Process to produce a tryptophan-enriched lysozyme hydrolysate
09770493 · 2017-09-26
Assignee
Inventors
Cpc classification
A61P25/28
HUMAN NECESSITIES
C12P21/06
CHEMISTRY; METALLURGY
A61K31/405
HUMAN NECESSITIES
A61K38/47
HUMAN NECESSITIES
International classification
A61K31/405
HUMAN NECESSITIES
C12P21/06
CHEMISTRY; METALLURGY
A61K38/47
HUMAN NECESSITIES
Abstract
The present disclosure relates to a composition, and method thereof, which comprises tryptophan whereby 10 to 90%, preferably 20 to 80% of the tryptophan is present as free tryptophan or peptide-bound tryptophan and 10 to 90%, preferably 20 to 80% of the tryptophan is present as polypeptide-bound tryptophan.
Claims
1. A process to produce a tryptophan-enriched lysozyme hydrolysate comprising the steps of enzymatically hydrolyzing lysozyme under alkaline conditions to prepare hydrolysate having a degree of hydrolysis (DH) of between 5 and 45, wherein the resulting hydrolysate a) has a tryptophan yield of more than 30% on protein tryptophan bases, and b) is a water soluble peptide composition comprising more than 50 molar % di- and tri-peptides; c) has a level of free tryptophan less than 1% of the total tryptophan present; and enriching the hydrolysate for tryptophan-containing peptides.
2. The process according to claim 1, wherein the lysozyme is hen egg lysozyme.
3. The process according to claim 1, wherein the hydrolysate is enriched for tryptophan-containing peptides by ion exchange chromatography.
4. The process according to claim 2, wherein the hydrolysate is enriched for tryptophan-containing peptides by ion exchange chromatography.
5. The process according to claim 3, wherein the ion exchange chromatography is conducted at a pH of about 3.
6. The process according to claim 4, wherein the ion exchange chromatography is conducted at a pH of about 3.
Description
LEGENDS TO THE FIGURES
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(10) Lane 1: lysozyme as such; lane 2: lysozyme after pepsin digestion; lane 3: whey protein as such; lane 4: whey protein after pepsin digestion; lane 5: pepsin as such.
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MATERIALS AND METHODS
(12) Materials
(13) Subtilisin under the commercial name of “Protex 6L” was obtained from Genencor (Leiden, The Netherlands), pepsin from Sigma and the mixture of trypsin/chymotrypsin (Porcine PEM) from Novozymes (Bagsvaerd, Denmark). Lysozyme was obtained either as Delvozyme L (22% dry matter) or as the dry Delvozyme G granulate from DSM Food Specialties (Delft, The Netherlands).
(14) Casein hydrolysate (“REF”) was obtained essentially as described by Edens et al (J Agric Food Chem, 53(20)7950-7957, 2005). Sodium caseinate was extensively hydrolysed with Protex 6L and, after lowering the pH to 4.5, with a proline specific endoprotease to reach a DH>20%. Following ultrafiltration, the permeate was heat treated to inactivate any remaining enzymatic activities and finally spray dried. Intact alpha-lactalbumin (“ALAC”) was obtained as “Biopure” (>90% alpha-lactalbumin) from Davisco Foods International, Inc. (Le Seuer, Minn.); tryptophan-enriched lysozyme hydrolysate (“WEPS”) was obtained as described in Example 4; the synthetic Ser-Trp dipeptide (“SYN”) was obtained as described in Example 5; pure L-tryptophan (“TRP”) was obtained as L-tryptophan-400 from Orthica, Almere, The Netherlands.
(15) Test for Protease Resistancy of Tryptophan-Containing Polypeptides, Especially Intact Proteins
(16) To test its digestibility in the human stomach, a 5% (w/w) solution of the intact protein was incubated with pepsin (Sigma; 1% w/w pepsin to intact protein) for 2 hours at 37 degrees C. in a Mc Ilvane buffer (0.2 M citric acid plus Na2HPO4) pH 4.0. The degree of protease resistancy is defined as the percentage of the protein that is not affected by the pepsin incubation. “Not affected” meaning that the molecular weight of the protein has not changed as a result of the pepsin incubation; “percentage of the protein” meaning the area under the curve after digestion times 100, divided by the area under the curve prior to digestion; “area under the curve” is the area of the protein having the initial molecular weight as provided by the quantitative analysis method used (see further).
(17) Molecular weights are compared according to SDS-PAGE followed by staining according to the protocol specified hereunder. After staining of the gel, a digital image is prepared using the OptiGo imaging system (Isogen Life Science; www.isogen-life-science.com) followed by the quantitative analysis of selected protein bands using Totallab TL 100, version 2006 software (Nonlinear Dynamics Ltd; www.nonlinear.com) running under Windows XP. A protein is protease resistant according to the present text if more than 50% of the protein with the original molecular weight is still present after pepsin incubation.
(18) SDS-PAGE
(19) The purity of the lysozyme preparations used was checked by SDS-PAGE. All materials used for SDS-PAGE and staining were purchased from Invitrogen (Carlsbad, Calif., US). Samples were prepared using SDS buffer according to manufacturers instructions and separated on 12% Bis-Tris gels using MES-SDS buffer system according to manufacturers instructions. Staining was performed using Simply Blue Safe Stain (Collodial Coomassie G250). Prior to hydrolysis the lysozyme appeared as a single band with a molecular weight of approx. 14 kDa on the gel.
(20) LC/MS/MS Analysis
(21) HPLC using an ion trap mass spectrometer (Thermo Electron, Breda, the Netherlands) coupled to a P4000 pump (Thermo Electron, Breda, the Netherlands) was used to determine the presence of tryptophan containing peptides (mainly di- and tri peptides) in the enzymatic protein hydrolysates produced by the process according to the invention. The peptides formed were separated using an Inertsil 3 ODS 3, 3 μm, 150*2.1 mm column (Varian Belgium, Belgium) in combination with a 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) for elution. The gradient started at 100% of Solution A, kept here for 10 minutes, increasing linear to 20% B in 25 minutes and immediately going to the starting conditions, and kept here 15 minutes for stabilization. The injection volume used was 50 microliters, the flow rate was 200 microliter per minute and the column temperature was maintained at 55° C. The protein concentration of the injected sample was approx. 50 micrograms/milliliter. Identification of the peptides of interest is based on the retention time, protonated molecule and by using dedicated MS/MS for the peptides of interest, using optimal collision energy of about 30%. Quantification of specific tryptophan containing peptides is performed by using an external standard method.
(22) The tetra peptide VVPP (M=410.2) was used to tune for optimal sensitivity in MS mode and for optimal fragmentation in MS/MS mode, performing constant infusion of 5 μg/ml, resulting in a protonated molecule in MS mode, and an optimal collision energy of about 30% in MS/MS mode, generating a B- and Y-ion series.
(23) Prior to LC/MS/MS the enzymatic protein hydrolysates were centrifuged at ambient temperature and 13000 rpm for 10 minutes and the supernatant was diluted 1:100 with demineralised water filtered through Millipore water filtration equipment (MilliQ water).
(24) Amino Acid Analyses
(25) The amino acid profiles in plasma were analyzed by HPLC according to van Eijk et al (J. Chromatogr. 1993: 620:143-148) as described in Example 6 or Example 11.
(26) Other amino acid analyses were carried out 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 samples were dried and directly derivatised using phenylisothiocyanate. The derivatised amino acids present were quantitated using HPLC methods as described. As during the usual acid hydrolysis Trp and Cys are destroyed, special methods were used to quantitate these two amino acids. To prevent Cys degradation during hydrolysis, this amino acid is first oxidized to cysteic acid using hydrogen peroxide and then quantitated. The analysis of tryptophan is based on a slightly modified Waters procedure. In this procedure an aliquot of the peptide solution is dried under vacuum and then hydrolysed during 1 hour at 150 degrees C. under nitrogen in 4M methane sulphonic acid containing 0.2% tryptamine. The reaction product is directly quantitated using HPLC equipped with an Alltech Altima C18 column and fluorescence detection.
(27) Degree of Hydrolysis
(28) The Degree of Hydrolysis (DH) as obtained during incubation with the various proteolytic mixtures was monitored using a rapid OPA test (Nielsen, P. M.; Petersen, D.; Dambmann, C. Improved method for determining food protein degree of hydrolysis. Journal of Food Science 2001, 66, 642-646).
(29) Kieldahl Nitrogen
(30) Total Kjeldahl Nitrogen was measured by Flow Injection Analysis. Using a Tecator FIASTAR 5000 Flow Injection System equipped with a TKN Method Cassette 5000-040, a Pentium 4 computer with SOFIA software and a Tecator 5027 Autosampler the ammonia released from protein containing solutions was quantitated at 590 nm. A sample amount corresponding with the dynamic range of the method (0.5-20 mg Nil) was placed in the digestion tube together with 95-97% sulphuric acid and a Kjeltab subjected to a digestion program of 30 minutes at 200 degrees C. followed by 90 minutes at 360 degrees C. After injection in the FIASTAR 5000 system the nitrogen peak is measured from which the amount of protein measured can be inferred.
(31) Molecular Weight Distribution of Peptides and Proteins Present in Hydrolysates.
(32) Analysis of the peptide size distribution of protease treated protein samples was done on an automated HPLC system equipped with a high pressure pump, an injection device able to inject 10-100 microliter sample and a UV detector able to monitor the column effluent at 214 nm.
(33) The column used for this analysis was a Superdex Peptide HR 10/300 GL (Amersham) equilibrated with 20 mM Sodium Phosphate/250 mM Sodium Chloride pH 7.0 buffer. After injecting a sample (typically 50 microliter) the various components were eluted from the column with buffer in 90 min at a flow rate of 0.5 ml/min. The system was calibrated using a mixture of cytochrome C (Mw 13 500 Da), aprotinin (Mw 6510 Da) and tetra-glycine (Mw 246 Da) as molecular weight markers.
(34) The following Examples illustrate the invention further.
EXAMPLES
Example 1
Hen Egg Lysozyme is not Cleaved by Either Pepsin or Trypsin/Chymotrypsin
(35) To test its digestibility in the human gastrointestinal tract, hen egg lysozyme was incubated in vitro with pepsin as well as with a mixture of trypsin and chymotrypsin. Both incubations were carried out under pH conditions that are prevalent in the stomach (pepsin) and duodenum (trypsin/chymotrypsin). To that end, a 5% (w/w) lysozyme solution was incubated with the enzymes (1% w/w enzyme to lysozyme protein) for 2 hours at 37 degrees C. To prevent major pH changes as the result of the ongoing protein hydrolysis, incubation was carried out in a Mc Ilvane buffer (0.2 M citric acid plus Na2HPO4). The low DH's values that are obtained after the two hours hydrolysis at 37 degrees C. (see Table 1), demonstrate that the lysozyme molecule cannot be degraded under conditions that mimic digestion conditions in the stomach and in the duodenum and jejunum because successful proteolysis can be expected to lead to a DH value of at least 10%. Therefore, tryptophan residues present in the intact hen egg lysozyme molecule will not be liberated in the gastro-intestinal tract hereby implying that tryptophan molecules present in intact hen egg lysozyme cannot contribute to plasma tryptophan levels shortly after consumption.
(36) TABLE-US-00001 TABLE 1 Lysozyme hydrolysis by pepsin and a trypsin/chymotrypsin mixture Enzyme pH start pH end DH start (%) DH end (%) Pepsin 2.8 2.4 = 0 2.4 Pepsin 3.6 3.2 <1 Pepsin 4.6 4.3 1.0 Trypsin/chymotrypsin 4.6 4.3 <1 Trypsin/chymotrypsin 5.9 5.5 <1 Trypsin/chymotrypsin 7.2 7.0 1.3
Example 2
Hen Egg Lysozyme is Efficiently Cleaved by Subtilisin at Elevated pH Values
(37) To test the susceptibility of lysozyme to enzyme hydrolysis under non-physiological pH and enzyme conditions, a lysozyme solution was incubated in vitro with a microbial subtilisin (EC 3.4.21.62) under alkaline pH conditions. To that end, a 5% (w/w) lysozyme solution was incubated at pH 7.0, 8.0 and 9.0 with 12.5 microliter of Protex 6L. per gram lysozyme protein present. The incubation was carried out for 3 hours at 60 degrees C. with a constant adjustment of the pH using 1M NaOH. The incubations yielded slightly turbid solutions without any significant precipitates. After a heating step to inactivate the subtilisin activity, the DH values of the various incubations were measured according to the protocol described in the Materials & Methods section. In contrast with the results obtained under physiological conditions (see Example 1), alkaline incubation conditions using subtilisin result in complete lysozyme hydrolysis. The pH 7.0 incubation yielded a DH of 6.3, the pH 8.0 incubation a DH of 11.2 and the pH 9.0 incubation a DH of 16.4. A subsequent SDS-PAGE analysis of the reaction products, indicated that the whole lysozyme molecule was degraded i.e. no large molecular weight fragments survived the subtilisin incubation. Furthermore, HPLC analysis of the hydrolysate on a Crownpak CR+ column (Daicel) revealed that no significant racemisation of tryptophan containing peptides took place, not even after prolonged heating at pH 9.0.
Example 3
Hydrolysing Lysozyme Using Protex and Identity of the Peptides Formed
(38) A solution containing 10% (w/w) pure lysozyme was adjusted to pH 8.2 using NaOH and heated to 52 degrees C. Hydrolysis was started by adding 25 microliter of Protex/g of protein present. Under continuous stirring and maintaining the pH at 8.2, the hydrolysis was continued for 5.5 hours to yield an almost clear solution without a visible precipitate. After a heating step to inactivate the Protex activity, a sample was taken for DH analysis. The DH of the solution turned out to be almost 30%. The heat treated solution was ultrafiltered over a 10 kDa filter to yield a completely clear liquid. This clear liquid was used for LC/MS analysis, for molecular weight distribution of peptides and proteins present as well as for ion exchange chromatography.
(39) To get an impression of the molecular weight distribution of peptides and proteins present, the clear liquid was subjected to a molecular size analysis as described in the Materials & Methods section. The results obtained (see
(40) LC/MS analysis was carried out according to the procedure as described in the Materials & Methods section. By selecting for those peptides containing a tryptophan (“W”), peptides AW, GNW, WIR, NAW, WVA, VAW, AWR, SLGNW and minor quantities of WW and SRWW could be detected. The level of free tryptophan in the hydrolysate after incubation was established to represent less than 1% of the total (lysozyme) tryptophan present.
(41) As di- and tripeptides are readily absorbed by peptide transporters present in the intestinal wall, there is little doubt that tryptophan residues present in such peptides will be rapidly absorbed and lead to increased plasma tryptophan levels upon oral intake of the present lysozyme hydrolysate.
Example 4
Increasing the Tryptophan Content of the Hydrolysate
(42) Lysozyme incorporates a surprising high amount of the basic arginine and lysine residues. Furthermore the lysozyme molecule incorporates a significant number of the acid glutamate and aspartate residues. This data has been used to devise an innovative and elegant purification route towards hydrolysates featuring high Trp/LNAA ratios. Essential requirement for this purification route is, however, that only very few of the tryptophan residues show up in peptides also containing either an arginine or lysine residue or a glutamate or aspartate residue. As shown in Example 3, the specific hydrolysis route used here yields only few tryptophan containing peptides containing an arginine residue and no peptides containing a lysine, glutamate or aspartate residue. Theory predicts that a maximal charge difference between peptides with and without a glutamate or aspartate residue can be achieved around pH 3. A maximal charge difference between peptides with and without an arginine or lysine residue, can be achieved around pH 5.
(43) To illustrate the selective power of this approach, a lysozyme hydrolysate was prepared according to the procedure specified in Example 3. Then, the pH of the hydrolysate was adjusted to pH 3.1 using acetic acid and approximately 0.5 gram of protein was applied to a 15 ml bed volume of SP Sepharose FF (GE Healthcare, Diegem, Belgium) column equilibrated with 20 mm sodium citrate pH 3.1. After washing the column with one column volume of the sodium citrate buffer to remove the majority of the peptides incorporating a glutamate or aspartate, the elution buffer was changed to a 20 mm sodium citrate buffer pH 5.1. During washing of the column with three column volumes of the latter buffer, a range of tryptophan containing peptides was eluted. According to LC/MS analysis, the dipeptide AW was present in large amounts as well as the tripeptides GNW, NAW, WVA, VAW and a small amount of the pentapeptide SLGNW. Amino acid analysis of the various pH 5.1 fractions showed that selective pooling yielded a solution having a molecular Trp/LNAA ratio of 1.75 and a tryptophan yield of almost 30%. A less selective pooling yielded a solution with a molecular Trp/LNAA ratio of 0.4 and a tryptophan yield of 70%. Subsequently, the column was washed with three column volumes 20 mM sodium citrate pH 7.1. According to the LC/MS data, this step eluted arginine containing peptides WIR, AWIR and, surprisingly, peptide WW. A final washing of the column with 1 M of NaOH, water and 1M of acetic acid prepared the column for a next run.
Example 5
Chemical Synthesis of Dipeptide Ser-Trp
(44) The dipeptide Ser-Trp was synthesized according to standard peptide technology. In a first step Z-Ser-OH and Trp-OMe were coupled via the carbonic anhydride methodology (J. Am. Chem. Soc. 1967, 5012) to yield the protected dipeptide Z-Ser-Trp-OMe. To that end Trp-OMe.HCl was suspended in tetrahydrofuran (THF) and subsequently N-methylmorpholine (NMM) was added. The mixture was stirred for one hour and subsequently added to a solution of Z-Ser in tetrahydrofuran/dimethylformamide (THF/DMF). A second equivalent of NMM was added and the mixture was cooled to −15° C. Isobutyl chloroformate is added at such a rate that the internal temperature does not exceed −15° C. Subsequently, the mixture was stirred for 3 hours, the temperature was allowed to rise to ambient temperature and the precipitated NMM.HCl was removed by filtration. The filtrate was kept at 4° C. overnight after which any additional precipitate was filtered and the filtrate is concentrated in vacuo. The residue was purified by column chromatography (SiO.sub.2, ethyl acetate/heptane). The combined fractions were concentrated, washed with water to remove any remaining DMF and concentrated in vacuo.
(45) In a second step, enzymatic hydrolysis of Z-Ser-Trp-OMe was accomplished using Alcalase 2.5L DX (Int. J. Peptide Protein Res. 1990, 52) and subsequent catalytic hydrogenolysis provided the desired peptide as an off-white solid. To that end the purified Z-Ser-Trp-OMe was dissolved in tBuOH and water and Alcalase 2.5 L DX (Novozymes, Bagsvaerd, Denmark) was added. The mixture was stirred until (almost) all starting material was consumed. The mixture was then concentrated in vacuo and the residue taken up in water of pH 7. The aqueous mixture was extracted with ethyl acetate to remove any remaining starting material and subsequently the aqueous phase was acidified. The desired product, i.e. Z-Ser-Trp-OH, was isolated by extraction with ethyl acetate; the extract was dried over sodium sulphate and concentrated in vacuo.
(46) In a third step, dipeptide Ser-Trp-OH was obtained. To that end the concentrated Z-Ser-Trp-OH was dissolved in MeOH and water (1:1), Pd/C was added and the mixture was stirred under a positive hydrogen atmosphere (5 bar). Upon completion of the reaction, the catalyst plus the majority of the product was removed by filtration and the filtrate discarded. The filter was washed extensively with milliQ water and the filtrate concentrated in vacuo, yielding the dipeptide Ser-Trp-OH as a white to off-white solid. Additional purification was achieved by stirring the product in a mixture of acetone-water and isolation of the peptide by filtration. This yielded a product suitable for oral consumption.
Example 6
Effects of Different Tryptophan Sources on Plasma Trp/LNAA Ratios and Mood in Healthy Volunteers
(47) The aim of the present study was to investigate in healthy volunteers plasma Trp/LNAA profiles and mood after the consumption of different tryptophan containing preparations. The following preparations were tested: intact alpha-lactalbumin (see Materials & Methods) hydrolyzed caseinate (DH>20%; see Materials & Methods) a Trp-enriched lysozyme hydrolysate with a high Trp/LNAA ratio (see Example 4) a synthetic SW dipeptide (Example 5) free L-tryptophan (see Materials & Methods).
(48) Eighteen healthy students (9 males and 9 females: age between 18-30 years) participated in the study. Exclusion criteria for participation were chronic and current illness, history of psychiatric or medical illness, use of medication or drugs, alcohol consumption (>2 units/day), metabolic-, hormonal- or intestinal diseases and irregular diets or deviant eating habits (assessed by health and life-style questionnaires). Subjects participating in the experiment were in the normal range for the Body-Mass Index (BMI in kg/m.sup.2 between 20-25) and female subjects are matched for contraception. Women participated during their mid-late follicular phase (day 4-10), while women using contraception participated when they actually used the contraception pill. Participants were non-smokers and did not use any alcohol before and during the study. All subjects participating in the experiment signed an Informed Consent Form. This study was conducted according to the EC principles of Good Clinical Practice (GCP) adopted by the 52.sup.nd WMA General Assembly, Edinburgh, Scotland, October 2000.
(49) Subjects were instructed to fast overnight; only water or tea without sugar was permitted. During five experimental morning sessions, subjects visited the laboratory to monitor plasma Trp/LNAA concentrations and mood following the intake of a drink containing different Trp or LNAA concentrations. The order of presentation of the various drinks was counterbalanced and the four experimental days were separated by a one-week period. On each experimental morning, a 312 ml drink was provided containing different tryptophan (Trp) or LNAA concentrations (Table 2). All drinks contained 0.10 g sweetener (acesulfame) and were filled up with plain water in order to reach 312 mL. A research assistant blind to the dietary conditions conducted the administration of the different drinks.
(50) TABLE-US-00002 TABLE 2 Protein/amino acid composition of drinks used Trp- enhanced Protein Casein Intact lysozyme source Hydrol. Alpha-lac hydrol. Ser-Trp Free L-Trp Code REF ALAC WEPS SYN TRP used grams 20 15 300 ml 1.20 0.82 solution Trp (g) 0.40 0.80 0.80 0.80 0.80 Trp/LNAA 0.04 0.10 1.1 ∞ ∞ (molar)
(51) Blood samples were collected in duplicate before and 15, 30, 60, 90, 120, 180 and 210 minutes after ingestion in 5 ml vacutainer tubes containing sodium heparine and were then centrifuged at 5000 rpm for 5 min at 4° C. The resulting supernatants were mixed with sulfasalicyl acid (4 mg/100 microliter) and directly stored at −80° C. until analysis. Plasma amino acid analysis was conducted with HPLC, making use of a 2-3 μm Bischof Spherisorb ODS II column as described by van Eijk et al (J. Chromatogr. 1993: 620: 143-148). The plasma Trp/LNAA ratios were calculated by dividing the plasma molar tryptophan concentration by the sum of the plasma molar concentrations of the large neutral amino acids valine, isoleucine, leucine, tyrosine and phenylalanine. Statistical analysis took place by means of repeated measures multivariate and univariate analyses of variance (MANOVA and ANOVA) using the General Linear Model (GLM: SPSS 12.0 for Windows). All statistics were evaluated at a significance level of P=0.05.
(52) Plasma Trp/LNAA Values
(53) A first repeated measures analysis of variance with Condition and Time as within-subjects factors on the plasma Trp/LNAA ratio revealed a main significant effect of Time and Condition and a significant interaction Condition× Time. The highest significant increases in plasma Trp/LNAA ratio were found (see
(54) The 255% rise in Trp/LNAA as found with “WEPS” considerably exceeds the 50-70% increases as previously found with intact alpha-lactalbumin (Markus et al., 2000; Booij et al., 2006) and all earlier reported 20-45% increases with other foods like carbohydrates (Markus, 2003). While a 40-50% variation in plasma Trp/LNAA is thought to be sufficient to change Trp levels and 5-HT synthesis and release in the brain (Markus et al., 2000), this 255% rise is expected to cause a much larger rise in available brain Trp and 5-HT and therefore may also result in a greater release of functionally active brain 5-HT.
(55) Profile of Mood States (POMS).
(56) Mood changes of the various participants were measured using a paper-and-pencil version of the Dutch shortened version of the Profile of Mood States questionnaire (Wald and Mellenbergh, Ned Tijdschr Psycho) 1990: 45: 86-90) as a VAS scale ranging from ‘strongly disagree’ to ‘strongly agree’. The POMS comprises five different subscales for mood; ranging from Anger, Depression, Fatigue and Tension that refer to a negative mood state, to Vigor concerning a positive mood.
(57) Repeated measures analysis of variance with Condition and Time as within-subjects factors on the total mood scores revealed a significant effect of Time and a significant interaction of Condition×Time; indicating that mood changes across time significantly differed between conditions. Comparable improvements of mood were found 60 min after the intake of “WEPS” and “TRP”, but only with “WEPS” mood further improved until 210 min after intake as compared with “TRP”. In contrast, no mood changes were found after the intake of “REF” and “ALAC”. The absence of a mood effect after intact alpha-lactalbumin is comparable with previous studies showing mild beneficial effects on mood after intact alpha-lactalbumin and only in stress-vulnerable subjects under acute stress exposure (Markus et al., 2000; Markus et al., 2000, Markus, 2003). Although mood also seemed to improve after intake “SYN”, this effect was not significant in this experimental set up.
(58) These current results suggest that a large 255% increase in plasma Trp/LNAA may be sufficient for an improved mood in normal non-stress-vulnerable subjects. Based on previous findings it is expected that these beneficial effects of the Trp-enhanced lysozyme hydrolysate on mood will be even greater in stress-vulnerable subjects under high mental stress conditions (Markus, 2003). Contrary to our expectations, there were no significant improvements in mood after intake of the synthetic dipeptide. This unexpected result may be attributable to the current experimental set up or to differences in tryptophan bioavailability from these various sources.
(59) TABLE-US-00003 TABLE 3 Changes in plasma amino acid concentrations (μmol/l) in time after ingestion of casein hydrolysate (“REF”), intact alpha-lactalbumin (“ALAC”) or Trp-enhanced lysozyme hydrolysate (“WEPS”). Amino Con- Time (min) acid dition 0 30 60 90 120 180 210 Isoleucine REF 0.07 0.10 0.18 0.15 0.12 0.09 0.08 ALAC 0.08 0.12 0.20 0.22 0.18 0.12 0.11 WEPS 0.07 0.09 0.09 0.14 0.09 0.08 0.09 Leucine REF 0.12 0.19 0.31 0.26 0.22 0.17 0.16 ALAC 0.13 0.22 0.37 0.38 0.28 0.21 0.20 WEPS 0.13 0.14 0.14 0.13 0.13 0.13 0.14 Phenyl- REF 0.06 0.08 0.10 0.08 0.08 0.06 0.06 alanine ALAC 0.07 0.09 0.11 0.10 0.09 0.07 0.07 WEPS 0.07 0.07 0.07 0.06 0.10 0.06 0.07 Tyrosine REF 0.06 0.07 0.12 0.11 0.09 0.07 0.07 ALAC 0.06 0.08 0.12 0.12 0.10 0.08 0.08 WEPS 0.06 0.07 0.07 0.06 0.06 0.06 0.06 Valine REF 0.24 0.28 0.45 0.42 0.38 0.32 0.30 ALAC 0.26 0.30 0.38 0.42 0.35 0.29 0.28 WEPS 0.26 0.27 0.25 0.25 0.25 0.25 0.26 Tryp- REF 0.06 0.07 0.08 0.08 0.07 0.06 0.05 tophan ALAC 0.07 0.09 0.18 0.23 0.19 0.13 0.12 WEPS 0.07 0.13 0.21 0.23 0.20 0.14 0.13 LNAA REF 0.52 0.67 1.14 1.01 0.86 0.73 0.65 ALAC 0.60 0.82 1.14 1.22 1.10 0.86 0.82 WEPS 0.62 0.60 0.65 0.60 0.68 0.55 0.64 Trp/ REF 0.11 0.09 0.08 0.08 0.08 0.08 0.08 LNAA ALAC 0.12 0.12 0.15 0.18 0.2 0.18 0.17 WEPS 0.11 0.19 0.36 0.39 0.35 0.25 0.22
(60) TABLE-US-00004 TABLE 4 Changes in plasma amino acid concentrations (μmol/l) in time after ingestion of free L-Trp (“TRP”) or the synthetic dipeptide SW (“SYN”). Amino Con- Time (min) acid dition 0 30 60 90 120 180 210 Isoleucine TRP 0.07 0.07 0.07 0.06 0.07 0.07 0.07 SYN 0.06 0.07 0.06 0.06 0.06 0.06 0.07 Leucine TRP 0.13 0.13 0.12 0.12 0.12 0.12 0.13 SYN 0.11 0.14 0.12 0.12 0.12 0.12 0.13 Phenyl- TRP 0.07 0.07 0.06 0.06 0.06 0.06 0.06 alanine SYN 0.06 0.07 0.06 0.06 0.06 0.06 0.06 Tyrosine TRP 0.06 0.06 0.06 0.05 0.06 0.05 0.05 SYN 0.05 0.06 0.05 0.05 0.05 0.05 0.05 Valine TRP 0.25 0.25 0.23 0.22 0.23 0.22 0.23 SYN 0.21 0.26 0.22 0.22 0.22 0.21 0.23 Tryp- TRP 0.07 0.07 0.17 0.18 0.18 0.13 0.11 tophan SYN 0.06 0.13 0.21 0.18 0.15 0.11 0.10 LNAA TRP 0.62 0.59 0.55 0.50 0.58 0.52 0.53 SYN 0.50 0.58 0.48 0.47 0.45 0.48 0.54 Trp/ TRP 0.11 0.12 0.29 0.31 0.32 0.24 0.20 LNAA SYN 0.11 0.22 0.40 0.37 0.31 0.22 0.19
Example 7
Large Scale Lysozyme Hydrolysis
(61) In larger scale lysozyme hydrolysis procedures, essentially the procedure as described in Example 3 was followed with some minor modifications. A solution containing 7.3% (w/w) pure lysozyme was heated to 65 degrees C. after which the pH was adjusted to pH 8.2 using NaOH. Hydrolysis was started by adding 25 microliter of Protex 6 L/g dry matter. Under continuous stirring and maintaining the pH at 8.2 and the temperature at 53 degrees C., the hydrolysis was continued for 2 hours. Then the pH value was increased to 9.0 and incubation was pursued for another 3.5 hours to yield a solution with some precipitate. Then the pH of the solution was lowered to 4.5 and the solution was cooled to below 4 degrees C. To obtain a completely clear product, the liquid was filtered over a Z 2000 filter (Pall) and subsequently excess water and salt was removed via nanofiltration. The resulting concentrate was then subjected to an UHT treatment of 7 seconds at 120 degrees C., evaporated and finally spray dried to obtain the lysozyme hydrolysate in a dry form. The product thus obtained has a molar Trp/LNAA ratio of about 0.19.
Example 8
Preparing a Beverage Incorporating the Lysozyme Hydrolysate
(62) The following recipe illustrates the preparation of an fat-free, lysozyme hydrolysate containing strawberry drink. To 10 grams of lysozyme hydrolysate powder (prepared according to Example 7), 40 grams of glucose, 2.4 grams of citric acid, 0.38 grams of malic acid, 0.15 grams of sucralose and 0.5 grams of strawberry flavor (Buteressence, Zaandam, The Netherlands) were added. This mixture of powders readily dissolves in 1 liter of water to obtain a ready-to-drink beverage with a high Trp/LNAA and a high Tyr/BCAA ratio. The powder mixture is suitable for e.g. sachet filling. Packaged liquid products can be produced using various known technologies.
Example 9
Effects of Lysozyme Hydrolysate on Post-Stress Performance in Stress-Susceptible and Stress-Resistant Healthy Volunteers
(63) The aim of the present study was to compare the effects of a lysozyme hydrolysate prepared according to the procedure described in Example 7, with a placebo (casein protein hydrolysate; see Example 6) in terms of plasma Trp/LNAA levels and its consequences on post-stress performance tasks. The performance tests used are known to address “vigilance” and “eye-motor control” aspects of individuals.
(64) Forty individuals, of which twenty males and twenty females, participated in the present study. Based on a pre-study questionnaire, one half of this group was classified as stress-resistant, the other half as stress-susceptible. The in- and exclusion criteria for the individuals as well as the general study conduct, were the same as described in Example 6. A flow diagram of the design of the study is given in
(65) On the experimental mornings, subjects arrived fasted at the laboratory. Upon arrival, they were given either a drink containing the lysozyme hydrolysate, or the placebo i.e. the drink containing the casein hydrolysate. The composition of test drink and placebo drink is outlined in Table 5.
(66) TABLE-US-00005 TABLE 5 Composition of drinks used. Protein source Casein hydrolysate Lysozyme hydrolysate abbreviation plc Trp-hydr g powder/300 ml 13.6 14.4 Water 286 g 285 g Sweetener 0.1 g 0.1 g g Trp/300 ml 0.4 0.8 Trp/LNAA ratio 0.04 0.19 (molar)
Ninety minutes after consumption of the 300 ml drinks, a blood sample was taken to assess Trp/LNAA ratios (see Example 6). Subsequently, either the group of stress-resistant or the group of stress-prone subjects was exposed to a performance test followed by exposure to a stress. This stress consisted of an arithmetic task that had to be performed under noise stimulation. Subjects were led to believe that the presence or absence of the noise was depended on their performance in the test. In reality, the arithmetic tasks were manipulated in such a way that all subjects failed each trial. This set up is known to induce psychological stress and is perceived as highly uncontrollable (Peters, M. L., Godaert, G. L. R., Ballieux, R. E. et al. (1998). Cardiovascular and catecholamine response to experimental stress: effects of mental effort and controllability. Psychoneuroendocrinology. 23, 1-17). After the arithmetic task, the first performance test was repeated to quantify the effect of the stress on the performance under the influence of the blood Trp/LNAA ratios in force.
(67) The performance tests carried out were the Mackworth Clock test (Mackworth, N (1948) The breakdown of vigilance during prolonged visual search. Quart J Exp Psych. 1, 6-21)) and the Critical Tracking Task (Jex H R et al., (1966) A “critical” tracking task for man-machine research related to the operator's effective delay time. NASA Contract Rep NASA CR.:1-105).
(68) The Mackworth Clock Test is an extensively used test to measure “vigilance”, alertness and concentration over a sustained period of time. Subjects are seated in front of a computer screen displaying a circular arrangement of 60 dots simulating the second marks on a clock. Dots are briefly illuminated in a clockwise rotation at a rate of one per 500 ms. Usually, the rotation proceeds with a single (one-dot) jump. Subjects were instructed that rarely, at irregular intervals, the target proceeds with a double (two-dot) jump by skipping one of the dots in the normal sequence. This should prompt the subjects to press a button as quickly as possible. A total of thirty such occasions were presented in the 45-minute test. Ten occasions occurred within each successive 15-minute period, with intervals ranging from 8 seconds to 7.2 minutes.
(69) The Critical Tracking Task is used as a perceptual-motor performance task that measures the ability to control a displayed error signal in a first-order compensatory perceptual-motor coordination task. During this task, subjects have to control an unstable cursor on a computer screen by using a sensitive joystick. Errors will appear as horizontal deviations of the cursor from the midpoint on a horizontal linear scale. Subjects have to try to keep the unstable cursor in the center of the axis, to reduce deviations back to zero, by continuously making compensatory joystick movements. The frequency of cursor deviations increases as a stochastic, linear function of time, and therefore the subject is required to make compensatory movements with a progressively higher frequency. Also, the subject's compensatory responses increase in frequency with an increasing phase lag (a response adds to, rather than subtracts from, the error) and consequently control is lost. The frequency at which the subjects lose the control is the critical frequency. The test was performed five times; the average critical frequency was calculated without the lowest and highest score as the dependent variable of this test.
(70) The plasma Trp/LNAA ratios determined 90 minutes after consumption of the drinks, revealed a significant effect (P<0.0001) on plasma Trp/LNAA ratio changes across the experimental conditions as applied. Ingestion of the lysozyme hydrolysate (“Trp-hydr”) increased plasma Trp/LNAA value to 0.25 μmol/l. Ingestion of the casein hydrolysate (“plc”) to a Trp/LNAA ratio of 0.08 μmol/l (
(71) TABLE-US-00006 TABLE 6 Amino acid concentrations (μmol/l) following ingestion of the placebo (“plc) or the lysozyme hydrolysate (“Trp-hydr”). Trp/ Tyr Val Ile Phe Leu Trp LNAA LNAA plc 90 315 107 63 168 60 744 0.082 Trp-hydr 73 266 120 58 152 167 670 0.250
(72) After ingestion of the casein hydrolysate, the performance of both groups of individuals subjected to the Mackworth Clock Test was significantly impaired by exposure to stress. However, ingestion of the Trp-rich lysozyme hydrolysate prevented such an impaired performance in the stress-resistant group. Quite surprisingly, the Trp-rich hydrolysate did not prevent such an impaired performance in the stress-prone group. The data obtained are graphically represented in
(73) In the Critical Tracking Task, the lambda CT value indicates the final level of complexity that is reached by the subjects. The higher the lambda CT value, the better the control. The data obtained in the present experiment show that after exposure to stress, the lambda CT value was significantly higher when the Trp-rich hydrolysate was consumed. Among the stress-resistant individuals, a 16% increase could be scored relative to the placebo treatment. Quite surprisingly, also in this test, the lambda CT values in the stress-prone group showed no significant differences between the Trp-rich hydrolysate and the placebo.
Example 10
Protease Resistancy of Lysozyme and Alpha-Lactalbumine
(74) Together with beta-lactoglobulin, alpha-lactalbumin forms the major protein constituent of whey. Because of its high Trp/LNAA ratio, isolated alpha-lactalbumin fractions as well as alpha-lactalbumin hydrolysates have gained popularity for enhancing plasma tryptophan levels. Although alpha-lactalbumin and hen egg lysozyme both have unusually high Trp/LNAA ratios, in other respects these two molecules are quite different. According to the present application, a pepsin-resistant molecule with a high Trp/LNAA ratio is essential to maintain high plasma Trp/LNAA ratios longer term. Here we demonstrate that, unlike hen egg lysozyme, alpha-lactalbumin is not pepsin-resistant. This was illustrated in the following experiment. To imitate conditions in the human stomach, 5% (w/w) solutions of lysozyme and whey protein (Bipro from Davisco) were incubated with pepsin (1% weight Sigma pepsin/weight lysozyme or whey proteins) for 2 hours at 37 degrees C. at pH 4 in a Mc Ilvane buffer (0.2 M citric acid plus Na2HPO4). After incubation both solutions were heated for 5 minutes at 80 degrees C. to terminate the reaction and small samples were subjected to SDS-PAGE (see Materials & Methods) to test the integrity of the various pepsin treated molecules.
(75)
Example 11
Prolonging High Trp/LNAA Levels by Combining Lysozyme Hydrolysate with the Intact Molecule
(76) To demonstrate the advantage of a combination of the peptide-bound and polypeptide-bound tryptophan composition, a study was carried out involving 15 healthy individuals. Exclusion criteria were: chronic and current illness, at the discretion of the investigator; history of psychiatric disorders; use of selective serotonin reuptake inhibitors (SSRI); use of supplements targeting the central nervous system, such as supplements containing tryptophan, ephedrine, or St John's wort; egg allergy; drug abuse; participation in any other study involving investigational or marketed products concomitantly; intolerance to artificial sweeteners; any (history of) gastrointestinal disease that interferes with gastrointestinal function, at the discretion of the investigator; use of medication targeting the gastro-intestinal tract, such as antacids. Finally, for women, pregnancy or the use of a medically not accepted contraceptive method is an exclusion criterion as well. All subjects participating in the experiment signed an Informed Consent Form.
(77) Procedure
(78) The study was performed according to a randomized, double-blind, crossover design with a washout period between the intake of the different treatments of three days at least.
(79) During three experimental morning sessions, subjects visited the laboratory to monitor their plasma Trp/LNAA concentrations following the intake of a drink containing either 6 grams of intact lysozyme, 6 grams of lysozyme hydrolysate or 6 grams of a mixture of hydrolysate and intact product. The drinks were presented as sterile products in bottles with straws. The intact lysozyme was obtained as Delvozyme G. The hydrolysate was prepared as described in Example 7 and the mixture incorporated 30 mol percent of Trp as hydrolysate and 70 mol percent of Trp as intact lysozyme. All drinks contained 6 grams of lysozyme derived protein, 0.10 g sweetener (acesulfame) and were filled up by plain water in order to reach a 300 mL drink. A research assistant blind to the dietary conditions conducted the administration of the different drinks.
(80) The subjects visited the site between 8 and 9 am, having fasted for at least 8 hours. A flexible cannula for blood drawings was inserted in their non-dominant forearm. After ingestion of one of the three experimental drinks blood samples were taken before (t=0) and at t=15, 30, 60, 90 120, 180, 210 and 240 minutes after ingestion to measure plasma Trp/LNAA ratios. The intake of any food or drinks other than water was prohibited during these 240 minutes.
(81) Plasma Measurements
(82) Approx. 5 ml blood was collected in a lithium heparin blood tube, swung and put immediately on ice. The sample was subsequently centrifuged and 750 μl plasma was mixed with 5-SSA (4 mg/100 ml plasma).
(83) These solutions were centrifuged at 13.000 RPM for 5 minutes and to 20 μl supernatant, 40 μl internal standard was added (160 mg Alpha-amino-adipic acid in 2 liter 1.2 mM HCl). 50 μl Borate buffer (included in Waters AccQ. Tag kit art nr. 186003836), 40 μl 0.4M NaOH, and 20 μl reagent (included in Waters AccQ. Tag kit art nr. 186003836) were added, mixed, and heated for 10 minutes at 55° C. Subsequently, 1 μl was injected onto the column and the analysis was proceeded as described by van Eijk et al (J. Chromatogr. 1993: 620: 143-148)
(84) Results
(85) The plasma Trp/LNAA ratio as a variable of time at the three different treatments is depicted in
(86) In a “repeated measures” analysis, all three treatments show a significantly different treatment by time interaction (P<0.001), indicating that all three curves have significantly different shapes. Noteworthy is that all three products produce exactly the same “area-under-the-curve” values indicating that both lysozyme hydrolysate and intact lysozyme are completely digested and taken up into the blood.
Example 12
Combining Lysozyme Hydrolysate with Intact Lysozyme Improves the Taste of the Final Product
(87) Upon mixing the lysozyme hydrolysate with the intact molecule, a considerable change in the taste impression of the final product was noted. The following ratio's of hydrolysate and intact molecule were prepared in 4 gram/200 ml water end concentrations: 100% lysozyme hydrolysate 70% lysozyme hydrolysate-30% Lysozyme 50% lysozyme hydrolysate-50% Lysozyme 30% lysozyme hydrolysate-70% Lysozyme.
(88) In all these combinations, the molar end concentrations of tryptophan were exactly the same. The hydrolysate was prepared as described in Example 7 and for the intact lysozyme the Delvozyme G granulated product was used.
(89) Whereas the taste of the hydrolysate as such is slightly bitter, adding the non-hydrolysed product increasingly masks this bitter note and compensates it with a lingering, slightly sweet taste impression. Taste-wise an experienced test panel preferred the hydrolysate/intact lysozyme mixtures over the pure hydrolysate.