (DH) of an enzymatic modification of casein was developed to assess the DH in a packed-bed column reactor by directly monitoring the pH value of the modified protein system. It was demonstrated that the linear DH range and the k value of the equation were dependent on the reactor type and the specificity of the proteolytic enzymes immobilized on chitin used in the present study, but no effect of the substrate casein concentration on the linear DH range was observed. Since DH and ΔpH values of the modified casein correlated with the flow rate in a packed-bed column reactor, it was suggested that the DH value, in a considerably wide range of casein modification with a certain immobilized protease in a column reactor, could be controlled by adjusting the flow rate of the substrate and monitored by a pH-meter. This relationship might be used as a basis for scale-up and long-term operation of enzymatic modification of proteins by immobilized protease in a column reactor.     

Hydrolysis of Lipid-Extracted Chlorella vulgaris by Simultaneous Use of Solid and Liquid Acids

Microalgal biomass was hydrolyzed using a solid acid catalyst with the aid of liquid acid. The use of solid acid as the main catalyst instead of liquid acid was to omit subsequent neutralization and/or desalination steps, which are commonly required in using the resulting hydrolysates for microbial fermentation. The hydrolysis of 10 g/L of lipid-extracted Chlorella vulgaris containing 12.2% carbohydrates using 7.6 g/L Amberlyst 36 and 0.0075 N nitric acid at 150°C resulted in 1.08 g/L of mono-sugars with a yield of 88.5%. For hydrolysis of higher concentrations of the biomass over 10 g/L, the amount of Amberlyst 36 needed to be increased in proportion to the biomass concentration to maintain similar levels of hydrolysis performance. Increasing the solid acid concentration protected the surface of the solid acid from being severely covered by cell debris during the reaction. A hydrolysate of lipid-extracted C. vulgaris 50 g/L was used, with no post-treatment of desalination, for the cultivation of Klebsiella oxytoca producing 2,3-butanediol. Cell growth in the hydrolysate was found to be almost the same as in the conventional medium with the same monosaccharide composition, confirming its fermentation compatibility. It was noticeable that the yield of 2,3-butanediol with the hydrolysate was observed to be 2.6 times higher than that with the conventional medium. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2729, 2019     

Production of lactic acid and ethanol by Rhizopus oryzae integrated with cassava pulp hydrolysis

Cassava pulp was hydrolyzed with acids or enzymes. A high glucose concentration (>100 g/L) was obtained from the hydrolysis with 1 N HCl at 121 °C, 15 min or with cellulase and amylases. While a high glucose yield (>0.85 g/g dry pulp) was obtained from the hydrolysis with HCl, enzymatic hydrolysis yielded only 0.4 g glucose/g dry pulp. These hydrolysates were used as the carbon source in fermentation by Rhizopus oryzae NRRL395. R. oryzae could not grow in media containing the hydrolysates treated with 1.5 N H₂SO₄ or 2 N H₃PO₄, but no significant growth inhibition was found with the hydrolysates from HCl (1 N) and enzyme treatments. Higher ethanol yield and productivity were observed from fermentation with the hydrolysates when compared with those from fermentation with glucose in which lactic acid was the main product. This was because the extra organic nitrogen in the hydrolysates promoted cell growth and ethanol production.     

Fed-batch cultivation for the production of clavulanic acid by an immobilized Streptomyces clavuligerus mutant

In order to obtain high productivity of clavulanic acid, a newly-introduced carrier, polyurethane pellet (PUP) Z97-020 was used for the immobilization process. In a stirred-tank bioreactor, batch cultivation by Streptomyces clavuligerus KK immobilized on PUP Z97-020 gave about 3100 mg of clavulanic acid per litre, representing an increase of 200% in productivity compared with that by fed-batch cultivation of free cells (1500 mg/l). However, the clavulanic acid produced rapidly decomposed due to the pH change during batch cultivation. Fed-batch cultivation by immobilized S. clavuligerus KK gave an excellent level of clavulanic acid up to 3250 mg/l, a productivity increase of 220% compared with that by fed-batch cultivation of free cells. These results suggest that immobilization with PUP Z97-020 is a more effective process for the production of clavulanic acid and that the maintenance of pH by fed-batch cultivation with glycerol as a limiting substrate prevents the clavulanic acid from decomposing during the fermentation.     

Extracellular acid phosphatase as a minor enzyme during the production of proteinases by Humicola lutea 120–5

Extracellular acid phosphatase was studied as a minor enzyme of the fungal strain Humicola lutea 120–5 having a clear relation to the secretion of acid proteinases. A medium lacking in mineral orthophosphates ensured a fivefold higher yield of phosphatase while the proteinase production was reduced. An acid phosphatase fraction free of proteinase activity was isolated demonstrating a maximum hydrolysis of 4-nitrophenyl-phosphate at a pH of 4.0 and 50°C. The phosphatase catalyzed a partial dephosphorylation of up to 30% of casein at a pH of 3.0 causing a complete substrate precipitation. Both proteinase and phosphatase biosynthesis increased twofold when natural casein was replaced by partially dephosphorylated casein in the cultivation medium.     

Hydrolysis of whey by whole cells of Kluyveromyces bulgaricus immobilized in calcium alginate gels and in hen egg white

Summary Whey hydrolysis was compared in column reactors containing whole yeast cells immobilized in Ca-alginate or in hen egg white in relation to cell -galactosidase activity, flow rates, temperature and time. With cells of 1.3 U/mg dry weight (ONPG method) immobilized in Ca-alginate, 80% hydrolysis was obtained at 4° and 20° C with, respectively 0.50 and 1.65 bed volume/H; the values were 0.2 and 0.74 with cells entrapped in hen egg white. When the flow rate was expressed as ml/H/g wet yeast, no significant difference was observed between both matrices and 80% hydrolysis was reached with a flow rate 1.7 and 5 according to the temperature. The best performance was achieved by the yeast egg white reactor. At 4°C, hydrolysis decreased by 10% after 13 days; by 20% after 17 days. The presence of lactose transport inhibitors in whey did not significantly influence lactose hydrolysis.M. Decleire et al.: Hydrolysis of whey by immobilized whole cells of Kluyveromyces bulgaricus     

Hydrolysis of Chlorella biomass for fermentable sugars in the presence of HCl and MgCl2

When Chlorella biomass was hydrolyzed in the presence of 2% HCl and 2.5% MgCl₂, a sugar concentration of nearly 12%, and a sugar recovery of about 83% was obtained. Fermentation experiments demonstrated that glucose in the Chlorella biomass hydrolysates was converted into ethanol by Saccharomyces cerevisiae with a yield of 0.47 g g⁻¹, which is 91% of the theoretical yield. This chemical hydrolysis approach is thus a novel route for the hydrolysis of biomass to generate fermentable sugars.     

Effect of external mass transfer of activation energy of hydrolysis of BAEE and casein by trypsin immobilized on molecular sieve type 4A

In this investigation, the activation energies of the hydrolysis of N-(α)-benzol-L -arginine ethyl ester (BAEE) and casein have been determined using trypsin immobilized on molecular sieve type 4A. There is a complete absence of intraparticle diffusion in the system, and the temperature dependence of the reaction has been studied only under external diffusional limitation. While the hydrolysis of BAEE by bound trypsin in found to be controlled by external diffusion, that of casein is kinetically controlled.     

Functional,bioactive and antioxidative properties of hydrolysates obtained from cod (Gadus morhua) backbones

Fish protein hydrolysates (FPH) have good and well documented functional properties. Peptides obtained from various fish protein hydrolysates have also shown bioactive and antioxidative activities.The aim of this study was to evaluate how storage and preparation of cod (Gadus morhua) backbones influence the yield, functionality, bioactivity (CGRP and gastrin/CCK related molecules) and antioxidative properties of fish protein hydrolysates. A series of hydrolysis trials have been carried out using backbones from cod that were initially fresh or frozen and further hydrolysed for different times (10, 25, 45 and 60 min). Use of fresh raw material significantly increased yield of dry FPH, gave lighter and less yellow powders with better emulsification properties. Longer time of hydrolysis gave higher FPH yield, increased degree of hydrolysis and decreased water holding capacity of the powders. Among the hydrolysis times tested, 25 and 45 min hydrolysis demonstrated the best emulsification properties.FPH have potential to enhance product stability by preventing oxidative deterioration. The DPPH scavenging activity showed that antioxidative activity of hydrolysates could be due to the ability to scavenge lipid radicals. The ability of hydrolysates to inhibit iron induced lipid oxidation was not influenced by time of hydrolysis.This work also shows that it is possible to obtain bioactive molecules from cod backbones by protein hydrolysis. The content of bioactive peptides (gastrin/CCK- and CGRP-like peptides) could make the cod hydrolysates useful for incorporation in functional foods.     

Kinetic and thermodynamic properties of purified alkaline protease from Bacillus pumilus Y7 and non‐covalent immobilization to poly(vinylimidazole)/clay hydrogel

The characterization of the hydrogel was performed using Fourier‐transform infrared spectroscopy, X‐ray diffraction, and scanning electron microscopy. Purified Bacillus pumilus Y7‐derived alkaline protease was immobilized in Poly (vinylimidazole)/clay (PVI/SEP) hydrogel with 95% yield of immobilization. Immobilization decreased the pH optimum from 9 to 6 for free and immobilized enzyme, respectively. Temperature optimum 3°C decreased for immobilized enzyme. The K_m, V_m, and k_cat of immobilized enzyme were 4.4, 1.7, and 7.5‐fold increased over its free counterpart. Immobilized protease retained about 65% residual activity for 16^th reuse. The immobilized protease endured its 35% residual activity in the material after six cycle's batch applications. The results of thermodynamic analysis for casein hydrolysis showed that the ΔG^≠ (activation free energy) and ΔG^≠_E‐T (activation free energy of transition state formation) obtained for the immobilized enzyme decreased in comparison to those obtained for the free enzyme. On the other hand, the value of ΔG^≠_ES (free energy of substrate binding) was observed to have increased. These results indicate an increase in the spontaneity of the biochemical reaction post immobilization. Enthalpy value of immobilized enzyme that was 2.2‐fold increased over the free enzyme indicated lower energy for the formation of the transition state, and increased ΔS^≠ value implied that the immobilized form of the enzyme was more ordered than its free form.