Debittering effect of Actinomucor elegans peptidases on soybean protein hydrolysates

Effects of the enzymes in Actinomucor elegans extract and the enzyme Alcalase 2.4L on debittering the soybean protein hydrolysates were investigated. When the protein was treated only with the latter, a strong bitterness formed; but it decreased if the protein was treated with both the enzymes. The more the enzymes were used, weaker was the bitterness tasted. SDS-PAGE profile and ESI-MS spectrum of the hydrolysates evidenced that the Alcalase could convert the protein into peptides rapidly, while the enzymes in the A. elegans extract were able to further degrade some peptides which were difficult or unable to be hydrolyzed by the Alcalase. Further systematic analysis of the peptidases showed that the Alcalase exhibited a significant endopeptidase activity towards NBZ-Phe-pNA substrate (p < 0.01), whereas many exopeptidases in the A. elegans extract had the carboxypeptidase activity towards N-CBZ-Ile-Leu (p < 0.01). It is concluded that those exopeptidases presented in the A. elegans extract can benefit by decreasing the bitterness of the soybean protein hydroysate. They are also capable of being used with the Alcalase in a single-step enzymatic reaction to prepare the bitterless protein hydrolysate, which may be an efficient application for food industry.     

Debittering of protein hydrolyzates

Enzymatic hydrolysis of proteins frequently results in bitter taste, which is due to the formation of low molecular weight peptides composed of mainly hydrophobic amino acids. Methods for debittering of protein hydrolyzates include selective separation such as treatment with activated carbon, extraction with alcohol, isoelectric precipitation, chromatography on silica gel, hydrophobic interaction chromatography, and masking of bitter taste. Bio-based methods include further hydrolysis of bitter peptides with enzymes such as aminopeptidase, alkaline/neutral protease and carboxypeptidase, condensation reactions of bitter peptides using protease, and use of Lactobacillus as a debittering starter adjunct. The causes for the production of bitter peptides in various food protein hydrolyzates and the development of methods for the prevention, reduction, and elimination of bitterness as well as masking of bitter taste in enzymatic protein hydrolyzates are presented.     

Preparation of hydrolysates from tobacco stalks and ethanolic fermentation by Saccharomyces cerevisiae

Chipped tobacco stalks were subjected to steam pretreatment at 205 °C for either 5 or 10 min before enzymatic hydrolysis. Glucose (15.4–17.1 g/l) and xylose (4.5–5.0 g/l) were the most abundant monosaccharides in the hydrolysates. Mannose, galactose and arabinose were also detected. The hydrolysate produced by pretreatment for 10 min contained higher levels of all sugars than the 5 min-pretreated hydrolysate. The amounts of inhibitory compounds found in the hydrolysates were relatively low and increased with increasing pretreatment time. The hydrolysates were fermented with baker's yeast. Ethanol yield, maximum volumetric productivity and specific productivity were used as criteria of fermentability of the hydrolysates. The fermentation of the hydrolysates was only slightly inhibited compared to reference solutions having a similar composition of fermentable sugars. The ethanol yield in the hydrolysates was 0.38–0.39 g/g of initial fermentable sugars, whereas it was 0.42 g/g in the reference. The biomass yield was twofold lower in the hydrolysates than in the reference. The fermentation inhibition caused by the tobacco stalk hydrolysates was less than that caused by sugarcane bagasse hydrolysates obtained under the same hydrolysis conditions.     

Nutritionally rich marine proteins from fresh herring by-products for human consumption

Marine oils and proteins are valuable compounds, but under unfavorable conditions these components are easily oxidized and enzymatically degraded. We have designed an industrial process for producing high quality human grade proteins from fresh herring by-products. Two different processes were tested in semi-industrial scale: (i) thermal extraction to separate oil and proteins and (ii) enzymatic hydrolysis with different commercial proteases to produce fish protein hydrolysates (FPH) and separate oil. Both stick water from thermal extraction and fish protein hydrolysates, after hydrolysis, are nutritionally rich fractions and yielded approx. 18 and 25% of dry material in these fractions respectively. Neither season nor enzymes used influenced the color of hydrolysate powders, but time and temperature of the processes are important tools for controlling the color. Fishing seasons did not influence bitterness of hydrolysates and stick water samples, but both the process conditions and applied enzymes played an important role for the formation of bitterness. Insoluble fractions after both processes had significantly higher protein efficiency ratio: 3.08 and 2.93 compared to soluble fractions: 2.62 and 2.76 after hydrolysis and thermal extraction respectively. Both stick water and FPH showed antioxidative activity against both iron (15 μM of Fe³⁺) and hemoglobin (80 ppm) induced oxidation. Herring proteins (1.25 mg/ml) were able to reduce iron induced oxidation by 50–70%, while Hb induced oxidation was reduced by 70–80% using 4 mg/ml proteins concentration.     

The use of chitosan solutions for defatting and clarification of protein hydrolysates]

The possibility of the use of small amounts of chitosan for defatting and clarification of protein solutions prepared by enzymatic hydrolysis was tested. The following treatment conditions were shown to be optimal: chitosan concentration range, from 1.0 to 1.5 g per kg raw weight; pH of precipitation medium, from 8.0 to 8.5; and duration of incubation of protein hydrolysate solution with chitosan, less than 1 h. The hydrolysate defatting grade was found to depend on the degree of chitosan deacetylation. A possible mechanism of the chitosan-induced effects was suggested. The use of chitosan allows the mass fraction of enzyme protein hydrolysates to be reduced fourfold to fivefold.     

Defatting and Clarification of Protein Hydrolysates by Using Chitosan Solutions

The possibility of the use of small amounts of chitosan for defatting and clarification of protein solutions prepared by enzymatic hydrolysis was tested. The following treatment conditions were shown to be optimal: a chitosan concentration range, from 1.0 to 1.5 gram per kilogram raw weight; pH of the precipitation medium from 8.0 to 8.5; and duration of the incubation of the protein hydrolysate solution with chitosan, less than 1 h. The hydrolysate defatting grade was found to depend on the degree of chitosan deacetylation. A possible mechanism of the chitosan-induced effects was suggested. The use of chitosan allows the mass fraction of enzyme protein hydrolysates to be reduced fourfold to fivefold.     

Determination of radioactivity of proline and hydroxyproline in tissue hydrolysates after the elimination of interference by primary amino acids by deamination

A simple method for the determination of radioactivity of proline and hydroxyproline, particularly of small amounts, in hydrolysates of tissues is described. Specificity is assured by eliminating primary amino acids from the hydrolysates by deamination and then extraction before separation of proline from hydroxyproline by paper chromatography. Six to eight tissue samples may be compared simultaneously. The efficiency and reproducibility are good, as indicated by the use of labeled l-proline, labeled dl-hydroxyproline, a hydrolysate of a protein in which the amino acids (and proline) were labeled, and hydrolysates of tissues cultured in media containing radioactive l-proline. The method is particularly useful when ion-exchange column chromatography of amino acids is not in routine use.     

Studies on a Model of Bitter Peptides Including Arginine,Proline and Phenylalanine Residues. II. Bitterness Behavior of a Tetrapeptide (Arg-Pro-Phe-Phe) and Its Derivatives

In order to investigate the production of a strong bitter taste of the tetrapeptide, Arg-Pro-Phe-Phe (1), we synthesized 16 kinds of analogs and tasted them. From the results, it was clarified that all the constituent amino acid residues in Arg-Pro-Phe-Phe (1) were necessary for its strong bitter taste. For a further increase in bitterness potency, it was found that the bitterness production units necessary should be concentrated together. In addition, Arg-Pro-Gly-Gly (6) and Gly-Gly-Arg-Pro (7) were found to have no bitterness. This will be very useful not only for studies on debittering of food but also for basic studies on the taste production mechanism.     

Production of a novel wheat gluten hydrolysate containing dipeptidyl peptidase-IV inhibitory tripeptides using ginger protease

Wheat gluten is a Pro-rich protein complex comprising glutenins and gliadins. Previous studies have reported that oral intake of enzymatic hydrolysates of gluten has beneficial effects, such as suppression of muscle injury and improvement of hepatitis. Here, we utilized ginger protease that preferentially cleaves peptide bonds with Pro at the P₂ position to produce a novel type of wheat gluten hydrolysate. Ginger protease efficiently hydrolyzed gluten, particularly under weak acidic conditions, to peptides with an average molecular weight of <600 Da. In addition, the gluten hydrolysate contained substantial amounts of tripeptides, including Gln-Pro-Gln, Gln-Pro-Gly, Gln-Pro-Phe, Leu-Pro-Gln, and Ser-Pro-Gln (e.g. 40.7 mg/g at pH 5.2). These gluten-derived tripeptides showed high inhibitory activity on dipeptidyl peptidase-IV with IC₅₀ values of 79.8, 70.9, 71.7, 56.7, and 78.9 μM, respectively, suggesting that the novel gluten hydrolysate prepared using ginger protease can be used as a functional food for patients with type 2 diabetes.     

Detoxification of wood hydrolysates with laccase and peroxidase from the white-rot fungus Trametes versicolor

Fermentation of wood hydrolysates to desirable products, such as fuel ethanol, is made difficult by the presence of inhibitory compounds in the hydrolysates. Here we present a novel method to increase the fermentability of lignocellulosic hydrolysates: enzymatic detoxification. Besides the detoxification effect, treatment with purified enzymes provides a new way to identify inhibitors by assaying the effect of enzymatic attack on specific compounds in the hydrolysate. Laccase, a phenol oxidase, and lignin peroxidase purified from the ligninolytic basidiomycete fungus Trametes versicolor were studied using a lignocellulosic hydrolysate from willow pretreated with steam and SO₂. Saccharomyces cerevisiae was employed for ethanolic fermentation of the hydrolysates. The results show more rapid consumption of glucose and increased ethanol productivity for samples treated with laccase. Treatment of the hydrolysate with lignin peroxidase also resulted in improved fermentability. Analyses by GC-MS indicated that the mechanism of laccase detoxification involves removal of monoaromatic phenolic compounds present in the hydrolysate. The results support the suggestion that phenolic compounds are important inhibitors of the fermentation process. Received: 3 November 1997 / Received revision: 4 February 1998 / Accepted: 6 February 1998     

Enzymatic protein hydrolysis in a membrane reactor related to taste properties

Potato, Vicia faba and soybean proteins were hydrolysed enzymatically in a substrate feed membrane reactor system. Alkaline proteolytic enzymes and PM-10 membranes were used for the hydrolysis of potato protein. The taste of the ultrafiltrates, which was unpleasantly bitter and potato-like, was improved by application of gelatin. Also using PM-10 membranes, Vicia faba protein isolate was hydrolysed by alkaline and acid proteolytic enzymes. The bitterness of the ultrafiltrate decreased with the formation of an isoelectric precipitate, which was probably due to association of hydrophobic peptides. The reactor equipped with a cellulose acetate membrane delivered an acceptable enzymatic hydrolysate of Promine D during the first hours of ultrafiltration. This was not the case when similar processes were performed using non-cellulosic DM-5 membranes. The usefulness of ultrafiltration for obtaining bland protein hydrolysates seems to be limited to short-term processes with cellulose acetate membranes.     

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.     

Ethanolic fermentation of hydrolysates from ammonia fiber expansion (AFEX) treated corn stover and distillers grain without detoxification and external nutrient supplementation

External nutrient supplementation and detoxification of hydrolysate significantly increase the production cost of cellulosic ethanol. In this study, we investigated the feasibility of fermenting cellulosic hydrolysates without washing, detoxification or external nutrient supplementation using ethanologens Escherichia coli KO11 and the adapted strain ML01 at low initial cell density (16 mg dry weight/L). The cellulosic hydrolysates were derived from enzymatically digested ammonia fiber expansion (AFEX)-treated corn stover and dry distiller's grain and solubles (DDGS) at high solids loading (18% by weight). The adaptation was achieved through selective evolution of KO11 on hydrolysate from AFEX-treated corn stover. All cellulosic hydrolysates tested (36-52 g/L glucose) were fermentable. Regardless of strains, metabolic ethanol yields were near the theoretical limit (0.51 g ethanol/g consumed sugar). Volumetric ethanol productivity of 1.2 g/h/L was achieved in fermentation on DDGS hydrolysate and DDGS improved the fermentability of hydrolysate from corn stover. However, enzymatic hydrolysis and xylose utilization during fermentation were the bottlenecks for ethanol production from corn stover at these experimental conditions. In conclusion, fermentation under the baseline conditions was feasible. Utilization of nutrient-rich feedstocks such as DDGS in fermentation can replace expensive media supplementation.     

瓜尔豆种皮酶解提取物抗氧化性研究

以3种蛋白酶对瓜尔豆种皮活性肽进行酶解分离,通过总抗氧化能力测定,筛选出木瓜蛋白酶水解提取物总抗氧化能力最强,分别为碱性蛋白酶和中性蛋白酶水解提取物的1．95倍和3．34倍。在清除超氧阴离子自由基的测定中,木瓜蛋白酶水解提取物也表现出较强的清除能力,清除率随水解溶液浓度的增加呈正量效关系,当溶液浓度为5．45mg/mL时,清除率达43．37％。通过实验证实瓜尔豆种皮酶解提取物与大豆多肽一样有较强的抗氧化能力。     

Simultaneous separation of acid and basic bioactive peptides by electrodialysis with ultrafiltration membrane

beta-Lactoglobulin (beta-lg), one of the major whey components, can release by enzymatic hydrolysis different bioactive peptidic sequences according to the enzyme used. However, these protein hydrolysates have to be fractionated to obtain peptides in a more purified form. The aim of the present work was to evaluate the feasibility of separating peptides from a beta-lg hydrolysate using an ultrafiltration (UF) membrane stacked in an electrodialysis (ED) cell and to study the effect of pH on the migration of basic/cationic and acid/anionic peptides in the ED configuration. Electrodialysis with ultrafiltration membrane (EDUF) appeared to be a selective method of separation since amongst a total of 40 peptides in the raw hydrolysate, only 13 were recovered in the separated adjacent solutions (KCl 1 and KCl 2). Amongst these 13 migrating peptides, 3 acid/anionic peptides migrated only in one compartment (KCl 1), while 3 basic/cationic peptides migrated only in the second compartment (KCl 2) and that whatever the pH conditions of the hydrolysate solution. Furthermore, the highest migration was obtained for the ACE-inhibitory peptide beta-lg 142-148, with a value of 10.75%. The integrity of the UF membrane was kept and EDUF would minimize the fouling of UF membrane.     

Isolation of protein hydrolysates with given properties (a review)

Methods for obtaining protein hydrolysates with certain given properties are considered. It is shown that, besides food proteins, various yeast proteins can be used. Advantages and disadvantages of different types of hydrolysis, hydrolysis conditions, methods of hydrolysate purification and prevention of labile amino acid destruction are described.     

HPLC determination of xylulose formed by enzymatic xylose isomerization in lignocellulose hydrolysates

Summary In the concentration range appropriate for enzymatic xylose isomerization, xylulose was measured in a lignocellulose hydrolysate using HPLC with two hydrogen loaded ion exchange columns in series. Spent sulphite liquour (SSL) was used as a model for lignocellulose hydrolysates. In buffer the separation took 22 minutes and in SSL the analysis time was 47 minutes due to the presence of ethanol. The enzymatic isomerization of xylose to xylulose was followed directly in SSL, providing a method for the direct determination of xylose isomerase activity in lignocellulose hydrolysates.     

Debittering of protein hydrolysates using immobilized chicken intestinal mucosa

Enzymatic hydrolysates of casein and soybean were treated with alginate immobilized chicken intestinal mucosa, as an aminopeptidase source, to bring about debittering. The mucosa was hygienised by irradiation (20 kGy) which brought about a complete decontamination of the tissue accompanied by a 20% loss in aminopepidase activity. The effectiveness of the process was demonstrable by a higher acceptability and a marked reduction in bitterness scores for casein (from 4.4 to 2.5) and soybean (from 3.8 to 2.2) in organoleptic analysis. The action of aminopeptidases to bring about this change was corroborated by a concomitant increase in free amino acids and a decrease in average peptide length of the samples after treatment. The RP HPLC profiles of casein and soybean protein hydrolysates before and after treatment showed a higher content of peaks in the hydrophilic region suggesting a decrease in hydrophobic peptides, responsible for bitter taste, in both the samples. Immobilization of the mucosal tissue in alginate afforded an increased pH and temperature tolerance to the enzymes. The possibility of the system for continuous operation over extended time periods is also discussed.     

Production of 2,3-butanediol by Klebsiella pneumoniae BLh-1 and Pantoea agglomerans BL1 cultivated in acid and enzymatic hydrolysates of soybean hull

We investigated the production of 2,3-butanediol by two enterobacteria isolated from an environmental consortium, Klebsiella pneumoniae BLh-1 and Pantoea agglomerans BL1, in a bioprocess using acid and enzymatic hydrolysates of soybean hull as substrates. Cultivations were carried out in orbital shaker under microaerophilic conditions, at 30°C and 37°C, for both bacteria. Both hydrolysates presented high osmotic pressures, around 2,000 mOsm/kg, with varying concentrations of glucose, xylose, and arabinose. Both bacteria were able to grow in the hydrolysates, at both temperatures, and they efficiently converted sugars into 2,3-butanediol, showing yields varying from 0.25 to 0.51 g/g of sugars and maximum 2,3-butanediol concentrations varying from 6.4 to 21.9 g/L. Other metabolic products were also obtained in lower amounts, notably ethanol, which peaked at 3.6 g/L in cultures using the enzymatic hydrolysate at 30°C. These results suggest the potential use of these recently isolated bacteria to convert lignocellulosic biomass hydrolysates into value-added products.     

Amino acid and soluble protein cocktail from waste keratin hydrolysed by a fungal keratinase of Paecilomyces marquandii

Waste bovine hooves and horns were enzymatically hydrolysed into soluble products intended for foliar fertilizer. With the powdered keratin at 50°C and pH 8 between 34 to nearly 60% of nitrogen was solubilized in 5 h, depending on the enzyme concentration. The reaction could further be improved by steam pretreatment of the keratin, resulting in 98% solubilisation of the nitrogen. The products of hydrolysis consisted of a mixture of soluble proteins, peptides, and free amino acids. Among the latter, 18 common amino acids were detected. Several of them were previously recognized to have a positive effect on plants. Nonpolar neutral, basic, and sulphur amino acids were present in relatively large amounts, while proline and tryptophan were not found. Comparison with other protein hydrolysates aimed for fertilizer suggests that keratin degradation products, obtained by enzymatic hydrolysis, have potential to be used for foliar fertilization, alone or in a combination with another complementary hydrolysate of a different source, such as skin or plant proteins.