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US20050256057A1 - Protein hydrolysate rich in tripeptides - Google Patents

Protein hydrolysate rich in tripeptides Download PDF

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Publication number
US20050256057A1
US20050256057A1 US10/516,983 US51698304A US2005256057A1 US 20050256057 A1 US20050256057 A1 US 20050256057A1 US 51698304 A US51698304 A US 51698304A US 2005256057 A1 US2005256057 A1 US 2005256057A1
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protein
proline
tripeptides
hydrolysate
peptides
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Luppo Edens
Petrus Jacobus Dekker
Andre Roos De
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DSM IP Assets BV
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DSM IP Assets BV
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Publication of US20050256057A1 publication Critical patent/US20050256057A1/en
Priority to US12/039,973 priority Critical patent/US7972808B2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)

Definitions

  • the present invention relates to protein hydrolysate and the uses thereof.
  • bioactive peptides and protein hydrolysates containing such bioactive peptides have been described in a number of patent applications.
  • WO 97/00078 describes hydrolysates obtained by incubation with probiotic bacteria or enzymes obtained from such bacteria.
  • WO 99/16461 describes the inhibition of angiotensin-converting enzyme by specific tripeptides obtained by fermentation of Lactobacillus .
  • WO 01/32905 describes the preparation of a product containing antihypertensive peptides by fermenting casein with lactic acid bacteria.
  • WO 01/68114 describe the use of highly purified or chemically synthesized peptides for reducing blood pressure or treating diabetes, renal impairment or obesity.
  • the present invention provides a process to produce protein hydrolysate which is rich in tripeptides whereby the peptides preferably are rich in proline at one end of the peptide and preferably the peptide has a carboxy terminal proline.
  • the protein hydrolysate of the invention is non-bitter.
  • the hydrolysate may optionally comprise dipeptides.
  • the selected protein or proteinaceous substrate is contacted with a suitable endoprotease.
  • a suitable endoprotease is preferably a proline specific endoprotease (PSE or Endopro), a serine protease, a metalloendoprotease or an aspartic protease, more preferably a PSE is used.
  • PSE proline specific endoprotease
  • serine protease a serine protease
  • a metalloendoprotease a metalloendoprotease or an aspartic protease
  • TPAP tripeptidase
  • Such tripeptidases are defined as enzymes capable of releasing tripeptides from a polypeptide, either from the N-terminal side of the polypeptide hereby encompassing the socalled tripeptidyl-peptidases or from the C-terminal side of the polypeptide hereby encompassing the socalled peptidyl-tripeptidases.
  • the protein substrate is first fermented with a endoprotease, such as a serine protease, metalloendoprotease or an aspartic protease, to partly hydrolyse the protein.
  • a endoprotease such as a serine protease, metalloendoprotease or an aspartic protease
  • the process according to the invention involves a combination of one or more endoproteases with one or more tripeptidases.
  • the enzymes are used in an isolated form and in an endoprotease to tripeptidase protein ratio range between 1:0.05 and 1:50, preferably between 1:0.1 to 1:10.
  • the protein substrate or the partial hydrolysate formed can first be subjected to the suitable first endoprotease and subsequently the TPAP or mixture of TPAP's can be added.
  • a one step process may be preferred.
  • the TPAP used in the present process is a TPAP which after an incubation at pH5 of 1 hour at 50° C. shows at least 70% residual activity on a Ala-Ala-X-pNa substrate as measured in Example 1.
  • X may vary with the TPAP in question depending on the specifity of the TPAP.
  • X is an amino acid residue which gives rise to at least an significant activity of the TPAP (see for example FIG. 1 .)
  • an enzyme To be useful as processing aids in the preparation of food ingredients, an enzyme must preferably meet a number of strict economical and legislative criteria. To meet the legislative criteria the enzyme should be obtained from an unsuspect source, for example a food-grade microorganism. To meet the economical criteria, the enzyme should be secreted by the microorganism, producible in high yields and exhibit a number of biochemical characteristics such as a long term stability under industrial processing conditions. To minimise the risks of microbial infections under such non-sterile conditions, industrial processing often employs acidic pH conditions and a temperature of 50 degrees C. or higher. An enzyme used in the present invention advantageously meet these demands.
  • the present invention further provides a hydrolysate rich in tripeptides whereby preferably these tripeptides are rich in carboxy terminal proline.
  • Rich in tripeptides means that at least 20 molar %, preferably at least 25 molar %, more preferably at least 30 molar % or most preferably at least 35 molar % of the smaller peptides present in the hydrolysate, is present as tripeptide.
  • Smaller peptides are defined as peptides with a molecular weight of 200-2000 Da.
  • Rich in proline means that at least 20%, preferably at least 30%, more preferably at least 40% and even more preferably 50% of the proline present in the starting protein substrate, is present in the tripeptides, preferably as carboxy terminal proline.
  • Preferably 30% of the tripeptides or more preferably 35% of the tripeptides have a carboxy terminal proline residue, the values can be obtained with protein substrates that are rich in proline.
  • the hydrolysate produced according to the present invention has in general a degree of hydrolysis of between 10 and 40, preferably between 15 and 30.
  • the degree of hydrolysis is determined using the OPA method as described by Nielsen, P. M. et al (Journal of Food Science, Vol 66, No 5, PP 642-646, 2001).
  • the hydrolysates produced according to the process of the present invention can be fractionated if desired. For example separation techniques such as centrifugation or filtration (for example microfiltration and ultrafiltration) can be used to produce compositions which are further enriched in peptides having a molecular weight of 2000 Da or less.
  • composition comprising for at least 10 wt %, preferably for at least 20 wt %, more preferably for at least 30% and most preferably for at least 40 wt % of peptides having a molecular weight of 200-2000 Da, based on total amount of peptides present.
  • Whey protein represents a very suitable substrate for producing hydrolysates by the process of the invention.
  • Whey protein is relatively rich in “essential” and “branched chain” amino acids and has a high biological digestibility. Moreover, whey hydrolysates exhibit relatively low bitterness profile. Because whey has a relatively low proline content, the role of the tripeptidase in generating a mixture of easily assimilable peptides is important.
  • proteins like casein, wheat and maize gluten, soy, rice protein, chicken feathers and gelatin exhibit vastly different amino acid compositions.
  • some of these proteins potentially form the substrate of choice for the production of hydrolysates by the process of the invention.
  • wheat gluten is extremely rich in glutamine and rice protein is rich in arginine residues. Both amino acids are known to improve physical endurance and the recovery rate following high intensity exercise.
  • glutamine is not stable so that supply in a readily assimilable peptide is advantageous.
  • Maize gluten is a cheap substrate that is extremely rich in leucine and phenylalanine, it is known that these amino acids can modulate glucose and insulin responses upon oral consumption.
  • cysteine is a labile compound that is preferably supplied in the form of di- or tripeptides.
  • proline residues i.e. more than 6 grams free amino acid per 100 grams of protein.
  • proline confers an increased stability to peptides thereby increasing their potential significance in eliciting physiological effects such as decreasing blood pressure, acting as opioid agonists or antagonists, contracting smooth muscles and inhibiting platelet aggregation.
  • recent research has implicated specific proline containing sequences or a shortage of proline-specific proteases to immunological effects associated with psychological features. For example celiac sprue is a widely prevalent autoimmune disease induced by exposure to dietary gluten (Shan, L. et al; Science Vol 297, 2002, 2275-2279) and linked with behaviour change (Bernejo, M. and Polanco I, Rev Neurol 2002 Feb. 28; 34 Suppl 1: S24-33).
  • proline residues Up to now peptide bonds involving proline residues have been notoriously difficult to cleave using commercially available enzymes so that protein hydrolysates prepared from proline-rich substrates contain major fractions of large molecular weight material. Moreover, proline represents a very hydrophobic amino acid and yields extremely bitter hydrolysates. Thus, the production of acceptable hydrolysates from proline-rich substrates using existing technologies would lead to low yields and highly priced products.
  • the present invention discloses an enzyme mixture that would permit simple protocols to convert all relevant proteinaceous substrates into highly desirable hydrolysates with a good taste, an efficient gastrointestinal uptake, low allergenicity levels and, if required, a high content of bioactive peptides.).
  • This enzyme composition consisting of an endoprotease, preferably a proline specific endoprotease, and a tripeptidase when added to a suitable protein is able to produce the protein hydrolysate which is rich in tripeptides and optionally dipeptides whereby the di- and/or tripeptides are rich in proline at one end of the peptide.
  • these protein hydrolysates offer attractive advantages such as lowered allergenicities, facilitated gastro-intestinal uptake, less chemical deterioration of desirable amino acids like glutamine and cystein and finally, absence of proteinaceous precipitations in acid beverages during prolonged storage periods. All these advantages can be combined if the hydrolysate is prepared using a combination of an endoprotease, preferably a proline specific endoprotease, and one or more tripeptidases. According to the invention several useful tripeptidases are preferably used in a pure or isolated state. Pure tripeptidase can be obtained for example by overexpression of the enzyme is a suitable transformed host microorganism.
  • tripeptidases that exhibit a low selectivity towards the substrate to be cleaved, i.e. exhibit minimal amino acid residue cleavage preferences only. Combinations of tripeptidases that hydrolyse high percentages of the naturally occurring peptide bonds are preferred. Despite this high activity to naturally occurring peptide bonds, a total hydrolysis to free amino acids is prevented by the nature of the tripeptidases. Also tripeptidases that are optimally active between pH 4 to 8 and exhibit adequate temperature stability are preferred. Adequate temperature stability means that at least 40%, preferably at least 60%, more preferably between 70 and 100% of the initial hydrolytic activity survives after heating the enzyme together with the substrate for 1 hour at 50 degrees C. Tripeptidyl aminopeptidase is the preferred tripeptidase
  • Tripeptidyl aminopeptidases are enzymes that can release tripeptides from the N-terminus of an oligopeptide. Little is known on enzymes that can release tripeptides from the oligopeptide's carboxyterminus (tripeptidyl carboxypeptidases or peptidyl-tripeptidases). The various physiological advantages of the mixture of tripeptides that can be formed by such enzymes was illustrated above. Tripeptides offer a much wider sequence variation than dipeptides can hereby increasing the chance of an optimal fit with the receptors responsible for modulating biological activities. This is well illustrated by the documented number of bioactive peptides having a carboxyterminal proline residue (see for example WO 01/68114).
  • Tripeptidyl aminopeptidases (EC 3.4.14) have been isolated from mammalian as well as plant sources. Microorganisms from which tripeptidylpeptidases have been isolated are for example Streptomyces species (JP08308565, WO 95/17512 and U.S. Pat. No. 5,856,166)), Porphyromones gingivalis (WO 00/52147), Dictyostelium discoidum and Aspergillus species (WO 96/14404). To date, the occurrence of tripeptidyl carboxypeptidases (EC 3.4.15) has been demonstrated in mammalian cells and in the microorganism Clostridium histolyticum only.
  • tripeptidases and/or endoproteases in high quantities and in a pure or isolated form.
  • a preferred way of obtaining purified and isolated tripeptidases is via the overproduction using recombinant DNA techniques.
  • a more preferred method is the overproduction of acid stable tripeptidases exhibiting adequate stabilities under processing conditions of 50 degrees C. or higher using recombinant DNA techniques.
  • a particulary preferred method is the overproduction of such tripeptidases derived from Aspergillus and a most preferred method is the overproduction of such tripeptidases from Aspergillus niger.
  • a polypeptide used in the process of the invention which has endoprotease or tripeptidase activity may be in an isolated form.
  • an isolated polypeptide is an endogenously produced or a recombinant polypeptide which is essentially free from other polypeptides, and is typically at least 20% pure, preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, still more preferably at least 90% pure, or most preferably at least 95% pure, as determined by SDS-PAGE.
  • the polypeptide may be isolated by centrifugation, filtration (for example utrafiltration) or chromatographic methods, or any other technique known in the art for obtaining pure proteins from crude solutions.
  • polypeptide may be mixed with carriers or diluents which do not interfere with the intended purpose of the polypeptide, and thus the polypeptide in this form will still be regarded as isolated. It will generally comprise the polypeptide in a preparation in which more than 10%, for example more than 20%, 30%, 40%, 50%, 80%, 90%, 95% or 99%, by weight of the proteins in the preparation is a polypeptide for use of the process of the present invention.
  • the main aim of the hydrolysates of the invention is to minimize the allergenicity or immunoresponse of the product or to facilitate gastro-intestinal uptake.
  • the use of a proline specific endoprotease in combination with one or more tripeptidases is of special importance as these offer an efficient way for producing such hydrolysates.
  • the enzyme mixture according to the invention may comprise a tripeptidase or a mixture of tripeptidases.
  • the enzyme mixture my also comprise a endoprotease, such as a serine protease, a metalloendoprotease, an aspartic protease, or a proline-specific endoprotease (PSE or E.C. 3.4.21.26) which work together with the tripeptidase to provide a primary protein hydrolysate.
  • the endoprotease can be one or more different endoproteases which are incubated either simultaneously or consecutively with the protein substrate, for example the proteinaceous substrate may be first digested with an endoprotease preferably a serine protease, a metalloendoprotease or an aspartic protease and subsequently, digested with a second endoprotease, preferably PSE. Before adding the second endoprotease, the enzymes already present are optionally inactivated.
  • Serine proteases represent a well known class of alkaline endoproteases. Examples include subtilisin (E.C. 3.4.21.62) and chymotrypsin (E.C. 3.4.21.1) which prefer cleavage of the peptide chain at the carboxy terminal side of hydrophobic amino acids such as Tyr, Trp, Phe and Leu.
  • the enzyme mixture of the invention may contain chymotrypsin and/or subtilisin.
  • Subtilisin is produced by species of Bacillus , has a particularly broad substrate specificity and a broad, alkaline pH optimum. The enzyme is optimally active between 50° C. and 60° C.
  • Chymotrypsin may be obtained from animal pancreas, has a somewhat narrower substrate specificity at slightly more alkaline pH values than subtilisin and is optimally active below 50 degrees C.
  • the class of metalloendoproteases is wide spread in bacteria, fungi and higher organisms. They can be separated into the neutral and acid metalloproteases. Of these two subclasses only the neutral proteases exhibit the desirable cleavage preference i.e. cleaving the peptide chain on the carboxy terminal side of hydrophobic amino acid residues such as Phe and Leu.
  • Well known examples of the neutral metalloproteases are bacillolysin (E.C. 3.4.24.28) and thermolysin (E.C. 3.4.24.27) and either, or both of these, may be present in the enzyme mixture of the invention. Both enzymes are obtained from Bacillus species and exhibit maximum activity under neutral or slightly alkaline conditions.
  • the aspartic proteases feature an acidic pH optimum that can be advantageously used in combination with a proline-specific endoprotease and a tripeptidase that also have acidic pH optima.
  • the aspartic proteases especially pepsin is recognized as an effective endoprotease with a broad specificity. Suitable A. niger derived aspartic endoproteases have been specified in our copending application PCT/EP0201984.
  • the process according to the invention involves a combination of one or more endoproteases with one or more tripeptidases.
  • the enzymes are used in isolated form and in an endoprotease to tripeptidase protein ratio range between 1:0.05 and 1:50, preferably between 1:0.1 to 1:10
  • the substantially pure enzymes are subjected to SDS-PAGE analysis followed by a standard protein staining protocol using Coomassie Brilliant Blue. Quantification of the enzymes used is carried out using a spot densitometer measuring the integrated density values of the protein bands corresponding with the active enzymes.
  • denaturation is carried out by mixing the enzymes with a protease inhibitor, immersion of the mixture in a waterbath of 99 degrees C. for 5 minutes after which the required quantities of SDS and reducing compound are added.
  • Serine endoproteases are inhibited by mixing with Pefabloc, metalloproteases by mixing with phosphoramidon and aspartic proteases by mixing with pepstatin. All inhibitors plus working procedures are obtainable from Roche.
  • proline-specific endoprotease which, in conjunction with the prior art endoproteases, is able to generate non-bitter protein hydrolysates.
  • This proline-specific endoprotease is an enzyme capable of cleaving peptides or polypeptides at the carboxy-terminal end of proline residues.
  • Proline-specific endoproteases are widely found in animals and plants, but their presence in microorganisms appears to be limited.
  • proline-specific endoprotease have been identified in species of Aspergillus (EP 0 522 428 and WO 02/45524), Flavobacterium (EP 0 967 285), Aeromonas (J.
  • the proline-specific endoprotease is capable of extensively hydrolysing proline-rich proteins yielding relatively small peptides with a narrow size distribution. Because of the cleavage preference the proline-specific endoprotease, many of the peptides formed have a carboxyterminal proline residue. Furthermore, the processing of the hydrolysate is relatively simple as a debittering step by exoproteases is not involved so that only low levels of free amino acids will be formed.
  • a particulary preferred method is the overproduction of an Aspergillus derived proline-specific endoprotease and a most preferred method is the overproduction of an Aspergillus niger derived proline-specific endopeptidase.
  • the enzymes according to the invention may be used in an immobilized form so that large quantities of protein containing liquids can be treated. Ways to select appropriate support materials and suitable immobilization methods have been extensively described in the literature, for example in “Immobilization of Enzymes and Cells” (ed. Gordon F. Bickerstaff; ISBN 0-89603-386-4).
  • non-bitter hydrolysates from proteinaceous substrates with novel amino acid compositions.
  • novel amino acid compositions may offer serious benefits in certain food and medical applications. Examples are casein or wheat gluten or maize protein isolate with high levels of hydrophobic amino acid residues and, more specifically, proline residues present. Hitherto such substrates were of no practical use because of the objectional bitter tastes generated upon hydrolysis and the limited degrees of hydrolysis obtained using prior art methods.
  • new, non-bitter hydrolysates can be made available to be used in infant and clinical nutrition, in therapeutic diets as well as in consumer diets and sport nutrition.
  • Liquid enzyme formulations providing good shelf stabilities and suitable for oral consumption have been described in the prior art. Upon oral intake and the combination of the two acid stable enzymes will aid the digestion of proline rich proteins such as caseins or glutens hereby preventing or minimising the effects described for, for example, coeliac sprue.
  • the proline-specific endoprotease is used to generate peptides having a carboxyterminal proline residue.
  • Such peptides are desirable additions to various food or nutraceutical products as they have been implicated in anorectic, fibrinolytic, antithrombotic and antihypertensive effects, as well as in protection of the gastric mucosa and the prevention of rheumatoid arthritis.
  • proline-specific endoprotease should preferably exhibit an activity spectrum with an acidic pH optimum.
  • the invention demonstrates that the activity of an isolated, purified proline-specific endoprotease alone, i.e. without the substantial concomitant or subsequent activity of an exoproteolytic enzyme, is sufficient for significantly debittering a protein hydrolysate. Therefore the proline-specific endoprotease may comprise at least 5 units per gram protein of the enzyme preparation of the invention, preferably 10 u/g, more preferably 25 u/g and even more preferably 50 u/g.
  • hydrolysates produced according to the invention are enriched in peptides having a carboxy terminal proline residue.
  • An embodiment of the present invention provides the use of a proline-specific endoprotease, preferably isolated and/or purified, for the high yield production of protein hydrolysates having substantially low bitterness and low allergenic properties without the concomitant production of substantial levels of free amino acids in combination with a TPAP. All the enzymes may be added at the same time to the substrate or the enzymatic process can be performed in two phases, first the PSE hydrolysis followed by the TPAP hydrolysis.
  • Tripeptidases are the enzymes of choice for preparing easily assimilable protein hydrolysates. Not only can the peptides formed be directly translocated over the wall of the small intestine but, due to their small size these peptides combine a good water solubility with a lack of any allergenic potential. Moreover, vulnerable but indispensible amino acids like glutamine, cysteine and tyrosine are much more stable if present in the form of tripeptides rather than free amino acids. Thus, upon digesting selected proteinaceous substrates with a suitable endoprotease in combination with a tripeptidyl peptidase, hydrolysates are formed in which selected amino acid residues are present in a stable and yet easily assimilable form.
  • Conceivable products that can be conveniently produced using the enzyme mixture according to the invention are easily assimilable gluten hydrolysates supplying high levels of glutamine as well as hydrolysates obtained from keratin or lactalbumin-rich fractions from whey supplying high levels of cysteine.
  • hydrolysates containing tripeptides exerting an enhanced modulating, regulatory or hormone-like activity as the result of their increased stability for example tripeptides rich in proline or glycine residues, could be formed upon the digestion of substrates like gelatin or casein or maize protein.
  • the process of the invention is suitable for preparing hydrolysates of various protein fractions.
  • a protein substrate such as a milk protein
  • a TPAP may be incubated with an isolated, purified proline-specific endoprotease and a TPAP to produce a protein hydrolysate enriched in peptide fragments having a carboxy terminal proline.
  • the average length of the peptides in the hydrolysates is in general from 2 to 9 amino acids, preferably from 3 to 6 amino acids, more preferably from 3 to 5 amino acids. This average length is based on peptides having molecular masses from 200 to 2000 Dalton and can be calculated by taking the sum of the number of each peptide multiplied with the length of said peptide and dividing this sum by the total number of peptides.
  • peptides or peptide fragments it is meant peptides with molecular masses from 200 to 2000 Dalton. These peptides can be analysed according to the LC/MC analysis as described the “Materials and Methods” section.
  • protein substrate is substantially hydrolysed, preferably at least 20% (w/w) of the protein substrate is converted into peptides having molecular masses from 200 to 2000 Dalton. More preferably from 30 to 90% (w/w) and even more preferably from 40 to 80% (w/w) of the protein substrate is converted into such peptides.
  • Another embodiment of the invention is a protein hydrolysate enriched with a relatively high content of peptides having proline as the carboxy terminal amino acid residue. Since enzyme preparations typically utilized in the genesis of protein hydrolysates are not capable of generating peptides bearing proline residues at carboxy terminii, protein hydrolysates that are relatively rich in such peptides are desired.
  • Substrates for hydrolysis by an enzyme mixture of the invention include whole milk, skimmed milk, acid casein, rennet casein, acid whey products or cheese whey products.
  • Industrially obtainable fractions as for example fractions enriched in lactalbumine are also useful.
  • the Aspergillus derived proline specific endoprotease does not only cleave at the carboxy-terminal side of proline residues but also at the carboxy-terminal side of hydroxyproline residues which makes other, collagen based animal proteins such as gelatine as well as bones or fish-bones containing residual meat interesting substrates for the enzyme.
  • milk protein hydrolysates produced according to the invention may be used with or without additional filtration or purification steps in various speciality foods such as hypoallergenic hydrolysates for infant nutrition, basic hydrolysates for enteral and dietetic nutrition, as well as protein concentrates for various forms of health food.
  • protein hydrolysates of the invention may be used to produce foodstuffs having low antigenicity, such as infant formula or requiring facilitated gastro-intestinal uptake, such as various medical or health related products.
  • enzyme preparations according to the invention may be used to reduce bitterness in foods flavored by at least one protein hydrolysate, even when the protein hydrolysate is present in large amounts.
  • foods may comprise between 5% and 10% (w/v) of a protein hydrolysate and still have their bitterness reduced using an enzyme preparation of the invention.
  • the present invention preferably uses an isolated or purified proline-specific endoprotease with an acidic pH optimum in a combination with one or more isolated tripeptidases exhibiting acid pH optima for the preparation of a protein hydrolysate for various food applications.
  • an isolated, purified proline-specific endoprotease is defined to have at least 10 units of proline specific endoprotease activity per gram of proteinaceous material. These units should be measured using the synthetic peptide Z-Gly-Pro-pNA (Bachem, Switserland) at 37 degrees C. and pH 7.
  • the units should be measured at pH 5, as specified in the Materials and Methods section.
  • the enzyme mixture of the invention overcomes a number of disadvantages of enzyme mixtures previously known in the art.
  • the isolated, purified proline-specific endoprotease is key in the production of hydrolysates which combine a low allergenic potential, a high yield and a low bitterness profile.
  • the isolated tripeptidases are key in the generation of easily assimilable peptides without any allergenic potential and a specific, preferred amino acid composition.
  • hydrolysates produced with an enzyme mixture comprising this proline-specific endoprotease are relatively stable in the body, exhibit a surprising shelf stability upon their incorporation in acid products and contain very low levels of free amino acids, such that minimal off-tastes are generated during heating steps, such as spray drying or product sterilisation.
  • Hydrolysates according to the invention will contain less than 900 micromoles of free amino acids per gram dry weight, preferably less than 300 micromoles of free amino acids per gram dry weight more preferably less than 150 micromoles of free amino acids per gram dry weight, and even more preferably less than 50 micromoles per gram dry weight.
  • FIG. 1 Comparison of specifity of TPAP-A and TPAP-B using A-A-X-pNA substrates where X is all natural aminoacids, at pH 4.
  • FIG. 2 Composition of soluble peptides obtained by hydrolysing alfa-lactalbumin with enzyme combinations as indicated.
  • the enzymatic activity of proline specific endoproteases exhibiting pH optima above pH 6.0 are tested according to T. Diefenthal and H. Dargatz (World Journal of Microbiology &Biotechnology 11, 209-212 (1995)) on Z-Gly-Pro-pNA 0.26 mM in phosphate buffer 0.1M pH 7.0 at 25° C. The product was monitored spectrophotometrically at 410 nm. Proline specific endoproteases from Aspergillus was measured according to the method described in Japanese patent JP5015314 with minor modifications. In brief the enzymatic activity is tested on Z-Gly-Pro-pNA at 37 degrees C. in a citrate/disodium phosphate buffer pH 5.
  • pH 5.0 is chosen because in this test the pH optimum of the enzyme is below pH 6.
  • the reaction product was also monitored spectrophotometrically at 410 nM using a molar extinction coefficient of 10500 per mol/liter.
  • the activity of the purified tripeptidyl aminopeptidase as over produced by A. niger (TPAP-A) was measured in a similar way. However, in this case the synthetic substrate Ala-Ala-Phe-pNA (Bachem, Switzerland) was used in an incubation in 0.1 mol/litre citrate buffer at pH 4.0 and 60 degrees C.
  • the purified TPAP-A had an activity of 8 units/ml.
  • a unit is defined as the quantity of enzyme that provokes the release of 1 ⁇ mol of p-nitroanilide per minute under these conditions.
  • the Degree of Hydrolysis (DH) as obtained during incubation with the various proteolytic mixtures was monitored using a rapid OPA test (JFS, Vol 66, NO 5, 2001).
  • HPLC high performance liquid chromatography
  • Qtof-2 Micromass, Manchester, UK
  • mass spectrometer was used to separate the peptides formed during digestion with trypsin. 5 microliter of the peptide solution was trapped on a micro-precolumn, C18, 5*0.3 mm (MCA30-05-C18, LC Packings, Amsterdam, Netherlands) using Milli Q water containing 0.1% of formic acid at a flow-rate of 20 microliter/min.
  • the peptides were then eluted from the precolumn, using a fast gradient of 0.1% formic acid in Milli Q water (Millipore, Bedford, Mass., USA; Solution A) and 0.1% formic acid in acetonitrile (Solution B).
  • the gradient started at 100% of Solution A and increased to 60% of solution B in 20 minutes and was kept at the latter ratio for another 5 minutes.
  • the flow rate used during elution of the peptides was 200 nl/min.
  • partial amino acid sequences of the A. niger proline-specific endopeptidase could be determined, by de novo sequencing of suitable peptides.
  • HPLC using an ion trap mass spectrometer (Thermoquest®, Breda, the Netherlands) coupled to a P4000 pump (Thermoquest®, Breda, the Netherlands) was used in characterising the enzymatic protein hydrolysates produced by the inventive enzyme mixture.
  • the peptides formed were separated using a PEPMAP C18 300A (MIC-15-03-C18-PM, LC Packings, Amsterdam, The Netherlands) column in combination with a gradient of 0.1% formic acid+1 mM nonafluoropentaoic acid (NFPA) in Milli Q water (Millipore, Bedford, Mass., USA; Solution A) and 0.1% formic acid in acetonitrile (Solution B) for elution.
  • NFPA nonafluoropentaoic acid
  • the gradient started at 100% of Solution A and increased to 40% of solution B in 140 minutes and was kept at the latter ratio for another 5 minutes.
  • the injection volume used was 50 microliters, the flow rate was 50 microliter per minute and the column temperature was maintained at 30° C.
  • the protein concentration of the injected sample was approx. 50 micrograms/milliliter.
  • the mass range of the peptides formed starts at di- and tripeptides.
  • the volatile ion-pairing reagent NFPA in combination with reversed phase liquid chromatography also smaller and more hydrophilic peptides can be monitored ending up with a mass ranging from approx. 200 to 2000 Daltons, considered suitable for further analysis by MS sequencing.
  • LC/MS/MS can be used for the analysis of the C-terminus of a peptide.
  • an algorithm in which the peptide's molecular mass (analyzed with LC/MS) and its (partial) amino acid sequence (analyzed with LC/MS/MS) are linked with automatic search procedures within protein databanks complex peptide mixtures can be analyzed. These options have enabled us to quantify the incidence of peptides carrying a carboxy terminal proline residue.
  • Acid hydrolysis of the protein substrate to convert the proteins present into free amino acids was achieved by making a suspension of 100 milligrams of proteinaceous material in 2 milliliters 6 N HCl. Acid hydrolysis was carried out for 22 hours at 112 degrees C in an oxygen free atmosphere. After centrifugation the supernatant was diluted 10 times in dilute HCl. After this hydrolysis the amino acids were derivatised and analysed according to the Picotag method as specified in the operators manual of the Amino Acid Analysis System of Waters (Milford Mass., USA). The level of proline present was quantitated using HPLC methods.
  • the micromoles of proline present times 100 were divided by the sum of the micromoles of all amino acids present in the sample analysed. Since during acid hydrolysis Trp and Cys are destroyed, these two amino acids are not included in this sum of the micromoles of all amino acids.
  • a precisely weighed sample of the proteinaceous material was dissolved in dilute acid and precipitates were removed by centrifugation in an Eppendorf centrifuge.
  • Amino acid analysis was carried out on the clear supernatant according to the PicoTag method as specified in the operators manual of the Amino Acid Analysis System of Waters (Milford Mass., USA). To that end a suitable sample was obtained from the liquid, added to dilute acid and homogenized. From the latter solution a new sample was taken, dried and derivatised using phenylisothiocyanate. The various derivatised amino acids present were quantitated using HPLC methods and added up to calculate the total level of free amino acids in the weighed sample.
  • the sample is also subjected to acid hydrolysis followed by a quantification of the total free amino acids present as detailed above.
  • the enzyme encoded by gene 12 was overproduced in an A. niger host cell and chromatographically purified. Purification was carried out on a Resource Q column in 50 millimol/liter acetate pH 4.5. Elution by increasing the NaCl concentration yielded the enzyme in a sharp activity peak. Activity was measured by incubation with the synthetic peptide Ala-Ala-Phe-pNA. The solution with the purified enzyme contained 8 units/ml if tested on the synthetic tripeptide Ala-Ala-Phe-pNA at pH 4.0 and 60 degrees C. (see Materials & Methods section).
  • Casein hydrolysates subjected to a proline-specific endoprotease in combination with a tripeptidylaminopeptidase are non-bitter and contain a high proportion of tripeptides having carboxyterminal proline residues.
  • a 6% (w/w on protein) casein solution was prepared by dissolving sodium caseinate in water. After adjustment of the pH to 8.0 by NaOH, the serine protease Delvolase was added to a concentration of 4% (volume of the commercial enzyme product per weight of sodium caseinate) and the mixture was incubated for 2.5 hours at 60 degrees C. under non-pH-stat conditions. Then the reaction was stopped by lowering the pH to 5.0 using lactic acid followed by a heat treatment of 10 minutes at 90 degrees C. The solution was cooled down to 50 degrees C. and two samples were taken. The first sample (Sample A) served as a reference characterizing the material that has been subjected to the action of a broad spectrum serine protease only.
  • EndoPro refers to an overproduced and chromatographically purified proline specific endoprotease from A. niger as described in WO 02/45524
  • TPAP-A chromatographically purified proline specific endoprotease from A. niger
  • Samples A and B were sensorically evaluated by a trained panel. The two samples were tasted “blind” and then scored on a scale from 0 (non bitter) to 4 (very bitter) as described in the Materials & Methods section. Sample A was unanimously scored as “very bitter”, Sample B was unanimously scored as “non bitter”. This outcome confirmed the surprising debittering capacity of the EndoPro enzyme once more.
  • Sample B Part of Sample B was then incubated with 20 units of chromatographically purified TPAP-A per gram of casein protein during 5 hours at pH 4.0 and 60 degrees C. Like before the enzyme reaction was terminated by heating of the solution for 10 minutes at 95 degrees C. to yield Sample C.
  • Samples A, B and C were then subjected to LC/MS analysis (see Materials & Methods section) to determine the size distribution of major peptides present. From all hydrolysates at least 124 different peptides were analysed. The data obtained are shown underneath.
  • Nutramigen (Mead Johnson, containing 14 grams of casein hydrolysate per 100 gram powder) contains the highest (i.e. 22%) molar fraction of peptides carrying C-terminal proline.
  • Nutramigen Mead Johnson, containing 14 grams of casein hydrolysate per 100 gram powder
  • the molar fraction of casein derived di- to heptapeptides as present in Nutramigen accounts for 83% of all peptides detected. Furthermore the molar fraction of di- and tripeptides as present amongst all peptides detected in Nutramigen could be shown to amount to 18%. Among the tripeptides identified, a molar fraction of 23% could be shown to have a carboxyterminal proline residue.
  • the protein hydrolysate used represents a product which has probably been highly purified and selectively enriched by a number of techniques such as ultrafiltration and chromatography, the hydrolysate exhibits a low level of carboxyterminal proline residues which implies considerable bitterness and a limited fraction of protease resistant tripeptides only.
  • the reaction was stopped by lowering the pH to 5.5 using lactic acid followed by a heat treatment of 10 minutes at 90 degrees C. Then the mixture was cooled down to 50 degrees C. and a sample was taken for LC/MS/MS analysis.
  • a subsequent incubation with EndoPro was carried out by adding a chromatographically purified solution of the overproduced proline specific endoprotease from A. niger in a concentration of 20 units/gram protein. After incubating for 2 hours at 50 degrees C. under non-pH-stat conditions the EndoPro enzyme was inactivated by another heat treatment to yield another sample for LC/MS/MS analysis.
  • chromatographically purified TPAP-A (see Example 1) was added in a concentration of 4 units per gram substrate and the incubation was continued for 2 hours at 60 degrees C. and then inactivated by heating to yield another LC/MS/MS sample. Subsequent incubations were carried out on beta-casein without Delvolase using EndoPro and TPAP, either alone or in combination, under the above described conditions. The latter samples were also subjected to LC/MS/MS analysis. The data obtained are shown underneath.
  • Tripeptides Enzymes used to of (molar % of all (molar % of all (molar % of all (molar % of all prepare beta-casein peptides peptides peptides tripeptides hydrolysate analysed analysed) analysed) analysed) Subtilisin 93 0 12 6 Subtilisin + EndoPro 68 41 34 25 Subtilisin 69 36 45 36 +EndoPro + TPAP-A EndoPro 55 49 11 11 TPAP-A 1 0 100 100 EndoPro + TPAP-A 68 40 43 40
  • TPAP-B tripeptidylaminopeptidase TPAP-B (corresponding with the enzyme encoded by gene 10 as described in our copending application PCT/EP02/01984 and overproduced in an A. niger host cell).
  • enzyme TPAP-A the overproduced and secreted enzyme TPAP-B was first chromatogaphically purified; in this case on a Q sepharose FF colum (AA1188, Pharmadia) equilibrated in 20 millimol/l Bis Tris buffer, pH 5.5 and eluted with a gradient containing 1 mol/l NaCl in the same buffer.
  • the reaction was started by adding 200 microliter of the chromatographically purified tripeptidase to each well. Extinction development was followed at 405 nm using a Tecan Genios micro titre plate reader. Efficiencies of both enzymes towards the various substrates are presented underneath in which the activity towards the Ala-Ala-Ala-pNA substrate was used as the 100% value.
  • the various letters on the X-axis in the figure underneath refer to the international one letter symbols used to specify the amino acid residue “X’ in the Ala-Ala-X-pNA substrate.
  • proline-rich protein substrates such as caseins or glutens or collagen-based compounds
  • a proline-specific endoprotease and a tripeptidylpeptidase has been adequately demonstrated in the previous Examples.
  • the enzyme combination is also beneficially used in the hydrolysis of substrates with lower proline contents.
  • a crude lactalbumin fraction from bovine milk (Sigma) was suspended in water in a concentration of 20 grams/liter after which the pH was adjusted to 8.0.
  • the serine protease subtilisin (Delvolase) was added to a concentration of 4% (volume of the commercial enzyme product per weight of the substrate) and the mixture was incubated for 2 hours at 60 degrees C. under non-pH-stat conditions. Then the pH of the suspension was lowered to pH to 4.5 using citric acid and divided into 4 portions. One portion was heated to inactivate the Delvolase enzyme and then kept frozen until LC/MS/MS analysis.
  • chromatographically purified Endopro enzyme was added (1 unit/gram lactalbumin) or tripeptidylaminopeptidase (TPAP-A; 20 units/gram of lactalbumin) or a combination of EndoPro and TPAP-A (1 unit+20 units/gram lactalbumin; see Materials&Methods for unit definitions).
  • TPAP-A tripeptidylaminopeptidase
  • TPAP-A a combination of EndoPro and TPAP-A
  • LC/MS/MS data obtained are shown in FIG. 2 peptide length in amino acid residues is depicted on the X-axis and on the Y-axis the number of peptides analysed. Even without a recalculation of peptides of a specific length into percentages of the total peptides analysed, the benefits of an incubation with either an proline-specific endopeptidase or a tripeptidase or a combination of these two enzymes, become visible. Together with the results provided in the previous Examples the data obtained clearly demonstrate that the combination of a proline-specific endoprotease with a tripeptidase provides superior hydrolysates, be it on proline-rich or on other proteinaceous substrates.

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