WO1999005918A1

WO1999005918A1 - Glutamine enriched peptide products

Info

Publication number: WO1999005918A1
Application number: PCT/IE1998/000068
Authority: WO
Inventors: Richard Fitzgerald; Paula O'connor
Original assignee: Teagasc, The Agriculture And Food Development Authority
Priority date: 1997-07-31
Filing date: 1998-07-31
Publication date: 1999-02-11
Also published as: IES80880B2; IES970570A2; AU8643798A

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 °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 peπods of metabolic stress There is therefore a need for glutamine in dietary preparations. Glutamine enπched products are particularly required duπng peπods 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"

US patent nos 5,397,803, 5 ,607,975 and (later published) 5,684,045 describe a method ol treating catabohc, gut-associated pathological processes and other similar maladies in animals, by administering an effective amount ot glutamine or an analogue of glutamine to the animal These patents stress the importance of glutamine, in particular in the recovery processes, of the body

US patent no 5,122,515 discloses problems associated with the production of nutrition solutions with relatively high amounts of tyrosine and cysteine due inter aha to the presence of glutamine Glutamine is described as being heat labile Glutamine is also seen as an important amino acid in US patent no 5 ,189,016 which describes a nutrient composition comprising a dipeptide with at least one N-terminal amino acid selected form a group comprising glutamine US patent no 5 ,292,722 describes the use of glutamine or a glutamine equivalent as a supplement to an intravenous solution

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

Glutamine-enπched 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-enπched 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 lnjectable solutions for chronically ill patients in clinical feeding programs

According to the present invention there is provided a glutamine- rich 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 % ot molecules with 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 or substantially no reduction in heat stability when heated to about 80°C for about 10 minutes. The phrase "substantially no reduction in heat stability" as used herein refers to less than about a 2% drop in the overall amount of peptide bound glutamine in the preparation, and more desirably less than about a 1.5% drop in the level of peptide bound glutamine and preferably less than 1.25% Suitably heat stability is maintained at 100 °C for 30 mins 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 . Altenatively the present invention provides a process for the preparation of a glutamine-enriched peptide product comprising hydrolysing a substrate selected from gluten, glutenin or gliadin with a serine alkaline proteinase enzymewhich suitably iis derived from Bacillus licheniformis

Suitably the enzyme treatment step is carried out at a pH in the range 6.0 to 11.0 more and desirably at, or above, a pH of 7.0 suitably 7.0 to 8.0. In a preferred embodiment of the process of the invention, the pH of the reaction mixture is adjusted to 7.0 or below before the inactivation of the enzyme. Adjusting the pH of the reaction mixture in this manner allows for a substantial reduction in the amount of time required to subsequently heat inactivate the enzyme. Suitably the pH of the reaction mixture is adjusted to a value in the range of 5.0 to 7.0 before the inactivation of the enzyme and more preferably the pH of the reaction mixture is adjusted to a value in the range 5.8 to 6.2 before the inactivation of the enzyme. More particularly the pH of the reaction mixture may be adjusted to about 6.0.

The enzymes used in the hydrolysis are preferably selected from Proteinase DS,

Proteinase D1.5S 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 cut-off 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 heat treatment step may be carried out at between temperatures of 25° to 70°C more preferably between 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 is 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 glutamine- enriched 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.

Detailed description

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(Mbχi/alpha)(l/MpXl/h_(ot) 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 h_tot 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/mm with 30% Acetonitπle containing 0.1 % tπfluoroacetic acid (TFA). Hydrolysates were diluted in Mil Q water to 0.25 % (w/v) protein, filtered through a Whatman 0.2 μm syringe filter and 20 μl was applied to the column Eluate was continually assayed at 214 nm and results compared to a 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) tπchloroacetic acid (TCA) and sample which was allowed to stand for 10 minutes before centπfuging 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 μl ol 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 deπvatisation 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 μg 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 μm syringe filter and injected (5 μl) onto a Phenomenex (Phenomenex Ltd., Macclestield, Cheshire, England) C₁₈ reverse phase column (250 x 3.2 mm, 5 μm) 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 (μV) 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 μg) in 1 ml Milli Q water were mixed, filtered through a 0.2 μm filter and injected (5 μl) onto a Phenomenex C_1S 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 μV responses for PYG standards (Sigma Chemical Co. Ltd., Dorset, UK). Solvent 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, 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_{(After hydro},_ysis) - %Ammonia_(Free)= %Ammonia_{(Peptide bound)}

^%Ammonia _(PePti_{de bound)} x 7% = %Ammonia_{(Altrihuted t0 Asn)} %Ammonιa_{(Attπb to Gln)} / 17.03 x 148.13 = %Glutamιne_{(Peptlde bound)}

7% of Ammonia was taken to be due to asparagine and was subtracted from the Ammonιa_{(Peptlde bound)} to give a % Ammonιa_{(Attrlbuted to Gln)} 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-dned 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 peπpheral 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 centπfugation on Ficoll-Hypaque cushions Cells were washed twice with 0.9% NaCl and subsequently diluted in RPM1-1640 medium containing 10% heat-inactivated calf serum (Sigma, Deisenhofen, Germany) in an atmosphere containing 5% CO₂. After 24 h of differentiation, PBL were separated from adherent monocytes and diluted in fresh medium for further assays.

To quantify proliferation the colorimetπc Cell Proliferation ELISA (Boehringer Mannheim, Germany) was used according to manufacturer's instructions. Cells (2 x 105/200 μl) cultured in a 96-well microtiter plate in the presence of various hydrolysates were pulsed with 5-bromo-2'-deoxy-uπdιne (BrdU) for 12 h Incorporation of BrdU into the DNA was detected by the anti-B rdU antibody peroxidase conjugate (POD). The amount of POD retained in the immunocomplex was quantified b> a substrate reaction using TMB Results were read out on an ELISA spectrophotometer at 450 nm (reference wavelength 620 nm)

Commercially available glutamine-enπched 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 ghaden 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, ghaden and ^β-casein were obtained from Sigma Chemical Co., (Poole, Dorset, UK), and food-grade wheat gluten was obtained from Odiums Mills Ltd , (Cork, Ireland).

Example 2:

Assessment of commercially available enzymes for suitability for glutamine peptide hydrolysate production The enzymes studied were selected from:- Alcalase (a Seπne endopepUdase [Subtihsin A] from Bacillus licheniformis available from Novo Nordisk, Denmark), Bioproteinase N100L (proteolytic enzyme preparation from Bacillus subtihs available from Quest International, Co. Cork, Ireland), Corolase 7092 (fungal proteinase with endopepUdase and exopeptidase activity from Aspergillus cultures available from Rohm GmbH, Darmstadt, Germany), Flavourzyme (fungal proteinase/peptidase complex from Aspergillus oryzae, available from Novo Nordisk, Bagsvaerd, Denmark), HT Proteolytic 200 (neutral proteinase [endopeptida.se] from Bacillus amylohquefaciens , available from Solvay Enzymes, Hannover, Germany), Neutrase (neutral proteinase from Bacillus subtihs, available from Novo Nordisk, Bagsvaerd, Denmark), Panazyme 77A (fungal proteinase tvo 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, Pontypπdd, Wales), Protamex (proteinase complex from Bacillus available from Novo Nordisk, Bagsvaerd, Denmark), Proteinase DS or Proteinase D1.5S(alkahne proteinases from Bacillus licheniformis , available from Rhone Poulenc, Cheshire, United Kingdom), Proteinase L 660 (alkaline proteinase from Bacillus licheniformis , available from Solvay Enzymes, Hannover, Germany), and Proteinase 200 (neutral proteinase f rom Bacillus subtihs, available from Rhone Poulenc, Cheshire, United Kingdom)

25 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 The pH was kept constant by the automatic addition ot 0 5 N NaOH using a pH-Stat titration apparatus (Metrohm, Heπsau, 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 (l e. > 10 kDa) and high levels of low molecular mass mateπal (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

25 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, Hensau, Switzerland, Model 718). The reaction was stopped after 5 hours hydrolysis by heat inactivating the enzyme at 90°C for 20 minutes 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 (Centrιcon-10-membrane, Amicon, Inc Beverly MA 01915 USA). The 10 kDa filtrate was freeze-dπed.

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 O.) 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.O 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. After overnight storage, the reaction mixture was heated to 25°C and centrifuged using a Westfaha (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 diafrltered with 60 litres of R.O. 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).

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 Milli 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 lree pyroglutamate were achieved using the above ultrafiltration step (see Table 4)

Example 5:

Generation of glutamine-enriched hydrolysates from gliaden 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.

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 (lkDa 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 OGL660) 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). Example 8:

Heat stability of hydrolysates at pH 5

Two and a half grams of glutamine peptide hydrolysate(s) from Example 3 was resuspended in 150 ml of distilled water, the pH adjusted to 5.2 with 0.5 N HC1 and the volume made up to 250 ml with distilled water. The resulting 1 % solution was heated to 100°C for 30 minutes, cooled and then freeze-dried. The peptide bound glutamine content of the pH adjusted, heat treated powder was compared to the unheated hydrolysate samples. No decrease in the peptide bound glutamine content of the Proteinase L660 hydrolysate was observed while a slight drop (i.e. 1 .04%) was observed in the peptide bound glutamine content of the Proteinase DS hydrolysate. These results demonstrate the stability of the glutamine peptide products to heat treatment.

The conditions under which the stability of the hydrolyates is measured in this Example mimic the conditions of a conventional baking process such as is used for breads, and pastry products including confectionery products so that the glutamine-rich peptide preparations of the invention may be incorporated in such products. Example 9:

Industrial scale production of glutamine peptide

Gluten was reconstituted via an inline mixer (Jackson Switchgear Ltd., Dublin, Ireland) with reverse osmosis water to give a final concentration of 8 % (w/v) in a cylindrical, jacketed, stainless steel tank. Proteinase D1.5S was resuspended in RO water and added at a dosage rate of 0.4 % (or 1 % for Proteinase L 660) (w/w) of the protein substrate. The reaction mixture was preheated to 60 C (55 C for Proteinase L660) 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 constent by continuous addition of 7.5 N NaOH. After 5 hours incubation the pH was reduced to 6.0 using 4 N HC1 and the enzyme was heat inactivated at 90 °C by passing the hydrolysate through a Eurocal Type 28 MP heat exchanger (Ernst P. Fisher, Vienna, Austria) which was fitted with a 2 minute holding tube. The hydrolysate was then cooled to 8 °C and stored overnight at this temperature with gentle agitation in the jacketed stainless steel tank.

Following overnight storage, the reaction mixture was warmed to 25 C and centπfuged using a Westfaha (Model KNA3) centrifugal separator The retentate was discarded and the supernatant recentπfuged prior to ultrafiltration through a spiral wound membrane having a nominal molecular mass cut-off of 10 kDa (Koch International (UK) Ltd., Stafford, UK). The permeate from ultrafiltration was collected, evaporated and spray- dried.

The characteristics of the industπal scale hydrolysate are outlined in Table 8 and show that the hydrolysate maintains the favourable characteristics of the glutamine peptide hydrolysates described in the previous examples l e a high peptide bound glutamine content, a low free glutamine level, a low free pyroglutamate level, a low free amino acid content and a favourable molecular mass distribution profile

The process outlined above yields a reaction mixture which is suited for ultrafiltration following centrifugation. Also the step of reducing the pH of the reaction mixture before inactivating the enzyme as described in Example 9 allows a substantial reduction in the amount of time needed to inactivate the enzyme Both of these features are important to industrial scale preparation. The heat inactivation of the enzyme depends on parameters such as the temperature and duration at the elevated temperature however by adjusting the pH as above provides a more time efficient process

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 osmola ties and high peripheral blood lymphocyte stimulating abilities. The osmolality values of the different glutamine nch 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 n rnngc f food protein substrates.

σ>

* Glutamine is converted to glutamic acid during acid hydrolysis ** Cysteine is converted to cysteic acid during acid hydrolysis

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.

Enzyme Hydrolysis conditions E/S ratio (%) DH Molecular mass distribution'' Time/pH/Temperature (w/w protein) (%) >10 kDa <10 kDa >l Da <l kDa

DMV Gin Pep N/A N/A N/A 0.07 13.53 86.39

Alcalase 4h 7,0 37°C 2.74 21.03 17.42 35.36 47.6

Corolase 7092 4h 7,0 7°C 2.73 14.66 7.38 23.83 68.78

Flavourzyme 4h 7.0 37°C 2.61 13.98 2.70 5.43 91.87

Flavourzyme 3h 7.0 37°C 1.01 13.19 7.20 16.43 76.37

Neutrase 4h 6.0 37°C 2.73 0.61 21.62 47,99 30.39

Promod 278 4h 7.0 37°C 2.75 21.29 8.33 19.13 7.:94

Protamex 4h 7.0 37°C 2,89 12.96 13.78 34.00 52.21

AlcaJase Corolase 7092 4h 7,0 37°C 3.42/3.18 20.41 5.65 17,96 76.39

AIcalase Neutrasβ 4h 7.0 37°C 2,78/2,81 15.00 1 1.81 27.74 60.47

Alcalase Prornod 278 4h 7,0 37°C 2.73/2,69 14,25 4.98 14.57 80.43

Alcalase Protamex 4h 7,0 37°C 3,05/2.79 20.00 5.50 20. L3 74.37

Protainex/Corolase 7092 4h 7,0 7°C 2.70/2.72 22.67 4.19 15.34 80.46

Protamex/Neutrase 4h 7.0 37°C 2.91/2.65 12.89 12.07 30.54 56.69

Protamex/Promod 278 4h 7.0 37°C 2,85/2.72 14.16 5,78 14.96 79.25

* Values expressed as % total area for a gel permeation profile obtained at 214 nm

N/A = Not applicable

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

Table 4: Comparative characteristics of large scale glutamine enriched hydrolysate products.

v

* 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

Table 5 : Characteristics of glutamine enriched hydrolysates following ultrafiltration through 1 kDa cut off membranes

* Decrease expressed as a percentage in the starting material i.e. OGDS (Odiums gluten hydrolysed with

Proteinase DS) or OGL660 (Odiums gluten hydrolysed with Proteinase 660)

** Values expressed as % total area for a gel permeation profile obtained at 214 nm

ND = Not determined

Tnble 6: Characteristics of gliadin hydrolysates

* gliadin hydrolysed with Proteinase DS

** gliadin hydrolysed with Proteinase L660

ND = Not determined

Table 7: Characteristics of gliadin glutamine enriched hydroly»ates following ultrafiltration through 1 kDn cut off membranes

* Decrease expressed as a percentage in the starting material i.e. ODS (Sigma gliadin hydrolysed with

Proteinase DS) or GL660 (Sigma gliadin hydrolysed with Proteinase L660)

** Values expressed as % total area for a gel permeation profile obtained at 214 nro

ND = Not detennined

Table 8: Characteristics of the industrial scale glutamine enriched hydrolysate

Appendix T

Summary table of osmolality values for glutamine peptide hydrolysates

Claims

1. A glutamine-nch 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 kilodaltons.

2. A glutamine-nch peptide preparation as claimed in claim 1 having at least 31 % peptide bound glutamine.

3. A glutamine-nch peptide preparation as claimed in claim 1 having at least 37% peptide bound glutamine.

4. A glutamine-nch peptide preparation as claimed in any preceding claim having less than 1 % of molecules with a molecular weight greater than 10 kilodaltons

5. A glutamine-nch peptide preparation as claimed in any preceding claim having at least 70% of the molecules with a molecular weight oi less than 1 kilodalton.

6. A glutamine-nch peptide preparation as claimed in any preceding claim having less than 1.50% free pyroglutamate.

7. A glutamine-nch peptide preparation as claimed in any preceding claim showing no reduction in heat stability, or substantially no reduction in heat stability, when heated to about 80┬░C

8. A glutamine-nch peptide preparation as claimed in claim 7 showing no reduction in heat stability, or substantially no reduction in heat stability, when heated to about 80┬░C for about 10 minutes.

9. 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.

10. A process for the preparation of a glutamine-enriched peptide product composing hydrolysing a substrate selected from gluten, glutenin or gliadin with a se╧Çne alkaline proteinase enzyme.

11. A process according to claim 10 wherein the enzyme is derived from Bacillus licheniformis

12. A process according to any one of claims 9 to 11 wherein the enzyme treatment step is carried out at a pH in the range 6.0 to 11.0.

13. A process according to any one of claims 9 to 12 wherein the enzyme treatment step is carried out at, or above a pH of 7.0 preferably in the range 7.0 to 8.0.

14. A process according to any one of claims 9 to 13 wherein the pH of the reaction mixture is adjusted to 7.0 or below before the inactivation of the enzyme.

15. A process according to claim 14 wherein the pH of the reaction mixture is adjusted to a value in the range of 5.0 to 7.0 before the inactivation of the enzyme.

16. A process according to claim 15 wherein the pH of the reaction mixture is adjusted to a value in the range 5.8 to 6.2 before the inactivation of the enzyme.

17. A process as claimed in any one of claims 9 to 16 wherein the enzyme is selected from Proteinase DS, Proteinase D1.5S or Proteinase L660.

18. A process as claimed in any one of claims 9 to 17 wherein the product of enzyme hydrolysis is filtered through membranes having cut off values of between 5 and 30 kilodaltons.

19. A process as claimed in claim 19 wherein the product of enzyme hydrolysis is filtered through membranes having cut off values of between 5 and 10 kilodaltons.

20. A process as claimed in any one of claims 9 to 19 wherein the hydrolysate is filtered through a 1 kilodalton membrane and the retentate isolated.

21. A process any one of claims 9 to 20 wherein the enzyme treatment step is carried out at temperatures of between 25┬░ to 70┬░C more preferably between 37┬░C and 60┬░C.

22. A process as claimed in any one of claims 9 to 21 wherein the enzyme used in the hydrolysis step is inactivated by heating the reaction mixture to at least 80┬░C.

23. A process as claimed in claim 22 wherein the enzyme used in the hydrolysis step is inactivated by heating the reaction mixture to at least 80┬░C for about 20 minutes.

24. A glutamine-enriched peptide preparation whenever prepared by a process as claimed in any one of claims 9-23.

25. A foodstuff comprising a glutamine-rich peptide preparation as claimed in any of claims 1 to 8, or 24.