CA2383021C - Cereal .beta. glucan - probiotic compositions - Google Patents

Abstract

The invention describes the preparation of functional food products containing living bacteria, prepared by inoculation and fermentation of cereal flours (meal) or extracts. The resulting products have both nutrition and health benefit. Products include yoghurts (traditional and frozen), flummery, milks, and dried probiotic bacteria/cereal combinations. Compositions containing cereal flours (meals) and/or bran with .beta. glucan are disclosed that protect cultures and products containing bifidobacteria from competition with lactobacilli. This protection extends through the manufacturing process in addition to the shelf-life storage of the product and ensures that functional doses of probiotic bacteria are delivered to the end-user. The compositions contain soluble fibre from oats preferably at sufficient levels to comply with the US FDA requirements to make a health claim.

Description

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Cereal 13 glucan - probiotic compositions Background of the Invention Technical Field The present invention relates generally to probiotic cultures, cereal flour (meal), cereal bran, and B glucan. More particularly, the invention relates to methods and compositions for producing probiotic-supporting preparations derived from oatmeal, oat bran, and/or 13 glucan.

Background of the Invention The =,concept of probiotics is based ori the premise that there are microorganisms in the intestinal tract which have a beneficial health effect on the host. Some of these effects are, for example, antibiotic production, lactose digestion enhancement, cholesterol reduction, antimicrobial effects, duration of diarrhea limitation, and gut mucosal immune system stimulation. These effects are dependent on the strain of bacteria which affect the composition of the gastrointestinal flora. A significant number of publications have focused on the fact that high amounts of lactobacilli and bifidobacteria are antagonistic to many pathogenic and putrefactive bacteria in the human intestine, which in turn may reduce the risk of cancer. (See Rafter J.J. (1995) Sca_n_d. J. C*astroenterol.
135:497) Fermentation has been used to preserve perishable foods for centuries with milk receiving most of the attention. "Sour milk" or "yoghurt" are often referred to in historical literature (Tamine A Y and Robinson. R.K. (19$5). YQghu_rt Science and Technology.
Pergamon Press. Chapters 1. 8. & 10.). Yoghurt is milk that has peen transformed to a solid by the fermentative action of organisms such as Lactobacillus delbrueckii ssp.
Bulgaricus and Streptococcus salivarus ssp. Thermophilus. These microorganisms do not have the ability to colonize the intestinal tract in large number because they are not resistant to the gastric acidity and bile salts. Therefore, it has been proposed that the inclusion of probiotic foods in the diet may enhance the gut population of beneficial bacteria.

Presently some yoghurt is fermented with bacterial cultures of intestinal origin and is . . . . . .. ..;::.,: -...... :.. .... . : . :...... ... : :. ... ...~,:.:-called Bio-Yoghurt. In Bio-Yoghurt, the microorganisms must survive distribution and retailing and it is imperative to functionality that the bacterial count in the final product is high. To provide health benefits, the suggested probiotic concentration is 3106 CFU/ml.
See RohinsonL$K (1987) Suid Afikaanse TYdskrif Vir Suiwelkunde 19:25. However, studies have shown low viability of probiotics in current market preparations see Shah.
NP (1995) t. Datrv J 5:515 Traditional inoculated grain based products are known in Europe. In Scotland and Wales, flummery is prepared from oatmeal steeped in water and kept until it becomes sour.
Similarly, in the Scandinavian and Baltic nations, soured cereal grains have been described, the majority of which are.cooked after fermentation, an action that kills the useful microorganisms, and thus destroys probiotic benefits.

Formulations of lactobacilli grown in combination with oat bran or oatmeal are known.
An oat bran product inoculated with defined, living microbes has been described by Salovaara (EP00568530). This product has an inherent granular texture due to bran particles. The Salovaara product contains combinations of lactobacilli but does not describe the use of bifidobacteria nor does it present evidence on the protection of bifidobacteria from competition with lactobacilli. YosaTM sold in Finland is based on the technology described in Salovaara (EP00568530). Other publications have described that oat pastes proved to be a suitable growth media for yoghurt organisms (Salovaara. H...
Backstr¾m. K. Mantere-Ajhonen. S.. 1991. J. Dairy Sci. 49: 3 7).

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25. In US5190755 a nutrient health drink composition is described suitable for enteral feeding comprising a fermented cereal based product, enzyme,, and optionally further nutrient components, in combination with lactobacilli. Two stages of enzyme treatment are used in the preparation of an oat gruel, firstly incubation with a am,ylase and secondly incubation with P glucanase.

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r:~::...rt US5591428 discloses an oatmeal-based oral nutritional supplement fermented by Lactobacillusplantarum strain No. 299 (at a concentration of -] 010CFU/g of freeze-dried product) and a feeding formulation for enteral nutrition containing the same strain of Lactobacillus plantarum at a concentration of 1.5x10 CFU/ml of nutrition solution.
Importantly, the method described in US5591428 requires the preparation of oatmeal gruel with 1%w/w 0-glucanase, which would serve to convert 13 glucan to glucose. This requirement is not part of the present invention.

However, typical oat yogurt preparations do not support the survival of bf fidobacteria in conjunction with other bacteria and as a result the number of bifidobacteria declines whilst the numbers of lactobacilli are sustained in steady numbers (see figure 1). Of additional relevance is the usual decline in numbers of bifidobacteria at the acidic pH of -4 found in yoghurt.

13 glucans, for example, (1-3), (1-4) !3 D glucans are found in the endosperm cell wall of such cereals as barley and oat, appear to influence digestion, assist in glucoregulation, and lower serum cholesterol. Cereal 13 glucans are useful nutritional agents and have also been used as bulking agents in place of sucrose. !3 glucans have also been described as potent immune system stimulants and promote the, see Yun et al. Int. J.
Parasitol. (1997) 27=329-337= Es da et al (1997) Microbiol Immunol. 41:991-998; Williams et al.
(,19971 US 5.676.967.

Through the addition of (3 glucan to yoghurt, synergistic formulations of probiotics and soluble fibre are produced with both novel composition and health promoting functionality.

. . . - : - - . . .. .....^.,,:::i:.::::::..;:, -:': :h'i.::i;;;.eF:=eL i.t!!t Disclosure of the Invention The present invention describes the use of cereals and cereal derived products, including whole oat flour, or oatmeal prepared from oats or rolled oat grain, to..,%upport the survival ,ftdobacteria in probiotic products. The products contain high numbers of bi (>108CFU/ml) of viable bifidobacteria, as well as functional numbers (>106CFU/ml) of other lactobacilli. The yoghurt was proven to have a minimum shelf-life of 30 days with no indications of syneresis.

The products also contain significant quantities of soluble fibre. The amount of soluble 1.0 fibre may readily be adjusted in order to comply with the 0.75g of soluble dietary fibre from.'oats per serving that the US FDA requires in order to make a functional food health claim.

The `oat milk' formulation described is novel in both its composition a.pd its formulation method. The formulation method differs from other oat gruels in so far as the P glucan derived from the cereal source is protected from enzymatic degradation in the manufacturing process. The oat milk product is in itself of interest as a functional health drink or the basis of a fermented oat milk drink with probiotic benefits.

The products described, the yoghurt, flummery, drink, and dry probiotic/fibre cereal are non-milk containing and are lactose free making them suitable for special dietary needs.
Brief Description of the Figures .
Figure 1 is a graph of the typical survival of bacteria in yoghurt and the competitive loss of bifrdobacteria.
Figure 2 is a graph of the survival of bacteria, and the enhanced survival of bifidobacteria in oat yoghurt cultures.
Figure 3 is a graph of the survival of bacteria, and the enhanced siuvival of bifidobacteria in oat yoghurt cultures containing artificial flavouring.
Figure 4 is a graph of the survival of bacteria, and the enhanced survival of bifidobacteria in oat yoghurt cultures containing fruit preserves as flavouring.

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} CA 02383021 2002-04-23 Detailed Description of the Invention, The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, cereal chemistry, microbiology, and biochemistry, within the skill of the art. Such techniques are explained fully in the literature. See, e.g.

Oats: Chemistrv andT~chnologX ediWebster FH (American Association of Cereal Chemists. St. Paui_,J<N), Aledical microbioloQV edited by Samuel Baron:. 4th ed (,The iversity of Texas Medical Branch. Galveston. TX) and Tarnine, A.Y. and Robinson.
R K yughurt Science and TechnologyP r mon Press.

to All publications, patents, and patent applications cited herein, whether supra or infra, are incorporated by reference in their entirety.

As used in this specification and the appended claims, the singular forms "a,"
"an," and 1.5 "the" include plural references unless the content clearly indicates otherwise. Thus, the term "a ¾ glucan" can include more than one 13 glucan.

1. Definitions In describing the present invention, the following terms will be employed, and are 20 intended to be defined as indicated below.
By "cereal" is meant any of several grains such as, but not limited to, cultivars of barley, oat, wheat, rye, sorghum, millet, and corn.
By "B glucan" is meant a glucan with a(3(1--3)-linked glucopyranosyl backbone, or a P(1-4)-linked glucopyranosyl backbone, or a mixed (3(1-3) (1 -4)-linked glucopyranosyl 25 backbone.
A "cereal B glucan" or a `cereal R glucan extract" is a B glucan or B glucan extract, respectively, which is derived from a cereal source.
By "probiotic" is meani a live microbial feed supplement which beneficially affects the host animal by improving its intestinal balance.

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By "prebiotic" is meant non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon and thus improve the host's heAlth.
By "cereal milk" is meant an aqueous suspension of a cereal treated'with a-amylase and optionally containing a stabiliser.
By "oat milk" is meant an aqueous suspension oatmeal, bran or oat 13 glucan treated with a-amylase optionally containing a stabiliser.
By "oatmeal" is meant the product of milling or reducing in particle size kernels of whole oats (Avena sativa).
By "oat bran" is meant the food which is produced by grinding clean oat groats or rolled oats ;and separating the resulting oat f)our by sieving, bolting, and/or other similar means into fra.ctions such that the oat bran fraction is not more than 50%> of the starting material, and has a total P glucan content of at least 5.5% (dry weight basis) and a total dietary fibre content of at least 16.0% (dry weight basis), and such that at least one-third of the total dietary fibre is soluble fibre.

2. General Methods Oats and oat extracts are readily available, commercial suppliers include Quaker Oat Company (Chicago, USA), Ceapro Inc. (Edmonton,. Canada), Nutrinova AG
(Frankfurt, Germany).

Oat milli.ng and particle reduction methods are known to those skilled in the art. Milling methods are described in Oats: Chemistrv and 1'echnaloQV ed. Webster FH
(American Association of Cereal Chemists. St. Paul. MN and The Oat Crop: Production and Utilisation. ed Welch. RW(Chanm & Hall. London). For oatmeal preparation oats were ground iii'a Fitz Mill model "M" hammer mill (W.J. Fitzpatrick Co. Model M
Comminutator Series 17 Code 80). The oats were milled until the product passed through a 0.5 mm screen sieve.

Oat bran containing !3 glucan was prepared for use in the present invention by milling through the Fitz Mill as described above with the milled product passing through a , . . = .. . . . . .. . . . .. . ,. .... .. .....a.:.... ri::. . -.. ... .
:h,~,t?.li:!'!'t' 0.5mm screen. Of particular benefit is extruded oat bran containing supra-normal amounts of 13 glucan such as GlucaMaxTM 16% !3 glucan (Nutrinova: Frankfurt, Germany). The extruded nature of the GlucaMax product enhances the performance of a-amylase in starch digestion and the supra normal amounts of !3 glucan enhances the soluble fibre content of the oat yoghurt.

To enhance the funetionality of the oat yoghurt described in the present invention, 13 glucan from any of several known cereal sources can be used in the described process.
Such cereals include, without limitation, any of the cultivars of e.g. barley, oat, wheat, rye, com, sorghum, and millet, with barley and oat preferred because of their high 13 glucan content. Oat 13 glucan is available from commercial suppliers for example Ceapro Inc. (Edmonton, Canada) The !3 glucan content can be determined using a number of inethods, known to those skilled in the art. For example,l3 glucan content can be assessed colorimetrically and/or by standard analytical techniques.such as size exclusion chromatography and HPLC. See Wood et al Cereal Chem. (1977) 54=524LWood et al Cereal Chem (1991) 68=31 =
and Wood et al_. Cereal Chem. (1991) 68:530.13 glucan can also be analyzed enzymatically using commercially available kits, such as Megazyme (Ireland) employing the techniques of McCle= and Glennie-Holmes J Inst. Brew. (198 ) 9 Conventional methods of drying liquids are known to those skilled in the art.
These methods include, but are not limited to, vacuum evaporation and spray drying, and lyophilization. Different spray drying operating modes and configurations are described in Chem. Ing. Tech (1987) 59:112. Lyophilization methods are described in ,,nQwman.
JW nownstreain process= Equipment and Technig-ues (1988) 315 Alanl. Liss.
Bacterial Strains Bacterial strains are available from a number of depositories and collections, for example the American Type Culture Collection (ATCC) Rockville, USA and Collection National de Cultures de Microorganismes, Institut Pasteur, Paris, France.

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Bacterial Growth Media:
MRS broth (DeMan, Rogosa and Sharpe media) for culturing Lactobacillus bulgaricus.
Obtained from BDH Lot #V809761. Preparation: Suspend 52.2g in 1 litre of distilled water. Sterilized through autoclaving for 15 min. at 15 psi.
MRS++ broth (Modified De Man, Rogosa and Sharpe media) for culturing Bifidobacteria infantis. To obtain the modified version of MRS to the basic MRS broth add: 0.2 g/l of NaZCO3, 0.1 g/1 CaC1Z-2HzO and adjust pH to 7.0 before sterilization. After autoclaving and cooling add 10 ml of filter sterilized 5% (w/v cysteine-HC1 solution).

M-17 broth for culturing Streptococcus thermophilus. Obtained from Oxoid Ltd (Basingstoke UK). Preparation: suspend 37.25 g in 950 ml of distilled water.
Autoclave for 15 min. at 15 psi for sterilization. After cooling add 50 ml of a 10%
(w/v) sterilized Lactose solution.
BL-Agar (Glucose blood liver agar) for quantification of lactic acid bacteria.
BL-agar 1.5 was prepared to include defibrinated sheep blood.

Selection of bacterial strains Lactobacillus bulgaricus (ATCC 11842) and Streptococcus thennophilus (ATCC
19258) were selected because of their demonstrated ability to coagulate milk without addition of a stabilizer and their previous use as yoghurt starter culture. These two organisms provided a typical yoghurt aroma and flavour when used with regular milk.

Bifidobacteria selection was based on:
(i) ability to coagulate milk without stabilizer, (ii) human origin, (iii) resistant to an acidic envirot1rnent (pH 4.2 to 4.5), typical of a yoghurt product: Human Bifidobacteria strains tested were: Bffidobacterium longum (ATCC. 15707), Bifidobacterium breve (ATCC 15770), Bifidobacterium adolescentis (ATCC 15703), and Bifidobacterium infantis (ATCC 15697).

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PreDaration of bacterial inocula Inocula were prepared by growing bacteria aerobically: 39-40'C for Lactobacillus bulgaricus [ATCC 11842] in MRS and Streptococcus thermophilus [ATCC 19258] in M-17. Bifidobacterium infantis [ATGC i5697] was grown anaerobically at 39 C in MRS++. After growth the cells were centrifuged at 7970 G's using a Sorvall Model RCS5 (Rotor #10 at 7000 rpm for 20 min). The concentrated cells were washed twice and resuspended in 0.2M sterile potassium buffer pH 6.5 for their future use as inocula.
Inocula consisted of between 10$ and 109CFU/ml viable Bifidobacteria and between 10' and 10$ CFU/ml each of viable Lactobacilli and Streptococci.

Microbiology Counts Miciobiology counts were performed by plating samples in duplicate from T=0 to one month shelf life (t-28-30 days) using BL agar plates, incubated anaerobically at 39-40 C
for 48 hrs. TSA plates were incubated aerobically at 35 C for 48 hrs.

Titratable Acidity Assav Titratable acidity is accepted as a measure of the growth of lactic acid-producing bacteria.
The methodology was adapted from Marshall, R T 1993. Standard Methods for the Examination of Dairy Products 16' Edition. Cap 15, ny. 435-437. Briefly, a 9 g of sample of product was diluted with 18 g of distilled water. Approximately 0.5 ml of phenolphthalein solution was added and the mixture titrated with 0.1N NaOH
solution until first persistent (30 seconds) pink colouration appeared). Acidity was expressed as % of lactic acid by weight (1 ml 0.1N NaOH = 0.0090 g Lactic Acid). Duplicates of titrated samples were averaged before final calculation. For undiluted samples and samples reconstituted to original solids the calculatiqn was as follows:

% acidity =(ml NaOH) (M NaOH) (9g)/weight of sample (g) Svneresis assay Syneresis or whey exudates that appear on the surface of yoghurt are considered to be a defect. Syneresis was measured by making a hole in the base of the tub with a soldering iron and inverting the tub gently onto a fine mesh screen held over the top of a funnel . .. . _ _ ... ..... . . . .. . . . . . . .....: ;;, inserted into a graduated cylinder. This process was done in a cold room (5 C) and the measurement of syneresis was taken after 1 hotir draining time.

C. Experimental Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrat.ive purposes only, and are not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used (e.g.
amounts, temperatures, etc.) but some experimental error and deviation should, of course be allowed for.

Example 1. Production of Oat Milk The inability of the above-mentioned bifidobacteria to coagulate the water-based `oat .15 milk' required the addition of stabilizers to create a set-style yoghurt.
The stabiliser was used at a concentration of 0.5% to meet regulatory requirements for yoghurt manufacture.
The preferred stabilizer was determined to be 0.5% Benlacta 750CM carrageenan (Food Specialities, Mississauga, Canada) or alternatively 0.4% Benlacta 750CM
carrageenan with 0.1% monoglyceride. High molecular B glucan was also useful to enhance both viscosity and add additional fibre content to the yoghurt.

Oat milk containing 12 or 15% oatmeal, 0.5% (w/w) stabilizer, and 0.15% (w/w) a-amylase (Sigma No. A-2771 type VIII-A from barley malt) were resuspended in distilled water, with continuous agitation for 30 min. at room temperature. To obtain a manageable oat milk consistency from the oats in solution, different concentrations of a-amylase were tested. Addition of a-amylase ranged from 0%-0.45% with the optimum being 0.15% w/w with incubation at room temperature for 30 min. based on a minimum of 10% oatmeal solids.

The oat milk obtained was autoclaved for 15 min. at 15 psi (121 C) and cooled before inoculation.

Example 2. Production of oat based yoghurts The step-by-step production protocol and quality control processes are summarized in Table 1.

Table 1.
Step in process Quality control Bacterial Inoculum Microbial counts on BL and TSA agar T=0 pH, titratable acidity, microbial counts onBL and Inoculated Oat Milk TSA agar.
T=0 Option Addition of flavouring or fruit preserve T=4 h pH, titratable acidity After 4 h. incubation at 39-40 C or when H=4.7-4.8 T=24 h. pH, titratable acidity, microbial counts on BL agar After 24 h. in refrigeration 5 C and syneresis.
T=7 d, 14 d, 21 d, 28130 d in pH, titratable acidity, microbial counts on BL
agar Storage under refrigeration 5 C and syneresis.

Sterilized oat milk containing 12% oatmeal and 0.4% Benlacta 750CM carrageenan with 0.1 % monoglyceride was inoculated with 2% each of concentrated washed cells of Lactobacillus bulgaricus and Streptococcus thermophilus and 4% of Bifidobacterium longum concentrated cells. Following inoculation, 100 ml quantities of yoghurt were dispensed in tubs, capped, and incubated aerobically at 39-40 C for approximately 3.5-4.0 hours or until pH reached 4.7-4.8. The tubs of yoghurt were refrigerated at 5 C for one month.
Physico-chemical tests included measuring pH, developed acidity, and syneresis.
Microbial counts were monitored in the inoculum (T=0) and tmtil one month of shelf life (T=28-30`days). The microbial counts for B.longum indicated a consistent survival at >108CFU/ml. Bacterial counts for both L.bulgaricus and.S.thermophilus - remained at >106CFU/ml. A graph of the time course for bacterial cell numbers is given in Figure 2.

The yoglntrts all developed a pH of <4.7 within 24 hours and optimally within the initial 4 hour incubation period.

The titratable acidity assay indicated the rapid achievement of >0. 18% lactic acid within the initial 24 hours of cultivation. Over the following 28 days the titratable lactic acid continued to rise to a average level of 0.27%

Surprisingly, none of the cultures displayed syneresis.
Experiment 3. Production of flavoured oat yoghurts Sterilized oat milk containing 15% oatmeal with 0.5% Benlacta 750CM
carrageenan was prepared using flavouring of:

a. 0.5% vanilla, b. 0.5% cinnamon, c. 0.5% maple syrup, d. 0.5 % cherry, or e. Control of 0.5% sucrose.

The oat milk was inoculated with 2% each of concentrated washed cells of Lactobacillus bulgaricus and Streptococcus thermophilus together with 4% of Bifzdobacterium infantis concentrated cells. Following inoculation, 100 ml quantities of yoghurt were dispensed in tubs, capped, and incubated aerobically at 39-40 C for approximately 3.5-4.0 hours or until pH i=eached 4.7-4.8 and then the tubs were refrigerated at 5 C for one month.

The microbial growth profiles were found to be as described in Experiment 2 and are illustrated in Figure 3. The acid profile and absence of syneresis gave the yoghurts ideal properties for commercial products.

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Experiment 4. Production of flavoured oat yoghurts Sterilized oat milk was prepared containing 15% oatmeal, 0.4% Benlacta 750CM
carrageenan, and 0.1% monoglyceride. Following sterilization of the oat milk, and before inoculation and fermentation the fruit flavouring (preserves) were added to avoid loss of natural colours and flavours which become dull due to the effect of the heat treatment. This technique also avoids reduction of viscosity and limits syneresis (wheying-off) in the final product.

The following flavourings were used: -a. 10% maple syrup/2.5% sucroge, b. 10% peach jam/2.5% sucrose, c. 10% raspberry jam/2.5% sucrose, d. 10% blueberryjam/ 2.5% sucrose, or 1.5 e. Control of 5% sucrose.

The oat milk was inoculated with 2% each of concentrated washed cells of Lactobacillus bulgaricus and Streptococcus thermophilus together with 4% of Bifidobacterium longum concentrated cells. Following inoculation, 100 ml 210 quantities of yoghurt were dispensed in tubs, capped, and incubated aerobically at 39-40 C for approximately 3.5-4.0 hours or until pH reached 4.7-4.8 and then the tubs were refrigerated at 5 C for one month.

The microbial growth profiles were found to be similar as those described in 25 Experiment 2 and are illustrated in Figure 4. The acid profile and absence of syneresis gave the yoghurts ideal properties for commercial products.

Because an invention has been described with regard to preferred embodiments, however it will be understood to persons skilled in the art that a number of 30 variations and modifications can be made without departing from the scope of the invention as described in the following claims.

Claims (7)

1. A method of preparing a fermented cereal-based product, comprising:
inoculating a cereal milk with a bacterial culture, the bacterial culture being a mixture of Lactobacillus sp., Streptococcus sp. and Bifidobacteria sp., and incubating said inoculated milk aerobically for a sufficient amount of time to form the fermented cereal-based product.

2. The method of claim 1 wherein the bacterial culture is a mixture of Lactobacillus bulgaricus, Streptococcus thermophilus, and Bifidobacteriam sp.
selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis and Bifidobacterium infantis.

3. The method of claim 1 or 2, wherein the cereal milk comprises an aqueous suspension of a cereal treated with .alpha.-amylase.

4. The method of claim 3, wherein the cereal milk further comprises a stabilizer.

5. The method of any one of claims 1 to 4, wherein the cereal milk is an oat milk.

6. The method of claim 5, wherein the oat milk comprises oatmeal, oat bran or oat .beta.-glucan.

7. The fermented cereal-based product prepared by the method defined in any one of claims 1 to 6.