Pre-fermented symbiotic matrix based on a cereal suspension with encapsulated probiotics, manufacture process and corresponding utilization

Abstract

A pre-fermented symbiotic matrix based on a cereal suspension containing encapsulated probiotics and prebiotics, the manufacturing process and the corresponding use are disclosed. The invention complements the actual functional food market solving problems inherent to reduced shelf-life of foods due to loss of probiotic viability to values below the minimum limits needed to promote biological activity. The invention also improves the enzymatic process in the preparation of the cereal base and the fermentative process conditions at different levels, namely the ability to control the concentration of sugars in the cereal suspension without adding sugars, increase protein and fiber content, reduce fermentation time to reduce energy consumption during the process and reduce the risk of contamination as well as promote long term microbial stability maintenance. The invention is designed for cases where intolerance and/or allergy to dairy products occur, as wells for the pharmaceutical, cosmetic and food industries, including pet food.

Claims

1. A process of obtaining a fermented cereal product consisting of: preparing a cereal suspension with a cereal concentration between 5 to 50% (w/w), by mixing cereals in water at a temperature between 80° C. and 100° C.; carrying out liquefaction using α-amylases at temperatures controlled between 50° C. and 90° C. and pH between 6 and 8; carrying out saccharification of the liquefied content using glucoamylases at temperatures controlled between 50° C. and 90° C. and pH between 6 and 8; solubilizing using glutaminase at temperatures controlled between 40 and 60° C., or solubilizing using a soluble salt to obtain a suspension; carrying out filtration of the suspension and cooling until a range of temperature between 25° C. and 48° C. is reached thereby obtaining the cereal suspension with the cereal concentration between 5 to 50% (w/w); preparing macroencapsulated immobilized probiotic or non-probiotic microorganisms by preparing a cell culture of microorganisms resuspended in sodium chloride solution thereby obtaining a cellular suspension; preparing a polymer solution with continuous stirring at temperatures between 60° C. and 80° C., followed by cooling down to a temperature range of 35° C. and 45° C.; preparing an oil solution using a mixture of vegetable oil with polysorbate 80 and/or a protective agent; and preparing capsules by mixing the cellular suspension with the polymer solution, adding the oil solution to obtain a homogenized water-in-oil emulsion, and adding a salt solution at a temperature range of 4° C. to 8° C. to form the macroencapsulated immobilized probiotic or non-probiotic microorganisms; adding the macroencapsulated immobilized probiotic or non-probiotic microorganisms to the cereal suspension prepared in the previous steps, such that said microorganisms ferment the cereals and generate an aqueous suspension containing (i) fermented cereal suspension and (ii) macroencapsulated immobilized probiotic or non-probiotic microorganisms; separating the aqueous suspension generated in the previous step from the macroencapsulated immobilized probiotic or non-probiotic microorganisms, to obtain an aqueous fermented cereal suspension without the macroencapsulated immobilized probiotic or non-probiotic microorganisms; and incorporating into the aqueous fermented cereal suspension without the macroencapsulated immobilized probiotic or non-probiotic microorganisms obtained in the previous step, at least one other component selected from the group consisting of: free or encapsulated prebiotics; free or microencapsulated probiotic or non-probiotic microorganisms; and other food ingredients, free or encapsulated.

2. The process of obtaining a fermented cereal product according to claim 1, wherein the cereals are flakes, bran or flour.

3. The process of obtaining a fermented cereal product according to claim 1, wherein the cereals include oats.

4. The process of obtaining a fermented cereal product according to claim 1, wherein the cereals are combined with at least one of the following elements: one or more other cereals applied in food industry; or one or more legumes.

5. The process of obtaining a fermented cereal product according to claim 1, wherein in the step of preparing the cereal suspension the water used therein is previously obtained through an osmosis process in order to remove minerals that cause off-flavours in the water.

6. The process of obtaining a fermented cereal product according to claim 1, wherein the step of preparing the cereal suspension is carried out until a sugar content, in terms of glucose, is at least 150 g/L and at least 25±5° Brix.

7. The process of obtaining a fermented cereal product according to claim 1, wherein the α-amylase is used on a concentration between 0.01% and 2% of cereal concentration, glucoamylase is used on a concentration between 0.01% and 1% of cereal concentration and glutaminase is used on a concentration between 0.01% and 1% of cereal concentration.

8. The process of obtaining a fermented cereal product according to claim 1, wherein the macroencapsulated immobilized probiotic or non-probiotic microorganisms are generally recognized as safe.

9. The process of obtaining a fermented cereal product according to claim 1, wherein the macroencapsulated immobilized probiotic or non-probiotic microorganisms are from the Bifidobacterium or Lactobacillus genera.

10. The process of obtaining a fermented cereal product according to claim 1, wherein the amount of free or microencapsulated probiotic or non-probiotic microorganisms incorporated into the above-described fermented cereal suspension is at least 10.sup.8-10.sup.10 CFU/g.

11. The process of obtaining a fermented cereal product according to claim 1, wherein the macroencapsulated immobilized probiotic or non-probiotic microorganisms are encapsulated in a coating material selected from proteins, polysaccharides, lipids or hydrocolloids.

12. The process of obtaining a fermented cereal product according to claim 1, wherein in the step of adding macroencapsulated immobilized probiotic or non-probiotic microorganisms to the cereal suspension prepared in the previous steps, such that said microorganisms ferment the cereals, is carried out by cereal suspension in a reactor with macroencapsulated immobilized probiotic or non-probiotic microorganisms.

13. The process of obtaining a fermented cereal product according to claim 1, wherein the soluble salt is sodium bicarbonate or sodium hydroxide.

14. The process of obtaining a fermented cereal product according to claim 1, wherein in the step of preparing capsules, the salt solution is KCl or Ca.sub.2Cl.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The present invention reports a cereal symbiotic matrix, preferentially oatmeal, pre-fermented with encapsulated probiotic and prebiotic compounds, its process of manufacture and its use in several applications, especially in the food industry but also in the pharmaceutical industry or similar counterparts.

(2) The products obtained possess organoleptic characteristics that are identical to those produced by traditional fermentation processes.

(3) When encapsulated microorganisms are included, these products also have the advantage of increasing their viability/stability, either as a long shelf-life or during passage through the gastro-intestinal tract following ingestion.

(4) The matrices also present, as an additional advantage, an extended expiration date up to 40% to 60% higher than those presented by available products on the market.

(5) Through the use of this technology one obtains a pre-fermented product with residual quantities of free microorganisms and with the same organoleptic characteristics as those of a traditionally fermented product, being therefore a more valued product.

(6) The immobilization technique of microbial cells confers advantages in comparison to free cell systems, such as: (i) reduction in fermentation time up to 50 to 60%; (ii) increase of the microbial metabolism and stability; (iii) reduced risk of contamination; (iv) higher cell density; (v) stable product quality associated with a decrease of post-acidification risk due to probiotic action, for example; (vi) improved substrate use and (vii) long time cell reutilization due to constant cellular regeneration.

(7) The process of obtaining these products reveals a method for improvement of the fermentative process conditions at several levels such as, (i) continuous reutilization of the immobilized cells; (ii) fermentation time reduction contributes to energy saving throughout the process, (iii) reduction of contaminating risks and (iv) long term maintenance of microbial stability.

(8) 1. Preparation process of oatmeal suspension (concentrate of 5-50% (w/w) 1.1. mixture of flakes, bran and/or flour in water at temperatures ranging from 80-100° C. until achieving starch gelatinization; 1.2. liquefaction using α-amylases (of bacterial or fungi origin) at temperatures controlled between 50° C. and 90° C., pH between 6 and 8; 1.3. saccharification of liquefied oat content using glucoamylases at temperatures controlled between 50° C. and 90° C., pH between 6 and 8; 1.4. solubilisation using a glutaminase at temperatures controlled between 40 and 60° C.; or using a soluble salt such as sodium bicarbonate or sodium hydroxide; 1.5. filtration of the obtained suspension; 1.6. cooling of the mixture until a range of temperatures between 25 and 48° C.

(9) In one embodiment, the water to be used in step 1.1 is previously obtained through an osmosis process in order to standardize the flavour of the final product by removing the minerals that may cause off-flavours.

(10) The entire enzymatic process occurs in a tank during 65 minutes after starch gelatinization performed during the first 15 minutes at 90° C., leading to a process with a total duration of up to 80 minutes.

(11) In one embodiment, the enzymatic process described above ends when sugar content, in terms of glucose, achieves 150 g/L and 25±5° Brix.

(12) The enzymes used in the process described in the present patent application are introduced at the same stage according to a sequential addition respecting the function of each enzymes, viz.—BAN 480 L (α-amylase), AMG 300 L (glucoamylase) and glutaminase. All the enzymes were obtained commercially from NOVOZYMES.

(13) Having an enzyme (glutaminase) and/or salts (sodium bicarbonate) allow to control protein precipitation caused by the pH.

(14) In a particular embodiment, the α-amylase is used on a concentration between 0.01% and 2% of cereal concentration, glucoamylase is used on a concentration between 0.01% and 1% of cereal concentration and glutaminase is used on a concentration between 0.01% and 1% of cereal concentration.

(15) The above-mentioned steps have the advantage of allowing a higher concentration of the cereal to be introduced in the process, leading to commercial products with a cereal concentration up to 50% (w/w). This implies a higher content of protein and fiber due to the high cereal concentration. The steps described above also lead to an increased content of starches and carbohydrates, which are the substrate for the enzymatic process, which in turn leads to an increase of glucose and other oligosaccharides. The high concentration of sugars in the oat base to be fermented, leads to a higher bioavailability of oligosaccharides and simple sugars for the fermentative action of the microorganisms (which require a given content of sugars). This also allows to decrease the addition of the inoculum to a minimum of 0.1% (m/v). This way, the final product will not require any added sugars, which in turn makes the product more appealing to the consumer.

(16) Additionally, the liquefaction step, also enables the standardization of cereal viscosity values (<500 Cp) for industrial production.

(17) 2. Process of pre-fermentation in fluidized bed reactor associated with cell encapsulation by emulsion 2.1. Process of pre-fermentation The process of pre-fermentation is performed in a fluidized bed reactor with immobilized microorganisms by cells obtained in steps 2.1.1. through 2.4.3. The capsules are introduced in a column, with porosity smaller than the diameter of the capsules to induce the microorganisms-matrix interaction, inside the pressurized reactor with constant and controlled bi-directional nitrogen flow. The immobilized cells inside the column are reutilized in the fermentation process until they lose their metabolic properties. 2.1.1. Cell culture preparation 2.1.1.1. Preparation of the inocula from frozen cultures and consequent activation by two consecutive transfers in MRS Broth supplemented with L-cysteine-HCl 0.05% (w/v). 2.1.1.2. Inoculation of 1 to 20% (v/v) in 1000 mL of MRS Broth (Man Rogosa and Sharpe) supplemented with L-cysteine-HCl 0.05% (m/v) and subsequent incubation for 24 h at 37° C., under anaerobic conditions, for Lactobacillus acidophilus Ki and 48 h at 37° C. under anaerobic conditions for Bifidobacterium animalis Bo and Bb12, for example. 2.1.1.3. Centrifugation of the resulting cultures at 4000 rpm for 15 minutes, at 4° C., subsequent washing of pellet with, for example, NaCl 0.9% (w/v) solution, and resuspension in 100 mL of the same solution. 2.2. Polymer solution preparation 2.2.1. Preparation of a polymer solution, for example, k-carrageenan 1 to 5% (w/v), with continuous stirring, variable duration between 1 to 4 hours, temperatures between 60 and 80° C., followed by cooling down to a temperature range of 35 to 45° C. 2.3. Oil solution preparation 2.3.1. Mixture of vegetable oil with one of the following compounds: Tween 80 0.2% (v/v) and/or a protective agent, as for a non-limiting example, laurel sodium sulphate 0.5% (v/v). 2.4. Capsule preparation 2.4.1. Mixture of cellular suspension 1 to 20% (v/v) (see 2.1.) with the polymer solution 1 to 5% (w/v) (see 2.2.). 2.4.2. Addition of the resulting mixture to 75 to 98% of the prepared oil solution (see 2.3.). The obtained solution is homogenised forming a water-in-oil emulsion. 2.4.3. The capsule formation occurs after the addition of a solution of KCl 10 mM, for example, to the mixture at a temperature range of 4 to 8° C.

(18) 2.5. Pre-fermentation process operation conditions

(19) After attainment of the capsules with microorganisms for utilization in a fluidized bed reactor, the process of pre-fermentation is performed at a temperature between 20° C. to 52° C., during 4 to 8 hours, under sterile and anaerobic conditions (circulating nitrogen flux), resulting in a fermented matrix. This suspension is drained into the reactor where the incorporation of the remaining food ingredients occurs.

(20) 3. Microorganisms encapsulation process by emulsion and/or spray-drying

(21) The microorganisms' encapsulation is done using the encapsulation techniques: 3.1. Emulsion, as described in point 2; 3.2. Spray-drying: 3.2.1. Preparation of a cellular suspension with polymers (see points 2.2; 2.2.1; and 2.4.1); 3.2.2. Drying of 250 ml of the previous mixture under the constant conditions of inlet and outlet temperatures of 150-175° C. and 50-85° C., respectively; 3.2.3. Addition of the resulting powder into the pre-fermented oat suspension (point 2.5) in a proportion of 2-5% (w/v), in a way to guarantee 10.sup.8-10.sup.10 CFU in the matrix, per 100 g or 100 mL.

(22) 4. Food ingredients incorporation:

(23) Addition of ingredients to the matrix obtained in the previous process (point 3.2.3,), having as an example inulin, at a concentration range between 1-3%, maintaining, as a non limitative example sea-salt, among others.

(24) 5. Presentation forms of the matrix

(25) The pre-fermented symbiotic matrix based on an oat suspension with encapsulated probiotics can be presented either in a fresh form, lyophilized and/or frozen. The fresh matrix can be further presented either in gel or extruded form.

(26) 6. Microbiological/Shelf-life study

(27) When encapsulated microorganisms are included, the products also have the advantage of increasing their viability, stability, either as a long shelf-life or during passage through the gastro-intestinal tract following ingestion. The fermented cereal product of the present patent application also presents an extended expiration date up to 40% to 60% higher than those presented by available products on the market.

(28) Furthermore, the fermented symbiotic matrix is free from fermenting microorganisms and consequently post-acidification phenomenon does not occur, thereby increasing the shelf-life.

(29) The advantage conferred by the invention is demonstrated by Table 1, where the ability to enhance the time of shelf-life is compared to a fermented product such as yogurt. By definition, shelf-life is the period during which the product maintains its microbiological safety and suitability at a specified storage temperature and, where appropriate, specified storage and handling conditions (see, e.g. Code of Hygienic Practice for Milk and Milk Products—CAC/RCP 57-2004).

(30) Shelf life experiments with the fermented cereal product of the present application have been conducted in order to identify and compare the time that the product is stable and with the desired characteristics (microorganisms' quality in order to have beneficial effect on the final consumer).

(31) The main goal of this study was to follow the overall product microbiology evolution of two different samples/microorganisms (Lactobacillus acidophilus and Bifidobacterium lactis): i) the behavior of the residual microorganisms on the cereal suspension, ii) the behavior of the barrier/passage between the cereal and the microorganisms inside the capsules and iii) their own behavior inside.

(32) The medium used on this study was applied specifically to enumerate the microorganisms Lactobacillus acidophilus and Bifidobacterium lactis. The study range temperature used was to simulate the refrigeration temperature of the storage period on this kind of fermented product. The duration of the study and the sampling points were done to evaluate the product shelf-life and compare with similar fermented products.

(33) At the end of the study, the capsules' sample with Bifidobacterium lactis had enough microorganisms' enumerations to guarantee the minimum biological beneficial effect at the ingestion of the fermented product (at least 10.sup.8-10.sup.10 CFU/g). The sample with Lactobacillus acidophilus encapsulated can easily achieve the same doses on other productions because the level obtained of the inoculated microorganisms was similar (10.sup.7 to 10.sup.8 CFU/g).

(34) The report showed that the product obtained by the invention was suitable until, at least, 52 days which is a significantly higher duration than other regular commercial products, such as yogurt, which typically have a shelf-life of approximately 24 days. Thus, when compared with the shelf-life of the product obtained by the method of the invention the product indeed has 40-60% higher shelf life.

(35) TABLE-US-00001 TABLE 1 Shelf-life - Microbiological study: Data (results in CFU/g): Sample Name Initial Day 4 Day 8 Day 12 Day 20 Day 32 Day 36 Day 40 Day 52 Lactobacillus 2.7 e7 1.3 e7 2.4 e7 1.0 e7 1.0 e7 3.0 e7 2.2 e7 3.3 e7 6.5 e7 Capsule 1 Oat Slurry 1 <100 <100 600 <100 3600 1000 1500 <100 <100 Bifidobacterium 1.3 e8 1.2 e8 1.2 e8 1.1 e8 3.0 e8 1.0 e8 3.0 e8 2.0 e8 6.0 e8 Capsule 2 Oat Slurry 2 1400 3200 100  200 2400  700 <100  200  900 Lactobacillus Capsule 1 - Capsules with microorganisms Genera Lactobacillus Oat Slurry 1 - Oat suspension separated from capsules with microorganisms Genera Lactobacillus Bifidobacterium Capsule 2 - Capsules with microorganisms Genera Bifidobacterium Oat Slurry 2 - Oat suspension separated from capsules with microorganisms Genera Bifidobacterium

(36) To study microorganisms's enumeration in MRS Agar supplemented with L-cysteine-HCl 0.05% (w/v) using spread technique to determine lactic count for the capsules and oat suspension. The product was stored at 4° C.-6° C. and analyzed at the following timepoints: initial, 4, 8, 12, 20, 32, 36, 40 and 52 days. The first decimal dilution was −2.

EXAMPLES

Example I

(37) Oat concentrated base is obtained from 30% (w/w) oat bran aqueous extraction, which is submitted to an enzymatic treatment of partial hydrolysis and saccharification of starch, followed by a heat treatment.

(38) Produced from dehulled, cleaned and heat stabilized oats by flaking and cleaning followed by milling.

(39) Cereal Sources/Types:

(40) Oat bran

(41) Industrial Procedure:

(42) In this example oat bran is dispersed using a blend table into a tank that contains hot water at 90° C. Water used to mixture oat bran is previously obtained through an osmosis process in order to standardize the flavour of final product through the removal of minerals that can cause off-flavours.

(43) Oat dispersion is performed according to a factorial plan that involves percentage of cereal, enzymes concentration, time, temperature, sugar formation and Brix, according to the steps below.

(44) The process comprises three main steps: liquefaction using α-amylases at optimal operation conditions namely temperatures between 50° C. and 70° C. at pH maintained between 6 and 8; saccharification using alfa-glucoamylases at optimal operation conditions namely 50° C.≤T≤70° C. at neutral pH; and oat solubilization using a glutaminase at optimal operation conditions namely 40° C.≤T≤60° C. at a pH maintained between 6 and 8.

(45) For this example, the enzymes used are BAN 480 L at 0.30% of cereal concentration, AMG 300 L at 0.40% of cereal concentration and glutaminase at 0.10% of cereal concentration. All the enzymes are commercialized by NOVOZYMES.

(46) The enzymes used are introduced at the same stage according to a sequential addition respecting the function of each enzymes, viz.—BAN 480 L, AMG 300 L and glutaminase.

(47) The entire enzymatic process occurs in a tank during 65 minutes after starch gelatinization performed during the first 15 minutes at 90° C., reaching 80 minutes process.

(48) The enzymatic process ends when sugar content, in terms of glucose, achieves 150 g/L and 25±5° Brix.

(49) Decantation of oat slurry is applied when whole grain or bran is used in the formulation. Decantation can be omitted when insoluble fibers are considered a product specification.

(50) Product resulted from decantation viz.—oat base can be submitted to a fermentation process according the present invention.

(51) In this particularly example, the oat base/oat suspension is cooled from 70° C. to 43° C. through a heat exchanger. Fermentation is carried out into a jacketed tank with controlled pH, temperature and agitation (constant 50 rpm). Lactic acid bacteria (LAB) and bifidobacteria microorganisms are introduced by direct VAT into a tank with constant agitation together with isotonic solution to hydrate. The encapsulation process is performed with the addition of the polymer with oil and the inoculum together with the contact with Ca.sub.2Cl solution at 0.1 M to the capsule formation at 4 to 8° C. Capsules with YF L02, from CHR Hansen, are introduced at 0.10% inoculum to the fermenter tank, which leads to a 4 hours fermentation process, ending when pH of oat base is less than 4.55. After the fermentation there is a step of drain the base to continue the process ahead and the remain capsules stay at the fermenter in order to ferment the next round up to 3 times.

(52) Enzymatic activity is eliminated by a direct steam injection unit which comprise the following steps: a) pre-heating of oat base up to 90° C., direct steam injection, sterilization binomial time and temperature (130° C. for 30 seconds), cooling process followed by aseptic filling.

(53) The oat liquid prepared may be used as such or used as a basis for the preparation of different products with the addition of food ingredients namely encapsulated microorganisms.

(54) According with this example, the final product (symbiotic matrix) has the following tables (2, 3 and 4) with physic-chemical, organoleptic and microbiological characteristics:

(55) TABLE-US-00002 TABLE 2 PHYSICAL AND CHEMICAL CHARACTERISTICS Tolerances can be applied due to raw material natural variability. Characteristic Method Unit Target Minimum Maximum Tolerance pH Potentiometer pH unit 4.55 4.0 6.5 n.a. Brix Refractometer °Brix 25.0 20.0 30.0 n.a. Dry Matter Oven (105° C., % (w/w) 27.0 22.0 32.0 ±5 Content constant weight) Glucose Glucometer g/l 120 80 200 n.a. (rapid method) (dilution 1|300) Protein (F = 6.25) % (w/w) 3.0 2.5 5.0 n.a  Viscosity Brookfield cP n.a. n.a. <500 n.a. (20° C., RPM, spindle)

(56) TABLE-US-00003 TABLE 3 ORGANOLEPTICAL CHARACTERISTICS Direct Inspection/ Homogeneous product with characteristic Aspect appearance, color and smell. No dark specks or any other type of visual defects. Color Yellowish, without brown or strange colors. Odor Characteristic to cereal, without strange odors. Taste Characteristic to cereal, sweet, without off-flavors. Mouthfeel With a certain viscosity, non-watery sensation.

(57) TABLE-US-00004 TABLE 4 MICROBIOLOGICAL CHARACTERISTICS CFU/g Target* Total viable count at 30° C. Absence in 50 g Yeasts Absence in 50 g Moulds Absence in 50 g Spore forming bacteria Absence in 50 g LAB and Bifidobacteria (YF-L02) 10.sup.6 to 10.sup.7

Example II

(58) Oat concentrated base is obtained from 40% (w/w) whole grain oat flour aqueous extraction, which is submitted to an enzymatic treatment of partial hydrolysis and saccharification of starch, followed by a heat treatment. Produced from dehulled, cleaned and heat stabilized oats by flaking and cleaning followed by milling.

(59) Cereal Sources/Types:

(60) Whole grain oat flour

(61) Industrial Procedure:

(62) In this example oat bran is dispersed using a blend table into a tank that contains hot water at 90° C. Water used to mixture oat bran is previously obtained through an osmosis process in order to standardize the flavour of final product through the removal of minerals that can cause off-flavours.

(63) Oat dispersion is performed according to a factorial plan that involves percentage of cereal, enzymes concentration, time, temperature, sugar formation and Brix, according to the steps below.

(64) The process comprises three main steps: liquefaction using α-amylases at optimal operation conditions namely temperatures between 70° C. and 90° C. at pH maintained between 6 and 8; saccharification using α-glucoamylases at optimal operation conditions namely 50° C. T 70° C. at neutral pH; and oat solubilization using a soluble salt, for example, sodium bicarbonate at 0.15% (w/v).

(65) For this example, the enzymes used are TERMAMYL at 0.90% of cereal concentration and AMG 300 L at 0.30% of cereal concentration. All the enzymes are commercialized by NOVOZYMES.

(66) The enzymes used are introduced at the same stage according to a sequential addition respecting the function of each enzymes, viz. —TERMAMYL and AMG 300 L.

(67) The entire enzymatic process occurs in a tank during 45 minutes after starch gelatinization performed during the first 35 minutes at 90° C., reaching 80 minutes process.

(68) The enzymatic process ends when sugar content, in terms of glucose, achieves 150 g/L and 30±5° Brix.

(69) Decantation of oat slurry is applied when whole grain is used in the formulation. Decantation can be omitted when insoluble fibers are considered a product specification.

(70) Product resulted from decantation viz.—oat base can be submitted to a fermentation process according the present invention.

(71) In this particularly example, the oat base/oat suspension is cooled from 70° C. to 43° C. through a heat exchanger. Fermentation is carried out into a jacketed tank with controlled pH, temperature and agitation (constant 50 rpm). Bifidobacteria microorganisms are introduced by direct VAT into a tank with constant agitation together with isotonic solution to hydrate. The encapsulation process is performed with the addition of the polymer with oil and the inoculum together with the contact with Ca.sub.2Cl solution at 0.1 M to the capsule formation at 4 to 8° C. Capsules with Nu-trish® BY-01 DA, from CHR Hansen, are introduced at 0.10% inoculum to the fermenter tank, which leads to a 4 hours fermentation process, ending when pH of oat base is less than 4.55. After the fermentation there is a step of drain the base to continue the process ahead and the remain capsules stay at the fermenter in order to ferment the next round until 3 times. The capsules are reused at least three times in the next fermentation processes.

(72) Enzymatic activity is eliminated by a direct steam injection unit which comprise the following steps: a) pre-heating of oat base up to 90° C., direct steam injection, sterilization binomial time and temperature (130° C. for 30 seconds), cooling process followed by aseptic filling.

(73) The oat liquid prepared may be used as such or used as a basis for the preparation of different products with the addition of food ingredients namely encapsulated microorganisms.

(74) According with this example, the final product (symbiotic matrix) has the following tables (5, 6 and 7) with physic-chemical, organoleptic and microbiological characteristics:

(75) TABLE-US-00005 TABLE 5 PHYSICAL AND CHEMICAL CHARACTERISTICS Tolerances can be applied due to raw material natural variability. Characteristic Method Unit Target Minimum Maximum Tolerance pH Potentiometer pH unit 4.55 4.0 6.5 n.a. Brix Refractometer °Brix 35.0 30.0 40.0 n.a. Dry Matter Oven (105° C., % (w/w) 37.0 32.0 42.0 ±5 Content constant weight) Glucose Glucometer g/l 120 80 200 n.a. (rapid method) (dilution 1|300) Protein (F = 6.25) % (w/w) 3.0 2.5 5.0 n.a  Viscosity Brookfield cP n.a. n.a. <500 n.a. (20° C., RPM, spindle)

(76) TABLE-US-00006 TABLE 6 ORGANOLEPTICAL CHARACTERISTICS Direct Inspection/ Homogeneous product with characteristic Aspect appearance, color and smell. No dark specks or any other type of visual defects. Color Yellowish, without brown or strange colors. Odor Characteristic to cereal, without strange odors. Taste Characteristic to cereal, sweet, without off-flavors. Mouthfeel With a certain viscosity, non-watery sensation.

(77) TABLE-US-00007 TABLE 7 MICROBIOLOGICAL CHARACTERISTICS CFU/g Target* Total viable count at 30° C. Absence in 50 g Yeasts Absence in 50 g Moulds Absence in 50 g Spore forming bacteria Absence in 50 g Bifidobacteria (nu-trish ® BY-01 DA) 10.sup.6 to 10.sup.7

DESCRIPTION OF EMBODIMENTS

(78) Now, preferred embodiments of the present application will be described in detail.

(79) The present patent application describes a process of obtaining a fermented cereal product comprising the following steps: Preparation process of cereal suspension with a concentration between 5 to 50% (w/w), wherein the preparation step comprises the mixture cereals in water at temperature between 80° C. and 100° C.; liquefaction using α-amylases at temperatures controlled between 50° C. and 90° C. and pH between 6 and 8; saccharification of the liquefied content using glucoamylases at temperatures controlled between 50° C. and 90° C. and pH between 6 and 8; and solubilisation using glutaminase at temperatures controlled between 40 and 60° C. or using a soluble salt such as sodium bicarbonate or sodium hydroxide. Adding macroencapsulated immobilized probiotic or non-probiotic microorganisms to the cereal suspension prepared on the previous step, such that said microorganisms ferment the cereals and generate an aqueous suspension containing (i) fermented cereal suspension and (ii) macroencapsulated immobilized probiotic or non-probiotic microorganisms; Separating the aqueous suspension generated in the previous step from the macroencapsulated immobilized probiotic or non-probiotic microorganisms, to obtain an aqueous fermented cereal suspension without the macroencapsulated immobilized probiotic or non-probiotic microorganisms; and Incorporating into the aqueous fermented cereal suspension without the macroencapsulated immobilized probiotic or non-probiotic microorganisms obtained in the previous step, at least on other component selected from the group consisting of: Free or encapsulated prebiotics; Free or microencapsulated probiotic or non-probiotic microorganisms; Other food ingredients, free or encapsulated.

(80) In one embodiment, the cereals are selected from flakes, bran or flour.

(81) In one embodiment, the cereals include oats.

(82) In another embodiment, the cereals are combined with at least one of: One or more other cereals usually applied in food industry; or One or more legumes.

(83) In one embodiment, the water to be used in the first step is previously obtained through an osmosis process in order to standardize the flavour of the final product by removing the minerals that may cause off-flavours.

(84) In one embodiment, the enzymatic process described above ends when sugar content, in terms of glucose, achieves at least 150 g/L and at least 25±5° Brix.

(85) In a particular embodiment, the α-amylase is used on a concentration between 0.01% and 2% of cereal concentration, glucoamylase is used on a concentration between 0.01% and 1% of cereal concentration and glutaminase is used on a concentration between 0.01% and 1% of cereal concentration.

(86) In one embodiment the fermented cereal product has residual quantities of free microorganisms, and comprises encapsulated prebiotics, free or microencapsulated probiotic or non-probiotic microorganisms, and other food ingredients selected from the group consisting of antioxidants, fatty acids, vitamins, minerals, sweeteners, flavors, fruit pulp and enzymes.

(87) In one embodiment, the macroencapsulated immobilized probiotic or non-probiotic microorganisms are generally recognized as safe.

(88) In one particular embodiment, the macroencapsulated immobilized probiotic or non-probiotic microorganisms are from the Bifidobacterium or Lactobacillus genera.

(89) In one embodiment, the amount of free or microencapsulated probiotic or non-probiotic microorganisms incorporated into the above-described fermented cereal suspension is at least 10.sup.8-10.sup.10 CFU/g.

(90) In one embodiment, the macroencapsulated immobilized probiotic or non-probiotic microorganisms are encapsulated in a coating material selected from proteins, polysaccharides, lipids or hydrocolloids.

(91) In one embodiment, the fermentation comprises placing a cereal suspension in a reactor with macroencapsulated immobilized probiotic or non-probiotic microorganisms.

(92) The fermented cereal product obtained by the process described in the present patent application comprises: an aqueous fermented cereal suspension without macroencapsulated immobilized probiotic or non-probiotic microorganisms; at least one other component selected from a group consisting of: i. free or encapsulated prebiotics; ii. free or microencapsulated probiotics/non-probiotics; and iii. other food ingredients, free or encapsulated selected from the group consisting of antioxidants, fatty acids, vitamins, minerals, sweeteners, flavors, fruit pulp and enzymes; a glucose concentration of at least 150 g/L and at least 25±5° Brix.

(93) In one embodiment, the fermented cereal product has a water activity (aw) of at least 0.3.

(94) In one embodiment, said prebiotics comprise β-glucan soluble fiber, in biologically active quantities, preferably extracted from cereals or legumes. In a preferable embodiment, said prebiotics comprise a minimum of 0.75% (w/w) of β-glucan soluble fiber.

(95) In one embodiment, apart from β-glucan soluble fibres, other prebiotic compounds such as, non limiting examples, inulin, fructooligossacharides (FOS) and chitosans.

(96) A fermented cereal product, characterised by allowing the integration of other food ingredients, that apart from the prebiotic function can still allow for other functions, such as, non limiting, organoleptic functions (such as, non limiting, sweeteners, flavours and/or fruit pulp) and texture (such as enzymes).

(97) Additional embodiments according to the invention are provided below:

(98) Embodiment 1 is process of obtaining a fermented cereal product by carrying out the following steps: preparation process of cereal suspension with a cereal concentration between 5 to 50% (w/w), wherein the preparation step comprises the mixture cereals in water at temperature between 80° C. and 100° C.; liquefaction using α-amylases at temperatures controlled between 50° C. and 90° C. and pH between 6 and 8; saccharification of the liquefied content using glucoamylases at temperatures controlled between 50° C. and 90° C. and pH between 6 and 8; and solubilisation using glutaminase at temperatures controlled between 40 and 60° C. or using a soluble salt such as sodium bicarbonate or sodium hydroxide; adding macroencapsulated immobilized probiotic or non-probiotic microorganisms to the cereal suspension prepared on the previous step, such that said microorganisms ferment the cereals and generate an aqueous suspension containing (i) fermented cereal suspension and (ii) macroencapsulated immobilized probiotic or non-probiotic microorganisms; separating the aqueous suspension generated in the previous step from the macroencapsulated immobilized probiotic or non-probiotic microorganisms, to obtain an aqueous fermented cereal suspension without the macroencapsulated immobilized probiotic or non-probiotic microorganisms; and Incorporating into the aqueous fermented cereal suspension without the macroencapsulated immobilized probiotic or non-probiotic microorganisms obtained in the previous step, at least on other component selected from the group consisting of: Free or encapsulated prebiotics; Free or microencapsulated probiotic or non-probiotic microorganisms; Other food ingredients, free or encapsulated.

(99) Embodiment 2 is a process of obtaining a fermented cereal product according to embodiment 1, in which the cereals are selected from flakes, bran or flour.

(100) Embodiment 3 is a process of obtaining a fermented cereal product according to embodiment 1, in which the cereals include oats.

(101) Embodiment 4 is a process of obtaining a fermented cereal product according to embodiment 1, in which the cereals are combined with at least one of: One or more other cereals usually applied in food industry; or One or more legumes.

(102) Embodiment 5 is a process of obtaining a fermented cereal product according to embodiment 1, in which the water to be used in the first step is previously obtained through an osmosis process in order to standardize the flavour of the final product by removing the minerals that may cause off-flavours.

(103) Embodiment 6 is a process of obtaining a fermented cereal product according to embodiment 1, in which the enzymatic process described above ends when sugar content, in terms of glucose, achieves at least 150 g/L and at least 25±5° Brix.

(104) Embodiment 7 is a process of obtaining a fermented cereal product according to embodiment 1, in which the α-amylase is used on a concentration between 0.01% and 2% of cereal concentration, glucoamylase is used on a concentration between 0.01% and 1% of cereal concentration and glutaminase is used on a concentration between 0.01% and 1% of cereal concentration.

(105) Embodiment 8 is a process of obtaining a fermented cereal product according to embodiment 1, wherein the macroencapsulated immobilized probiotic or non-probiotic microorganisms are generally recognized as safe.

(106) Embodiment 9 is a process of obtaining a fermented cereal product according to embodiment 1, wherein the macroencapsulated immobilized probiotic or non-probiotic microorganisms are from the Bifidobacterium or Lactobacillus genera.

(107) Embodiment 10 is a process of obtaining a fermented cereal product according to embodiment 1, in which the amount of free or microencapsulated probiotic or non-probiotic microorganisms incorporated into the above-described fermented cereal suspension is at least 10.sup.8-10.sup.10 CFU/g.

(108) Embodiment 11 is a process of obtaining a fermented cereal product according to embodiment 1, in which the macroencapsulated immobilized probiotic or non-probiotic microorganisms are encapsulated in a coating material that ca be proteins, polysaccharides, lipids or hydrocolloids.

(109) Embodiment 12 is a process of obtaining a fermented cereal product according to embodiment 1, where the fermentation includes the placing of a cereal suspension in a reactor with macroencapsulated immobilized probiotic or non-probiotic microorganisms.

(110) Embodiment 13 is a fermented cereal product obtained according to process described in embodiment 1, which includes: an aqueous fermented cereal suspension without macroencapsulated immobilized probiotic or non-probiotic microorganisms; at least one other component selected from a group consisting of: i. free or encapsulated prebiotics; ii. free or microencapsulated probiotics/non-probiotics; and iii. other food ingredients, free or encapsulated selected from the group consisting of antioxidants, fatty acids, vitamins, minerals, sweeteners, flavors, fruit pulp and enzymes; a glucose concentration of at least 150 g/L and at least 25±5° Brix.

(111) Embodiment 14 is a fermented cereal product according to embodiment 13, wherein said prebiotics includes β-glucan soluble fiber, in biologically active quantities, preferably extracted from cereals or legumes. In a preferable embodiment, said prebiotics comprise a minimum of 0.75% (w/w) of β-glucan soluble fiber.

(112) Embodiment 15 is a fermented cereal product according to embodiment 14, wherein apart from β-glucan soluble fibres, other prebiotic compounds such as, inulin, fructooligossacharides (FOS) and chitosans are included.