IES80880B2 - Glutamine enriched peptide products - Google Patents

Abstract

A glutamine-rich peptide preparation and foodstuffs containing them the preparation having at least 28 % peptide bound glutamine and a gel permeation profile with least 90 % of the peptides having a molecular weight below 3 kilodaltons. The preparation may have at leave 31 % peptide bound glutamine and suitably less than 1 % of molecules with a molecular weight greater than 10 kilodaltons. The preparation may have less than 1.50 % free pyroglutamate and shows heat stability when heated to about 80 DEG C for about 10 minutes. A process for the preparation of a glutamine-enriched peptide product comprising hydrolysing a substrate selected from gluten, glutenin or gliadin with an alkaline proteinase enzyme derived from Bacillus licheniformis is also described.

Description

Glutamine enriched peptide products The present invention relates to a process for the preparation of a glutamine enriched peptide product and to a product produced by the process.

In general, glutamine is considered to be a non-essential nutrient as it can be synthesised by the human body. However glutamine cannot be synthesised as quickly as it is metabolised during periods of metabolic stress. There is therefore a need for glutamine in dietary preparations. Glutamine enriched products are particularly required during periods of heavy physical training. For example, it is believed that glutamine may stimulate muscle glycogen synthesis in humans so that a glutamine enriched product may have a function in restoring the muscle glycogen store after bouts of intensive physical exercise.

It is also known that in critically ill patients recovery time may be speeded up by glutamine administration. It is also believed that the immune system requires increased amounts of glutamine during stress such as infection or sepsis. Glutamine seems to have a function in helping wound repair and can improve recovery from infections generally.

Thus it is often considered that glutamine is conditionally essential.

EP-A-0 672 352 of Campina Melkunie B.V. discloses a process for the preparation of peptide mixtures having a high glutamine content in which enzymatic hydrolysis is carried out with an endopeptidase. The peptides produced by the process have peptide bound glutamine contents of up to 25%.

EP-A-0 540 462 of Sandoz Nutrition Ltd. discloses a composition containing L-glutamine of at least 20 wt%, the compositions being useful in the manufacture of preparations for avoiding excessively low plasma L-glutamine levels for use in endurance exercises or other physical activities. - 2 Glutamine-enriched peptide products are also available commercially from DMV International, Verghel, The Netherlands and Quest International, Zwijndrecht, The Netherlands.

It is thus an object of the present invention to provide a glutamine-enriched product having a high level of peptide bound glutamine and which is suitable for incorporation into dietary products such as sports formulae, food and drink products and invalid feedstuffs. The product of the present invention may also find application in injectable solutions for chronically ill patients in clinical feeding programs.

According to the present invention there is provided a glutaminerich peptide preparation having at least 28% peptide bound glutamine and a gel permeation profile with at least 90% of the peptides having a molecular weight below 3 kilodaltons. Preferably the preparation has at least 31% peptide bound glutamine, more preferably at least 37% peptide bound glutamine.

The preparation may preferably have less than 1% of molecules wifh a molecular weight greater than 10 kilodaltons and at least 70% of the molecules with a molecular weight of less than 1 kilodalton.

Suitably the peptide preparation has less than 1.50% free pyroglutamate.

In a particularly preferred embodiment the peptide preparation shows no reduction in heat stability when heated to about 80°C for about 10 minutes.

In a further aspect the invention provides a process for the preparation of a glutamine enriched peptide product comprising hydrolysing gluten, glutenen or gliadin with an alkaline proteinase derived from Bacillus licheniformis.

The enzymes used in the hydrolysis are preferably selected from Proteinase DS, available from Rhone Poulenc, Cheshire, U.K. and Proteinase L660, available from Solvey Enzymes, Hannover, Germany.

The product of the enzyme hydrolysis is then preferably filtered through membranes having cutoff values of between 5 and 30 kilodaltons (kDa), preferably between 5 and 10 kilodaltons. This serves to remove high molecular weight and undigested components.

Suitably the hydrolysate is also filtered through a 1 kilodalton membrane to remove low molecular weight material and remove free amino acids, the peptide bound glutamine content of the retentate being thus improved.

The enzyme treatment step may be carried out at between temperatures of 37° to 60°C.

The enzymes used in the hydrolysis step may be inactivated by heating the reaction mixture to at least 80°C for about 20 minutes.

Preferably the pH of the reaction mixture during enzyme treatment was kept constant. This is suitably-achieved by the addition of an alkaline solution such as sodium hydroxide, using a pH titration apparatus.

Preferably, following enzyme inactivation, the reaction mixture is centrifuged, and the supernatant ultrafiltered. In a preferred embodiment the retentate is diafiltered and the permeate collected to improve yield.

The invention also relates to a foodstuff comprising a glutamineenriched peptide as defined above or whenever prepared by a process as defined above. By foodstuff is meant a solid or semi-solid food composition or beverage.

Characterisation of glutamine peptide hydrolysates Degree of hydrolysis The degree of hydrolysis (DH%), defined as the percentage of peptide bonds cleaved was calculated from the volume and molarity of NaOH used to maintain constant pH (Adler-Nissen, 1986). The DH% was calculated as :DH% = B(Mb)(1/alpha)(1/Mp)(l/htQt) X 100 where B is the volume of NaOH consumed (ml), Mb is the molarity of NaOH, alpha is the average degree of dissociation of the alpha-NH2 groups at pH 8.0 and 60°C (or pH 7.0 and 55°C for Proteinase L 660), Mp is the mass of protein (g), and htQt is the total number of peptide bonds in the protein substrate (mequiv/g of protein).

Protein (Nitrogen) Total protein (N x 5.7) of substrates and hydrolysates was determined by MacroKjeldahl (IDF 20B:1993).

Protein (Nitrogen) solubility Hydrolysates (2% w/v) were adjusted to pH 2-8 with 1 N HC1 or 1 N NaOH, stirred continuously for 1 hour and centrifuged at 1,300 g (Mistral 6000 MSE Scientific Instruments, West Sussex, UK) for 15 minutes at 20°C. The supernatants were then filtered through a Whatman No. 1 filter paper. Nitrogen was determined as described above and expressed as a percentage of total nitrogen in the hydrolysate.

(IDF 88/1:1987).

Molecular mass distribution of peptides in glutamine peptide hydrolysates The size distribution of peptides in glutamine peptide hydrolysates was determined using a TSK 2000 SW (Beckman Instruments Ltd., UK) gel permeation column (7.5 nm x 60 cm) fitted to a Waters HPLC System. The column was eluted at a flow rate of 1 ml/min with 30% Acetonitrile containing 0.1% trifluoroacetic acid (TFA). Hydrolysates were diluted in Milli Q water to 0.25% (w/v) protein, filtered through a Whatman 0.2 pm syringe filter and 20 pi was applied to the column. Eluate was continually assayed at 214 nm and results compared to a - 5 calibration curve prepared from the average retention volume of standard proteins and peptides.

Free amino acid analysis of hydrolysates Hydrolysates were deproteinised by mixing equal volumes of 24% (w/v) trichloroacetic acid (TCA) and sample which was allowed to stand for 10 minutes before centrifuging at 14400 g (Microcentaur, MSE, UK) for 10 minutes. Supernatants were removed and diluted with 0.2 M sodium citrate buffer, pH 2.2, to give approximately 25 nmol of each amino acid residue per 50 jil of injection volume and then analysed on a 120 x 4 mm cation exchange column (Na+ form) using a Beckman 6300 amino acid analyser (Beckman Instruments Ltd., High Wycombe UK).

Results were expressed in percentage terms, i.e. g per lOOg powder product.

Free glutamine analysis of hydrolysates Free glutamine was measured after derivatisation with o-phthalaldehyde-mercaptoethanol (OPA-ME) and separation of the individual amino acids by means of reversed phase HPLC using a Shimadzu HPLC System (Shimadzu Corp., Analytical Instruments, Nakagyo-ku, Kyoto, Japan, Shih F.F., 1985). To a solution of 80 nmol amino acid or 24 |ig hydrolysate in 0.3 ml water were added 0.2 ml OPA-ME solution. After 1 minute at room temperature, 0.5 ml 0.1 M potassium phosphate (pH 4.0) was added followed by 3.0 ml methanol. The solution was mixed, filtered through a 0.2 jim syringe filter and injected (5 pi) onto a Phenomenex (Phenomenex Ltd., Macclesfield, Cheshire, England) Cjg reverse phase column (250 x 3.2 mm, 5 pm) equilibrated with solvent A (0.04 M Sodium Acetate buffer with 13.6% acetonitrile, pH 5.9-6.0). Isocratic elution was used and the flow rate was 0.3 ml/min. Eluate was continually assayed at 340 nm and results compared to microvolt (pV) responses for amino acid standards (Sigma Chemical Co. Ltd., Dorset, England). Solvent B (100% acetonitrile) was used to wash off remaining amino acids and the column was re-equilibrated with Solvent A for 15-20 minutes prior to another injection. Results were expressed in percentage terms i.e. g per lOOg powder product.

Free pyroglutamate analysis of hydrolysates Free pyroglutamate (PYG) levels of glutamine peptide hydrolysates were measured by means of reversed phase HPLC using a Shimadzu HPLC System (Shih, 1985). PYG (50 nanomoles) or hydrolysates (6 jig) in 1 ml Milli Q water were mixed, filtered through a 0.2 jim filter and injected (5 |il) onto a Phenomenex Cjg column (described above) equilibrated with solvent A (0.1% phosphoric acid). Isocratic elution was used and the flow rate was 0.3 ml/min. Eluate was continually assayed at 200 nm and the results compared to pV responses for PYG standards (Sigma Chemical Co. Ltd., Dorset, UK). SolvenL.B (100% acetonitrile) was used to wash off remaining hydrolysate followed by re-equilibration with solvent A for 15-20 minutes prior to another injection. Results were expressed in percentage terms i.e. g per lOOg powder product.

Peptide bound glutamine content of hydrolysates Peptide bound glutamine was indirectly measured by quantifying the amount of ammonia (%) released by acid hydrolysis of peptides and subsequently converting ammonia content to glutamine content (Wilcox,t 1967). Glutamine peptide hydrolysate (0.1 g) was heated to 110°C in 50 ml 2 N HC1 for 3 hours. After neutralisation with potassium hydroxide (KOH), ammonia was measured enzymatically with glutamine dehydrogenase using a Boehringer ammonia detection kit (Boehringer Mannheim, East Sussex, UK). Peptides (1 g/1) were also resuspended in Milli Q water and assayed for free ammonia content. Peptide bound glutamine was calculated using the following formula %Ammonia/Aft , . , - %Ammonia._ = %Ammonia.„ ... ..

(After hydrolysis) (Free) (Peptide bound) %Ammonia,_ ., , ., x 7% = %Ammonia.A, ., (Peptide bound) (Attributed to Asn) %Ammonia/A„ .. t _. . / 17.03 x 148.13 = %Glutamine._ ,.. . ..

(Attrib to Gin) (Peptide bound) 7% of Ammonia was taken to be due to asparagine and was subtracted - 7 from the Ammonia (Peplide to give a % Ammonia (AttributedtoGln) value (MacRitchie, 1979). Results were expressed in percentage terms i.e. g per lOOg powder product.

Clarity of hydrolysate solutions The clarity of glutamine peptide hydrolysates in solution was measured by % transmission at 600 nm on a Philips model PYE Unicam PU8610 uv/vis spectrophotometer (Philips Test and Measurement, Herts., UK).

One percent (w/v powder) solutions of hydrolysates (pH 2-8) were assayed for % transmission.at .600 nm. A solution was considered clear if the transmission in a 1 cm cuvette was at least 98%.

Sensory analysis of hydrolysates One percent (w/v protein) solutions of hydrolysates were presented to a taste panel trained to detect bitterness. Samples were compared to caffeine standards and scored (0-100%) for bitterness.

Determination of heat stability of hydrolysates Two % (w/v) powder of various hydrolysates were heat-treated at 80°C for 10 minutes, freeze-dried and then assayed for peptide bound glutamine content as described above.

Hydrolysate osmolality determination The osmolality of 1% (w/v) powder solutions of various hydrolysates was determined using an osmometer (Fiske & Associates, MA, USA).

Cell culture analysis Human peripheral blood lymphocytes (PBL) were isolated by a method described by Boyum (1974) as follows. The heparin-anticoagulated blood of normal subjects (buffy coat) was diluted 2-fold with PBS (pH 7.2), and PBL together with monocytes were recovered by centrifugation on Ficoll-Hypaque cushions. Cells were washed twice with 0.9% NaCl and subsequently diluted in RPM1-1640 medium containing 10% heatinactivated calf serum (Sigma, Deisenhofen, Germany) in an atmosphere containing 5% C02· After 24 h of differentiation, PBL were separated from adherent monocytes and diluted in fresh medium for further assays.

To quantify proliferation the colorimetric Cell Proliferation ELISA (Boehringer Mannheim, Germany) was used according to manufacturer's instructions. Cells (2 x_105/200 jil) cultured in a 96-well microtiter plate in the presence of various hydrolysates were pulsed with 5-bromo-2'-deoxy-uridine (BrdU) for 12 h. Incorporation of BrdU into the DNA was detected by the anti-BrdU antibody peroxidase conjugate (POD). The amount of POD retained in the immunocomplex was quantified by a substrate reaction using TMB. Results were read out on an ELISA spectrophotometer at 450 nm (reference wavelength: 620 nm).

Commercially available glutamine-enriched peptide products were obtained from DMV International, Verghel, The Netherlands, and Quest International, Zwijndrecht, The Netherlands.

Example 1: Assessment of a range of food protein substrates as starting material for the generation of a glutamine-enriched peptide preparation The amino acid composition of a range of substrates was determined and the results are summarised in Table 1. From Table 1 it is seen that gluten and gliaden are naturally rich in glutamic acid and as such were selected as good potential substrates for the preparation of glutamine-enriched peptide hydrolysates. Wheat gluten, gliaden and B-casein were obtained from Sigma Chemical Co., (Poole, Dorset, UK), and food-grade wheat gluten was obtained from Odiums Mills Ltd., (Cork).

Example 2: Assessment of commercially available enzymes for suitability for glutamine peptide hydrolysate production The enzymes studied were selected from:Alcalase (a Serine endopeptidase [Subtilisin A] from Bacillus lichenifomris available from Novo Nordisk, Denmark), Bioproteinase N100L (proteolytic enzyme preparation from Bacillus subtil is available from Quest International, Co. Cork, Ireland), Corolase 7092 (fungal proteinase with endopeptidase and exopeptidase activity from Aspergillus cultures available from Rohm GmbH, Darmstadt, Germany), Flavourzyme (fungal proteinase/peptidase complex from Aspergi1lus oryzae, available from Novo Nordisk, Bagsvaerd, Denmark), HT Proteolytic 200 (neutral proteinase [endopeptidase] from Bacillus amyloliquefaciens, available from Solvay Enzymes, Hannover, Germany), Neutrase (neutral proteinase from Bacillus subtilis, available from Novo Nordisk, Bagsvaerd, Denmark), Panazyme 77A (fungal proteinase from Aspergillus oryzae, available from Rhone Poulenc, Cheshire, United Kingdom), Profix (purified Papain extracted from the fruit Carica papaya, available from Quest International, Co. Cork, Ireland), Promod (selected proteinases from strains of Bacillus and Aspergillus combined with Papain, available from Biocatalysts, Pontypridd, Wales), Protamex (proteinase complex from Bacillus available from Novo Nordisk, Bagsvaerd, Denmark), Proteinase DS (alkaline proteinase from Bacillus lichenifomris, available from Rhone Poulenc, Cheshire, United Kingdom), Proteinase L 660 (alkaline proteinase from Bacillus lichenifomris, available from Solvay Enzymes, Hannover, Germany), and Proteinase 200 (neutral proteinase from Bacillus subtilis, available from Rhone Poulenc, Cheshire, United Kingdom). ml scale hydrolysates 2.0 g Gluten (71.51% (w/w) protein) was resuspended in 23 ml of Milli Q water in a 75 ml reaction vessel. The pH and temperature of the reaction were dictated by the enzyme under study and adjusted accordingly. Enzyme, 0.0286 g, (2% w/w protein) was mixed with 2 ml Milli Q water and added with constant stirring to the reaction vessel. - 10 The pH was kept constant by the automatic addition of 0.5 N NaOH using a pH-Stat titration apparatus (Metrohm, Herisau, Switzerland; Model 718). The reaction was allowed to proceed for 3-4 hours at which stage it was stopped by heat inactivation of the enzyme at 90°C for 20 minutes. The resulting hydrolysates were assessed for their molecular mass distribution profiles and free glutamine content. Table 2 summarises the DH% achieved and the molecular mass distributions in hydrolysates prepared at 37°C using a range of different enzymes preparations. Table 3 summarises the characteristics of gluten (Odiums) hydrolysates prepared at the optimum pH and temperature values for different enzyme preparations. This,Table also includes data on the DMV and the Quest products. From the Table, it is seen that Proteinase DS and Proteinase L660 result in low levels of high molecular mass material (i.e. > 10 kDa) and high levels of low molecular mass material (i.e. < 1 kDa). These are favourable characteristics with respect to the utilisation of glutamine-enriched peptide products. Further, it is seen in Table 3 that these enzymes yield gluten hydrolysates having low levels of free glutamine and free pyroglutamate. It is evident from Table 3 that some characteristics of the 25 ml hydrolysates are more favourable than those of the DMV or Quest products.

Example 3: Preparation of glutamine enriched peptide hydrolysates using Proteinase DS and Proteinase L660 ml/1 litre scale Gluten (Odiums Mills Limited, Cork) was resuspended in Milli Q water (8% w/v), pre-heated to 60°C (or 55°C for Proteinase L 660) and adjusted to pH 8.0 (or 7.0 for Proteinase L 660) with 0.5 N NaOH. Proteinase DS previously resuspended in water was added to the reaction mixture at a final enzyme to substrate (E/S) ratio of 1% (w/w protein). The pH was kept constant by the automatic addition of 0.5 N NaOH using a pH-stat titration apparatus (Metrohm, Herisau, Switzerland; Model 718). The reaction was stopped after 5 hours hydrolysis by heat inactivating the enzyme at 90°C for 20 minutes. - 11 The hydrolysate was cooled to room temperature, centrifuged at 2,500 g for 20 minutes, the supernatant decanted off and ultrafiltered through a 10 kDa cut off membrane (Centricon-10-membrane, Amicon, Inc. Beverly MA 01915 USA). The 10 kDa filtrate was freeze-dried. 150 litre scale (Pilot plant scale) Gluten was reconstituted using a Silverson mixer (Machines ltd., Bucks., U.K.) in 150 litres of reversed osmosis (R.0.) water to give a final concentration of 8% (w/v) in a cylindrical, jacketed, stainless steel tank. Proteinase DS (or Proteinase L 660) was resuspended in R.0. water and added at a dosage rate of 1% (w/w) of the protein substrate. The reaction mixture was pre-heated to 60°C (55°C for Proteinase L 660) and maintained at pH 8.0 (pH 7.0 for Proteinase L 660) and the reaction allowed to proceed for 5 hours. The pH was maintained constant by continuous addition of 7.5 N NaOH. After 5 hours incubation the enzyme was heat inactivated at 90°C for 20 minutes. The hydrolysate was cooled to 8°C and stored overnight with gentle agitation in the jacketed stainless steel tank.

A After overnight storage, the reaction mixture was heated to 25°C and centrifuged using a Westfalia (Model KNA3) centrifugal separator. The retentate was discarded and the supernatant was ultrafiltered through a spiral wound membrane system fitted with 10 kDa nominal molecular mass cut off (Koch International (UK) Ltd., Stafford, UK).

The retentate was diafiltered with 60 litres of R.0. water and the permeates collected, evaporated and spray-dried.

The characteristics of the large-scale hydrolysates are shown in Table 4. From this Table it is seen that both enzymes produce hydrolysates with favourable characteristics i.e. having high peptide bound glutamine contents (at least 28%), low free glutamine levels, low free pyroglutamate levels, low overall free amino acid contents, favourable molecular mass distribution profiles, high solubilities, good clarity, low osmolality and hydrolysates displaying good heat and acid stabilities. Similar results were obtained for the small scale (25 ml) hydrolysates (data not shown). - 12 Example 4: Enrichment of Proteinase DS and L660 gluten hydrolysates for peptide bound glutamine content Ten grams of glutamine peptide hydrolysate was resuspended in 200 ml of Mi 11i Q water. The 5% (w/v) solution was filtered through an Amicon stirred cell unit (model 202) (Amicon, Inc. Gloustershire, UK) fitted with a YMI (1 kDa nominal molecular mass cut off) Diaflo ultrafiltration membrane (Amicon, Inc. Gloustershire, UK) until 60-70 ml of permeate (equivalent to 15-20% by_weight of hydrolysate) was collected. Both retentate and permeate solutions were freeze dried and their peptide bound glutamine contents determined.

Table 5 summarised the characteristics of the retentates obtained from the above procedure. Using this procedure it is seen that the peptide bound glutamine content of the hydrolysates can be increased to > 30%. Furthermore, favourable reductions in osmolality, free amino acids, free glutamine and free pyroglutamate were achieved using the above ultrafiltration step (see Table 4).

Example 5: Generation of glutamine-enriched hydrolysates from glladen Proteinase DS and L660 hydrolysates of gliaden were prepared at 25 ml scale using the procedure as outlined in Example 3. The characteristics of the resulting products are outlined in Table 6.

Both products contain high levels of peptide bound glutamine (37%), low free glutamine levels, low free pyroglutamate levels, low overall free amino acid contents, favourable molecular mass distribution profiles, high solubilities, good clarity, low osmolality and hydrolysates displaying good heat and acid stabilities. It is evident therefore that the gliadin hydrolysates have superior characteristics to the DMV and Quest products. - 13 Example 6: Enrichment of proteinase DS and L660 gliadin hydrolysates for peptide bound glutamine content Ten grams of glutamine peptide hydrolysate(s) from Example 5 was resuspended in 200 ml of Milli Q water. The 5% (w/v) solution was filtered through an Amicon stirred cell unit (model 202) (Amicon, Inc. Gloucesterhire, UK) fitted with a YM1 (IkOa nominal molecular mass cut off) Diaflo ultrafiltration membrane (Amicon, Inc. Gloucestershire, UK) until 60-70 ml of permeate (equivalent to 15-20% by weight.of hydrolysate) was collected. Both retentate and permeate solutions were freeze dried and their peptide bound glutamine contents determined. Table 7 summarises the characteristics of the retentates obtained from the above procedure. Using this procedure it is seen that the peptide bound glutamine content of the hydrolysates can be increased to >37.5%. Furthermore, favourable reductions in free amino acid, free glutamine and free pyroglutamate levels were achieved using the above ultrafiltration step.

Example 7 Tissue culture analysis of glutamine-enriched peptide hydrolysates It can be seen from Figure 1 that the glutamine rich peptide products (OGDS and 0GL660) described herein (Table 4) bring about significant increases in the proliferative response of concanavalin A-activated peripheral blood cells. It is also seen that these hydrolysates mediate greater proliferative responses than the commercially available glutamine-enriched peptide products (Figure 1).

It is evident from the preceding examples that novel glutamine rich peptide products have been produced which have improved/high peptide bound glutamine contents, favourable molecular mass distribution profiles, low levels of free amino acids, low free glutamine levels, low pyroglutamate levels, high solubilities, good clarity, good heat and acid stabilities, low osmolalities and high peripheral blood lymphocyte stimulating abilities. The osmolality - 14 values of the different glutamine rich products are summarised in Appendix 1.

References Adler-Nissen J. (1986). Enzymatic hydrolysis of food proteins.

Elsevier Applied Science, Publishers, London.

Boyum A. (1974). -Tissue Antigens 4, 269.

International Dairy Federation Cicrulaire; 88/1; October 1987.

International Dairy Federation Cicrulaire; 20B; 1993.

MacRitchie F. (1979). A relation between gluten protein amide content and baking performance of wheat flours. Journal of Food Technology 14; 595-601.

Shih F.F. (1985) Analysis of glutamine, glutamic acid and pyroglutamic acid in protein hydrolysates in high performance liquid chromatography. Journal of Chromatography, 322: 248-256.

Wilcox P.E. (1967). Determination of amide residues by chemical methods. In: Methods of Enzymology. Academy Press Publishers, New York.

Table 1: Amino acid composition of a range of food protein substrates.

** Cysteine is converted to cysteic acid during acid hydrolysis S XJ £ - 16 Table 2: Degree of hydrolysis (DH %) achieved and molecular mass distributions of gluten (Sigma) hydrolysates prepared at 37° C with a range of commercially available enzymes.

B «J «> *© s O' Ό oo r- OV V w O' tr <5 r- S Ov m m r- 00 n TO «V CM cn V xt Τ*ϊ vo CM VO 00 vo O’.'— CM MD o o O vo O' oo VO ov r* CO t- yv *- Ό OO OO m VO «*1 m n O' co o Ό M co V XT »o cv oo xt xr Os V— o O' •o CO m Ov cn IO m in sO r- o Γ- XT © in o xr cn CM •V —l m t—i CM CM m t cm © v « · © η* OO Ο O CM CV OO « r> « ό o > V» OO © Ov.t— oo Ον «η ·— O CCM t — CM « Π Ό — Μ- >Λ Μ- Γ4 O CM Ov xr © CM Z ci ri 6 — C< © «O X· CM — — — CM —. ΟΪ — Γ» O' vo Ό OO — CM CM xf CM — — a u a•s ε « a * £ fc CQ Ή o e © Q ag ,>» ε Ό ta 4> a ε «J g Mi s j? s ^TTen»-ir— <*νι/νσνοο—..OvOvcMwc'i •^Γ-'Γ-ΌΟ^Ρ-αοΓ-οοΌΓ-^ΐΟί'' Z ci ci ci —‘ cm ci cm' j*j cm cm' cm cm cm' cm CS « m in O — Ό xr Γ Γ- Ο Γ- Οι oo C·» CM CM CO CM CM CM υ ο ο u υ υ u Q Ο Ο a a Ο © > r- t> r> ΓΩ CO CO CO u υ υ υ υ o q o o o o o o o r- r- t> r- r- r- o cn m co «*v <*» co cn r-~ r~ \o Γ- Γ Λ Λ JS .-j = -*= -O ii cr 5T M xr v Q U &, e- Z M CO W W β « «β as « s •a s ο o 3 3 o © © © © © © O i< c-‘ t> r-' t'- C JB -C -E JC JC XT V XT XT XT V XT ♦ Values expressed as % total area for a gel permeation profile obtained at 214 nm Table 3: Characteristics of hydrolysates prepared with gluten (Odiums) using a range of commercially available enzyme preparations. * values expressed as % total area for a gel permeation profile obtained at 214 nm N/A=Not applicable ND » Not determined -IBTable 4: Comparative characteristics of large scale glutamine enriched hydrolysate products.

• Odiums gluten hydrolysed with Proteinase DS ** Odiums gluten hydrolysed with Proteinase L660 Values expressed as % total area for a gel permeation profile obtained at 214 nm ND = Not determined ς Table 5: Characteristics of glutamine enriched hydrolysates following ultrafiltration through 1 kDa cut off membranes >» o J3 CO O O O H | t j nil 3 ·§ ! 3 5 5 tfi 1 g S S3 ο. o 3 S J « •ο O « S ° *8 S! w β» e ° 8 Q S 8 £ 8 e «2 8 2 £ Q2 ; * Qm * Not determined Tnble 6: Characteristics of gliadin hydrolysates CM Ό o -JU «3 o >» ua c -a -S -o •e « - 2L Table 7: Characteristics of gliadin glutamine enriched hydrolysates fallowing ultrafiltration through 1 kJDn cut off membranes s S’ .9 M o ss £ Ο» J 2 8 O « © '© C -5 E * « Έ -S S. c -x * 1 -9 3 re M __ I s. s. (3 O M \0 S 2 O § 5 jr w ® Q § 8 S Q g * cu Έ -S JS «X c q © oo re a £ M •s 1 -i ε i g· « φ -u CO Tx « -2 ,2 Z £ 11 -22Appendix I Summary table of osmolality values for glutamine peptide hydrolysates Test sample Osmolality (mOsm/kg) DMV 34 Quest 28 OGDS 20 OGL660 18 Ultrafiltrated Odiums gluten/Proteinase DS 17 Ultrafiltrated Odiums gluten/Protemase L660 15 GDS 28 GL660 26

Claims (5)

1. A glutamine-rich peptide preparation having at least 28%, preferably at least 31%, most preferably at least 37% peptide bound glutamine, and a gel permeation profile with at least 90% of the peptides having a molecular weight below 3 kilodaltons and preferably at least 70% of the molecules with a molecular weight of less than 1 kilodalton, and preferably less than 1% of molecules with a molecular weight greater than 10 kilodaltons.

2. A glutamine-rich peptide preparation as claimed in claim 1 having less than 1.50% free pyroglutamate.

3. A process for the preparation of a glutamine-enriched peptide product comprising hydrolysing a substrate selected from gluten, glutenin or gliadin with an alkaline proteinase derived from Bacillus licheniformis, the enzyme preferably being selected from Proteinase DS or Proteinase L660, and preferably the product of enzyme hydrolyis being filtered through membranes having cut-off values of between 5 and 30 kilodaltons, preferably between 5 and 10 kilodaltons.

4. A glutamine-rich peptide preparation substantially as described herein with reference to the Examples.

5. A food-stuff comprising a glutamine-rich peptide preparation as claimed in claim 1 or claim 2 or claim 4, or a peptide preparation whenever prepared by a process as claimed in claim 3. TOMKINS & CO. 3828R vi Figure 1: Proliferative response of ConcanavalinA-activated peripheral blood lymphocytes by glutamine peptide hydrolysate products in a glutamine free medium (I