# Patent

## Title
Liquid oat base

## Abstract

A process for preparing a liquid oat base or drink of improved soluble oat protein content from an oats material, in particular an oats material that has not been heat treated in a humid state, comprises solubilizing oat protein in an aqueous solvent by means of protein-deamidase. Also disclosed is a corresponding liquid oat base and uses thereof.

## Background
FIELD OF THE INVENTION
The present invention relates to a liquid oat base, in particular a liquid oat base for use as a milk substitute or a food additive, and to a method for its manufacture.
BACKGROUND OF THE INVENTION
Oat drinks (“oat milk”) for use as cow milk substitutes (EP 731646 B1; EP 1124441 B1; U.S. Pat. No. 6,451,369 B1) and as a raw material for other non-dairy milk products (U.S. Pat. No. 7,160,564 B2) are known in the art. They are preferred by many customers for various reasons, such as for their content of soluble β-glucan fiber beneficial to health, their lack of potentially allergenic proteins and of lactose, which cannot be digested by the majority of the global population. The soluble protein content of oat milk is about 0.5 to about 1.0% by weight.
In the prior art processes for preparing oat milk, the starting material, such as oat flour or oat bran or the whole oats from which it is made or an aqueous suspending or mixture of it is heated to a temperature and for a time sufficient to substantially prevent the development of endogenous enzymatic activity, in particular lipase/lipoxygenase activity, but also β-glucanase activity, during the respective process. Known oat drinks may be termed “oat bases” since, in addition to be used as drinks, in particular milk drinks, they can be used as a base for food other products, such as oat yogurt or oat batter, or be used as a food additive.
Due to the low fat content of oat milk (typically 0.5% by weight), fat in form of vegetable oil, such as rapeseed oil, is often added to the product.
In spite of the commercial success of oat drinks available on the market, there is room for further improvement, in particular in respect of increasing the protein content of the drinks. Processes for producing oat drinks known in the art do not adequately access the protein in oat raw material.
It is known to increase the content of water soluble protein in oat drinks by the use of proteinase in addition to amylase(s) in the enzymatic degradation of oat raw material. The use of proteinase, however, results in the formation of low-molecular peptides, which may change the organoleptic properties of the drinks.
EP 976 829 A1 discloses a protein deamidating enzyme and a process for its production. EP 1 371 734 A1 discloses a method of denaturating milk protein by a deamidating enzyme to improve its sensitivity to protease and its emulsifying, foaming and gelling characteristics. EP 1 839 491 discloses a dairy product and a method of its production by contacting milk with a deamidating enzyme to suppress acidic and bitter taste. WO 2008/138900 A2 discloses a method for producing an acidified milk drink by contacting raw or processed milk with a deamidating enzyme.
In addition to from deamidation by a deamidating enzyme glutamyl and asparagyl residues in peptides and proteins have been observed to undergo non-enzymatic deamidation in vitro and in vivo (Robinson N A, Protein Deamidation. Proc Nat Acad Sci, 99 (2002)5283-5288=http://www.pnas.org/content/99/8/5283.full and literature cited therein).
OBJECTS OF THE INVENTION
It is an object of the invention to provide an oat drink or base of the aforementioned kind, which has improved protein content.
Another object of the invention is to provide said improvements while maintaining or even improving the organoleptic properties of the drink.
A further object of the invention is to provide a process for producing the improved oat drink or base.
Additional objects of the invention will become evident from the following summary of the invention, a number of examples describing preferred embodiments thereof, and the appended claims.
SUMMARY OF THE INVENTION
According to the present invention, what is provided an oat base of the aforementioned kind having an improved content of soluble oat protein. “Improved protein content” means a higher protein content than obtainable by methods known in the art from a given oat raw material with the proviso that the improved content is not due to the use of protease (peptidase/proteinase).
The oat base of the invention is provided by degrading an oats material with one or more amylases and protein-deamidase.
According to one preferred aspect of the invention, the protein-deamidase is one capable of deamidating high-molecular oat protein, such as oat globulin.
According to a preferred aspect of the invention, the protein-deamidase does not comprise substantial protease (peptidase) activity. The protein-deamidase of the invention is preferably free from protease activity. Examples for protein-deamidases useful in the invention are disclosed in EP 976829 B1. A preferred amount of protein-deamidase is from 0.5-2.0 U/g oat protein.
According to another preferred aspect of the invention, deamidation is carried out in parallel amidolysis, that is, with starch degradation by amylase(s). “In parallel with amidolysis” is understood as simultaneous with the enzymatic degradation of starch by amylase(s). In the process of the invention, however, deamidation of oat protein may be continued even after amidolysis has ceased or substantially ceased.
The process of the invention can be stopped at a desired viscosity, such as at a viscosity of from 100 cP to 200 cP or from 50 cP to 100 cP or from 25 cP to 50 cP or from 10 cP to 25 cP (sp2/60 rpm/25±2° C.). The process of the invention is preferably stopped by heating to a temperature at which any enzymatic activity is destroyed within a short time, such as within ten seconds or one minute or five minutes, said temperature being >80° C., preferably greater than 90° C., in particular greater than 100° C., such as about 105° C., at which temperature heating for about 10 seconds is sufficient to destroy any enzymatic activity.
The improved oat base of the invention differs from prior art oat bases (oat drinks) by its increased content of soluble oat protein. In this application, “soluble” signifies “water soluble”. The improvement in soluble protein content obtainable by the method of the invention is 10 per cent by weight and up to 20 per cent by weight or more.
Thus, according to the present invention, the content of soluble protein in the oat base is not one increased by addition of soluble protein to the base or to the raw material from which it is made or during the process by which it is manufactured but by use of an appropriate oat raw material and an appropriate protein solubilization process. It is preferable to use a raw material with a high content of protein preserved in its natural state. “Preserved in its (a) natural state” signifies that the protein in the raw material has not been denaturated or has only been denaturated to a minor extent, such as by 10% by weight or 20% by weight.
Oats used for producing oat drinks is dry- or wet-heated prior to use as starting material for producing oat bases or drinks. The purpose with the heat treatment is twofold. On the one hand, the purpose is to destroy beta-glucanase present and/or to prevent it from being formed during starch hydrolysis so as to preserve water-soluble beta-glucans in their native state. Beta-glucans in their native state are high-molecular beta-glucans, such as of a molecular weight of 50,000 D or more. High molecular beta-glucans are considered to constitute a valuable health-promoting component of oat drinks. Inactivation of beta-glucanase by heat treatment is however only indicated if the oat drink to be manufactures is desired to contain substantial amounts of beta-glucans.
On the other hand and, in a more general manner, the purpose of the traditional heat treatment is to inactivate lipase and lipoxygenase. Inactivation of lipase and lipoxygenase is indicated to prevent the product from turning rancid. According to a preferred aspect of the invention, the need of inactivating lipase and lipoxigenase can be avoided by removing the lipids of the raw material, such as by extraction with ethanol or supercritical carbon dioxide. Preferably at least 90% and even at least 95% of the lipids are removed.
While the content of water-soluble protein in untreated oats is about 60% to about 70% weight of total protein, it is only about 30% weight in microwave-treated oats (Skånemöllan, Sweden) and in steam-treated (102° C. for 50 min, then air-dried (110° C.-120° C. min for 50 min) oats.
In the method of the invention, this kind of heat treatment, in particular steaming, should be avoided or at least be kept as short as possible and/or carried out at a temperature as low as possible to keep oat protein denaturation low. If avoided, the lipids should be removed from the oats. If heating is the preferred method of preventing the product from turning rancid and from preventing substantial degradation of β-glucan, a compromise between heating temperature and/or length of heating, at the one hand, and completeness of inactivation of β-glucanase and lipase/lipoxygenase, at the other hand is attempted.
A preferred raw material for use in the invention is dehulled or hulless/naked, dry milled oat flour that has not been heat treated, in particular steamed. However, wet milled oat flour that has not been heat treated or dry milled flour of any oats fraction can also be used. Particularly preferred is the use of dry milled non-heat treated oats, non-heat treated oat bran, and non-steamed oats.
According to the invention, it has been found that heating of oats in any form at a temperature of up to about 50° C. or even up to about 65° C. for a few hours, such as for one or two or even five hours, does not result in substantial denaturation. On the other hand, heating such oats material for a corresponding time period at a temperature of 80° C. or more does result in a substantial reduction of soluble protein, in particular if the material is in a humid state. Steaming of oats in any form results in substantial denaturation, such as denaturation of 30% or more and even of 50% of more. Consequently, steamed oats materials, such as, for instance, those disclosed in U.S. Pat. No. 6,165,365 A and U.S. Pat. No. 7,494,683 B2, are not preferred for use in the present invention.
According to one preferred aspect of the invention, the oat base of the invention is prepared by milling groats (dehulled oats) with water to obtain a mash containing from 8% by weight to 13% by weight dry substance, then adding amylase(s) and degrading the oat starch at a temperature of from 50° C. to 75° C. The amylase may be beta- and alpha amylase or a mixture thereof, the amylases being added as a mixture or their mixture in the mash being formed by their simultaneous or sequential addition.
The amylases are added in amount(s) sufficient for significant hydrolysis of starch over a time period of from 0.5 h to 4 hrs, in particular from about 1 h to about 2 hrs, hydrolysis of more than 50% by weight of the starch, in particular of more than 80% by weight or even more than 90% weight being considered significant.
Typically the amylase(s) are added in an amount to provide amylase activity of from 140 to 250 Betamyl-3 units and from 0.5 to 4 Ceralpha units per g of starch, in particular of about 180 Betamyl-3 units and about 1 Ceralpha unit per g of starch.
Also disclosed according to the invention is a liquid oat base prepared by the process of the invention and a liquid oat base comprising oat protein deamidated by protein deamidase. It is preferred for the oat base protein to comprise 10% by weight or 20% by weight or more of protein deamidated by protein deamidase.
According to the invention is furthermore disclosed the use of the liquid oat base of the invention as a food, a food additive or a starting material for production of a food, all intended for human consumption.


DESCRIPTION OF PREFERRED EMBODIMENTS
Material and Methods
Oat kernels: Dehulled, steam treated, wet ground or dry ground.
Oat bran (Frebaco Kvarn A B, LidköPing, Sweden): Prepared from steam treated Swedish oat grain by grinding in a rolling mill. Composition (% by weight): Protein 18, fat 7, carbohydrate 45, fiber 16%, water 9.5.
Enzymes: Protein-glutaminase “Amano 50”, 50 U/g (Amano Inc., Japan). Commercial alpha-amylase and beta-amylase are available from various commercial sources.
Alpha-amylase activity: One Ceralpha unit is defined as the amount of enzyme required to release one micromole of p-nitrophenol from BPNPG7 (non-reducing end blocked p-nitrophenyl maltoheptaoside) in one minute under defined assay conditions: http://secure.megazyme.com/files/BOOKLET/K-BETA3_1010_DATA.pdf
Beta-amylase activity: One BNPβ-G3 (p-nitrophenyl-β-D-maltotrioside) unit is defined as the amount of enzyme required to release one micromole of p-nitrophenol from PNPβ-G3 in one minute under defined assay conditions: http://secure.megazyme.com/files/BOOKLET/K-BETA3_1010_DATA.pdf
Protein-glutaminase activity: One activity unit (U) is defined as the quantity of enzyme producing one μmol of ammonia per min in the reaction with 10 mM aqueous benzylocarbonyl-L-glutaminylglycine (Cbz-Gln-Gly).
Viscosity: Measured with a Brookfield Visco DV-II+ instrument (http://www.brookfieldengineering.com/products/viscosimeters/laboratory-dv-ii.asp.
EXAMPLE 1
Pilot Scale Process for Producing the Improved Oat Base of the Invention
Dehulled, steam treated oat kernels (675 kg) were wet ground in a colloidal mill at a temperature of 54° C. and directly fed into a stainless steel enzyme treatment tank over a period of about 20 min. Stirring was started at a mash volume of about 100 L. About 7.5 L of an aqueous solution of alpha-and beta-amylase (1 Ceralpha unit per 180 Betamyl-3 units per g of starch) was used. Enzyme activity may vary depending on the commercial source of the enzymes; the total weight of amylases in this experiment was 432 g. The enzyme solution was fed into the tank in parallel with the mash over a period of about 12 min at the end of which about 3000 L of the mash had been fed into the tank. The rest of the mash was fed into the tank over a period of about 8 min to bring the total contents of the tank to about 5600 L. The temperature of the mash was kept constant at 56° C.
Protein-glutaminase (PG) dosing. PG (687.5 g) was dissolved in 1.5 L water at room temperature. The PG solution was added to the mash at a viscosity of 160.5 (sp2/60 rpm/25±2° C.). Stirring was continued for about 120 min at a temperature of about 56° C. to reach a mash viscosity of 35 (sp2/60 rpm/25±2° C.) and a pH of 6.6. Any enzyme activity was then destroyed by heating the product to 95° C. The mash was cooled to room temperature and decanted. Decantation can be omitted if a whole grain product is to be produced.
The thus produced oat base of the invention can be transferred into a formulation tank in which rapeseed oil, vitamins, sodium chloride, di- and tricalcium phosphate, and calcium carbonate is added. The thus obtained enriched oat drink has a viscosity (sp2/60 rpm/25±2° C.) of 17.5 cP and a pH of 6.8. The formulated oat drink or oat milk is transferred to a storage tank from which it is dispensed for UHT treatment and packaging.
Product analysis. Deamidation of product: 7.3% of total releaseable ammonia (by treatment with 2 N sulphuric acid at 100° C. for 4 h). Deamidation of control (non-enzymatic deamidation): 1.6% of total releaseable ammonia (same process in absence of PG). Soluble protein: 78% of total protein (product of the invention) v. 64% of total protein (control).
Instead of dehulled steam treated oat kernels also corresponding naked kernels may be used, for instance, as a starting material.
EXAMPLE 2
Modified and Down-scaled (1:105) Process of Example 1
Wet-milled oat slurry is heated to 60° C. under stirring. Alpha-and beta-amylase as well as protein glutaminase (1 U/g of oat protein) are added and reacted with the slurry under stirring at 60° C. for two hours. The slurry is the heated to 95° C. for 5 min. Insoluble matter is removed by pulse centrifugation (pulses of 1100 g) and analyzed.
Product analysis. Deamidation of product: 6.9% of total releaseable ammonia. Deamidation of control (non-enzymatic deamidation): 1.9% of total releaseable ammonia (same process in absence of PG). Soluble protein: 84% of total protein (product of the invention) v. 56% of total protein (control).
EXAMPLE 3
Modified and Down-scaled Process of Example 1
The process of Example 2 was repeated but with heat treated dry milled and sieved oat kernels, fraction size <0.5 mm mixed with water to a dry weight of 11%.
Product analysis. Deamidation of product: 6.1% of total releaseable ammonia. Deamidation of control (non-enzymatic deamidation): 1.5% of total releaseable ammonia (same process in absence of PG). Soluble protein: 59% of total protein (product of the invention) v. 48% of total protein (control).
EXAMPLE 4
Modified and Down-scaled Process of Example 1
The process of Example 2 was repeated but with non-heat treated dry milled and sieved oat kernels, fraction size <0.5 mm mixed with water to a dry weight of 11%.
Product analysis. Deamidation of product: 8.9% of total releaseable ammonia. Deamidation of control (non-enzymatic deamidation): 1.5% of total releaseable ammonia (same process in absence of PG). Soluble protein: 81% of total protein (product of the invention) v. 62% of total protein (control).
EXAMPLE 5
Deamidation of Oat Drink at Laboratory and Pilot Plant Scale by Protein-glutaminase
The oat base or drink used in this example was prepared according to the method disclosed in European patent no. 731 646. This oat drink is a commercial product manufactured by Oatly AB, Landskrona, Sweden. In Table 1 important features of a number of products according to the invention are shown. Also shown are corresponding features of deamidation products obtained from dry-milled heat-treated oats. The products were obtained in absence of deamidase (0 U) and in presence of deamidase at two deamidase addition regimes (1 U; 2×0.5 U/g oat protein). It is evident from Table 1 that the content of total protein is substantially increased in the presence of deamidase. It is also evident that, at otherwise identical conditions, a non-heat treated starting material yields a product with higher protein content than a corresponding heat-treated starting material.
It is furthermore evident that that, at otherwise identical conditions, sequential addition of deamidase (2×0.5 U) yields a product of higher protein content than obtained by a single addition of the same amount of amylase (1 U). A higher protein content of the product is paralleled by increased emulsion stability (reduced sedimentation rate) of the product.







TABLE 1







Deamidation of oat drink at laboratory and pilot plant scale














Protein-Glutaminase,

Soluble protein,
Total
Droplet



Oat raw
U/g of
Deamidation
g/100 g (% of
protein
size (μm),


material
Oat Protein
(%)
total)
g/100 g
1.5% fat
Sedimentation










Laboratory scale














Wet-milled
0
U
1.9
0.71 (57%)
0.84
3.2
14% UPH**



1
U
6.7
0.90 (72%)
0.92
1.7
2 white PH*



2 × 0.5
U***
6.9
1.06 (87%)
0.95
0.8
2 white PH


Dry-milled,
0
U
1.5
0.59 (46%)
0.64
4.5
17% UPH


heat treated
1
U
6.1
0.75 (59%)
0.81
3.6
No sediment



2 × 0.5
U***
8.6
0.80 (63%)
0.80
4.0
No sediment


Dry milled,
0
U
1.5
0.88 (64%)
0.80
2.6
38% UPH


non-heat
1
U
8.9
1.16 (81%)
0.92
1.2
30% UPH


treated
2 × 0.5
U***
9.5
1.26 (93%)
0.94
1.2
28% UPH


Oat bran
0
U
1.8
0.41 (17%)
1.25
7.1
13% UPH



1
U
5.9
1.05 (42%)
1.62
6.3
7% UPH



2 × 0.5
U***
6.1
1.09 (44%)
1.60
5.6
4% UPH







Small pilot scale














Wet-milled
0
U
2.3
0.65 (56%)
0.82
15.8
10% UPH



1
U
7.9
0.70 (67%)
0.80
15.8
2 white PH



2 × 0.5
U***
13.0
0.83 (72%)
1.01
17.8
No sediment







Large pilot scale














Wet-milled
0
U
1.6
0.70 (64%)
0.75
7.9
63% UPH



2 × 0.5
U***
7.3
1.00 (78%)
0.91
10.0
2 white PH





*PH = Phase;


**UPH = Upper phase;


***0.5 U added to each of two amylase enzymation steps

## Claims

1. A process for preparing a liquid oat base or drink of improved soluble oat protein content from an oats material comprising starch and oat protein, optionally enriched with at least one member selected from the group consisting of vegetable oil, sodium chloride, dicalcium phosphate, tricalcium phosphate, calcium carbonate, and vitamin, comprising:
providing the oats material, including the starch and the oat protein of the oats material, in an aqueous medium,
degrading the starch of the oats material with at least one amylase,
and solubilizing the oat protein of the oats material using protein-deamidase without use of protease, and wherein the protein-deamidase is glutaminase, wherein the amount of protein glutaminase used in the process is from 0.5 U/g of oat protein to 2 U/g of oat protein, wherein the amylase comprises β-amylase, and wherein the process is conducted to obtain a content of soluble protein from 10 per cent by weight and up to 20 per cent by weight, and wherein the oat protein solubilization and starch degradation is carried out at a temperature of from 50° C. to 60° C., and
wherein the oats material is at least one member selected from the group consisting of non-steamed wet milled oats, non-steamed dry milled oats, non-steamed oat bran, and non-steamed dehulled or hulless/naked dry milled oat flour, and
wherein the oat protein is solubilized by protein-deamidase concurrently with two steps of starch degradation, and wherein the beta amylase is added in an amount sufficient for the hydrolysis of at least 50% of the starch over a time period of from 0.5 hours to 4 hours.

2. The process for preparing a liquid oat base as a food, a food additive or a starting material for the production of food, for human consumption which comprises using the liquid oat base of claim 1 as said food, food additive or starting material.

3. The process of claim 1, further comprising UHT treating the resulting product.

4. The process of claim 1, wherein said two steps are separated by a period extending from 30 min to 90 minutes.