Microencapsulated bacterial consortium for the degradation of gluten into sourdough and method for producing said sourdough

Abstract

The present invention is related to a microencapsulated bacterial consortium for gluten degradation, which comprises: a) three different strains of commercially available lactic-acid bacteria; b) encapsulating agents; c) prebiotics; and d) trehalose; in combination with a proteolytic enzyme of bacterial origin and a proteolytic enzyme of fungal origin. Preferably, the microencapsulated bacterial consortium comprises: a) Lactobacillus plantarum ATCC 8014; b) Lactobacillus sanfranciscensis ATCC 27652; c) Lactobacillus brevis ATCC 14869; d) isolated protein from milk serum with 90% protein; e) maltodextrin with a dextrose equivalent of 10; f) arabic gum; g) maguey honey; and h) trehalose; in combination with a protease of bacterial origin and a protease of fungal origin. It also describes a process for obtaining the microencapsulated bacterial consortium, as well as the preparation of sourdoughs therefrom, and the use of said sourdoughs to obtain baking products.

Claims

1. A microencapsulated bacterial consortium for gluten degradation comprising: (a) the following three different species of lactic-acid bacteria: Lactobacillus plantarum ATCC 8014, Lactobacillus sanfranciscensis ATCC 27652 and Lactobacillus brevis ATCC 14869, and no other species of lactic acid bacteria; b) encapsulating agents; c) prebiotics; and d) trehalose; in combination with a proteolytic enzyme of bacterial origin and a proteolytic enzyme of fungal origin.

2. A microencapsulated bacterial consortium, according to claim 1, wherein the encapsulating agents are selected from the group consisting of isolated protein from milk serum with 90% protein, maltodextrin with a dextrose equivalent of 10, arabic gum, mesquite gum, sodium alginate, pectin, soy isolated protein, casein and combinations thereof.

3. A microencapsulated bacterial consortium, according to claim 2, wherein the encapsulating agents are isolated protein from milk serum with 90% protein, maltodextrin with a dextrose equivalent of 10, arabic gum and combinations thereof.

4. A microencapsulated bacterial consortium, according to claim 1, wherein the prebiotics are selected from the group consisting of polydextrose, inulin and maguey honey.

5. A microencapsulated bacterial consortium, according to claim 4, wherein the prebiotic is maguey honey.

6. A microencapsulated bacterial consortium, according to claim 1, wherein the proteolytic enzyme of bacterial origin and the proteolytic enzyme of fungal origin are selected from enzymes commonly used for baking.

7. A microencapsulated bacterial consortium, according to claim 1, comprising: a) Lactobacillus plantarum ATCC 8014; b) Lactobacillus sanfranciscensis ATCC 27652; c) Lactobacillus brevis ATCC 14869; d) isolated protein from milk serum with 90% protein; e) maltodextrin with a dextrose equivalent of 10; f) arabic gum; g) maguey honey; and h) trehalose; in combination with a proteolytic enzyme of bacterial origin and a proteolytic enzyme of fungal origin.

8. A microencapsulated bacterial consortium, according to claim 7, comprising: a) Lactobacillus plantarum ATCC 8014; b) Lactobacillus sanfranciscensis ATCC 27652; c) Lactobacillus brevis ATCC 14869; d) from 40 to 60% of isolated protein from milk serum with 90% protein; e) from 10 to 20% of maltodextrin with a dextrose equivalent of 10; f) from 20 to 80% arabic gum; g) from 1 to 10% of maguey honey; and h) trehalose; in combination with 0.005 to 0.03% of a proteolytic enzyme of bacterial origin and 0.001 to 0.02% of a proteolytic enzyme of fungal origin.

9. A process for producing a microencapsulated bacterial consortium for gluten degradation, comprising the steps of: a) separately reactivating each strain of lactic-acid bacteria; b) when each strain is active, separately culturing in a liquid culture medium until achieving each strain desired concentration; c) for each strain, removing the excess culture medium to concentrate the microorganisms, obtaining a pellet; d) separately, resuspending the pellet obtained for each strain in saline suspension and adjusting to the required volume; e) mixing the necessary amounts of each strain and taking to a final volume; f) dissolving the encapsulating agents in water, at the proper ratios to have 20 to 30% of dissolved solids; g) adding prebiotics and trehalose; h) inoculating with the mixture of the three strains of lactic-acid bacteria, such that about 10.sup.10 CFU of said mixture of three strains of lactic-acid bacteria per gram of powdered microencapsulated are obtained; and i) drying, to an inlet temperature of between 110 and 160 C., and an outlet temperature of between 60 and 80 C., with a feed rate between 20 and 50 mL/min.

10. A process for producing a microencapsulated bacterial consortium, according to claim 9, wherein the drying is carried out by a spray-dryer, at an inlet temperature of 150 C., and at an outlet temperature of 80 C., with a feed rate of 22 mL/min.

11. A process to make a sourdough from a microencapsulated bacterial consortium for gluten degradation, in combination with a proteolytic enzyme of bacterial origin and a proteolytic enzyme of fungal origin, comprising the following steps: i) mixing 30 to 60% flour with 40 to 80% water to obtain a dough with a dough yield (DY) from 150 to 475; ii) adding 0.005 to 0.03% proteolytic enzyme of bacterial origin and 0.001 to 0.02% proteolytic enzyme of fungal origin previously dissolved in kneading water; iii) adding the microencapsulated bacterial consortium such that about 10.sup.10 CFU/g flour is present; iv) mixing; and v) fermenting for 3 to 48 hours, at a temperature from 30 to 37 C., and a relative humidity from 70 to 92%.

12. A process to make a sourdough, according to claim 11, wherein the flour used in step i) is selected from wheat flour, rye flour and oatmeal flour.

13. A process to make a dough sour, according to claim 12, wherein the flour is wheat flour.

14. A process to make a sourdough, according to claim 11, wherein the fermentation is carried out during 31 hours, at a temperature of 35 C. and a relative humidity of 76%.

15. A process for producing a microencapsulated bacterial consortium, according to claim 9, wherein in step c) the excess culture medium is removed by centrifugation.

16. A process to make a sourdough, according to claim 11, wherein in step i) the dough obtained has a dough yield (DY) from 150 to 160.

17. A process to make a sourdough, according to claim 11, wherein in step v) the fermentation is carried out for 28 to 35 hours.

18. A process to make a sourdough, according to claim 11, wherein in step v) the fermentation is carried out at a relative humidity from 75 to 80%.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) During the development of the present invention, it was unexpectedly found that a microencapsulated bacterial consortium comprising three different strains of commercially available lactic acid bacteria; encapsulating agents to provide a high level of protection to the bacterial consortium, and fast dissolution in the wheat dough system so that its activity starts quickly; prebiotics to achieve the highest survival of the consortium; and, trehalose as a specific nutritional ingredient of the bacteria they integrate, when used in combination with a proteolytic enzyme of bacterial origin and a proteolytic enzyme of fungal origin, both also commercially available, is useful for gluten degradation during the sourdough fermentation process.

(2) Preferably, lactic-acid bacteria strains are Lactobacillus plantarum ATCC 8014, Lactobacillus sanfranciscensis ATCC 27652 and Lactobacillus brevis ATCC 14869.

(3) Encapsulating agents are selected from the group comprising isolated protein from milk serum with 90% protein, maltodextrin with a dextrose equivalent of 10, arabic gum, mesquite gum, sodium alginate, pectin, soy isolated protein, casein and combinations thereof, preferably isolated protein from milk serum with 90% protein, maltodextrin with a dextrose equivalent of 10, arabic gum and combinations thereof.

(4) On the other hand, prebiotics are selected from the group comprising polydextrose, inulin and maguey honey, preferably maguey honey.

(5) With respect to the proteolytic enzyme of bacterial origin and the proteolytic enzyme of fungal origin, these are selected from enzymes commonly used for baking.

(6) In a specific embodiment of the invention, the microencapsulated bacterial consortium comprises: (a) Lactobacillus plantarum ATCC 8014; b) Lactobacillus sanfranciscensis ATCC 27652; c) Lactobacillus brevis ATCC 14869; d) isolated protein from milk serum with 90% protein; e) maltodextrin with a dextrose equivalent of 10; f) arabic gum; g) maguey honey; and h) trehalose; in combination with a proteolytic enzyme of bacterial origin and a proteolytic enzyme of fungal origin.

(7) More preferably, the microencapsulated bacterial consortium comprises: a) Lactobacillus plantarum ATCC 8014; b) Lactobacillus sanfranciscensis ATCC 27652; c) Lactobacillus brevis ATCC 14869; d) from 40 to 60% of isolated protein from milk serum with 90% protein; e) from 10 to 20% of maltodextrin with a dextrose equivalent of 10; f) from 20 to 80% of arabic gum; g) from 1 to 10% of maguey honey; and h) from 0.5 to 5% of trehalose; in combination with 0.005 to 0.03% of a proteolytic enzyme of bacterial origin and 0.001 to 0.02% of a proteolytic enzyme of fungal origin.

(8) The microencapsulating is carried out by processes well-known in the state of the art, such as spray-drying. For example, the bacterial consortium microencapsulated can be obtained according to the following:

(9) a) separately reactivate each strain of lactic-acid bacteria;

(10) b) when each strain is active, separately culturing in a liquid culture medium until reaching each strain desired concentration;

(11) c) for each strain, removing the excess culture medium to concentrate the microorganisms, preferably by centrifugation, obtaining a pellet;

(12) d) separately, resuspending the pellet obtained for each strain in saline suspension and adjusting to the required volume;

(13) e) mixing the necessary amounts of each strain and take to a final volume;

(14) f) dissolve the encapsulating agents in water, at the proper ratios to have 20 to 30% of dissolved solids;

(15) g) adding prebiotics and trehalose;

(16) h) inoculate the mixture of three strains of lactic-acid bacteria, such that about 10.sup.10 CFU of said mixture of three strains of lactic-acid bacteria per gram of powder microencapsulated is obtained; and

(17) i) drying, preferably with a spray-dryer, to an inlet temperature of between 110 and 160 C., and an outlet temperature of between 60 and 80 C., with a feed rate between 20 and 50 mL/min.

(18) Preferably, in step e), Lactobacillus plantarum ATCC 8014 is mixed at a ratio from 10 to 50%, Lactobacillus sanfranciscensis ATCC 27652 in a ratio of 40%, and Lactobacillus brevis ATCC 14869 at a ratio from 10 to 50%.

(19) Preferably, the drying is carried out using a spray-dryer, at an inlet temperature of 150 C., and an outlet temperature of 80 C., with a feed rate of 22 mL/min.

(20) Another aspect of the invention considers a sourdough made from the microencapsulated bacterial consortium of the present invention, in combination with a proteolytic enzyme of bacterial origin and a proteolytic enzyme of fungal origin, and wherein the sourdough has a gluten concentration of less than 20 ppm, preferably less than 10 ppm, being suitable for the preparation of gluten-free baking products.

(21) Preferably, this gluten concentration in the sourdough is achieved after at least 3 hours of fermentation, more preferably after 31 hours of fermentation.

(22) The process of making sourdoughs of the present invention comprises the following steps:

(23) i) mixing 30 to 60% of flour with 40 to 80% of water to obtain a dough with a dough yield from 150 to 475, preferably from 150 to 160;

(24) ii) adding 0.005 to 0.03% of proteolytic enzyme of bacterial origin and 0.001 to 0.02% of proteolytic enzyme of fungal origin previously dissolved in the kneading water;

(25) iii) adding the microencapsulated bacteria consortium such that about 10.sup.10 CFU of the mixture of three strains of lactic-acid bacteria per gram of fluor is present;

(26) iv) mixing; and

(27) v) fermenting for 3 to 48 hours, preferably from 28 to 35 hours, at a temperature from 30 to 37 C., and a relative humidity from 70 to 92%, preferably 75 to 80%.

(28) The flour used in step i) is selected from wheat flour, rye flour and oatmeal flour, preferably wheat flour.

(29) Regarding fermentation, preferably this is carried out during 31 hours, at a temperature of 35 C. and a relative humidity of 76%.

(30) As noted above, the sourdoughs of the present invention, obtained by the process noted above, have a gluten concentration lower than 20 ppm, preferably lower than 10 ppm. Preferably, this gluten concentration in the sourdoughs is achieved after 3 hours of fermentation.

(31) From these sourdoughs it is possible to obtain baking products, preferably sweet bread, gluten-free, which are suitable for consumption by patients with celiac disease.

(32) The present invention will be better understood from the following examples, which are presented with illustrative purposes only to allow a proper understanding of the preferred embodiments of the present invention, without implying that there are no other non-illustrated embodiments that can be practiced based on the above detailed description made.

EXAMPLES

Example 1

Inoculum Production

(33) Strains of Lactobacillus plantarum ATCC 8014, Lactobacillus brevis ATCC 14869 and Lactobacillus sanfranciscensis ATCC 27652 were obtained through a supplier from the ATCC; the first two were preserved in a solid medium and the latter was lyophilized.

(34) In order to reactivate the strains, seeds in liquid Man Rogosa Sharpe media (MRS) (Difco, USA) were made in test tubes with a screw cap, incubating from 35 to 37 C. for 24 to 48 hours or until observing growth due to increased turbidity.

(35) When the cultures had this feature, they were transfered to solid MRS medium in Petri dishes, incubating at the same temperature also over 24 to 48 hours.

(36) Once the microorganisms grew up, they were transferred separately to a 125 mL Erlenmeyer flask with 50 ml of MRS medium, incubating for 8 to 12 hours at 37 C. with stirring at 50 to 100 rpm; this was the pre-inoculum.

(37) After the incubation time the culture was transferred again, in aseptic conditions, to a 2800 mL Fembach flask with 500 to 1000 ml of MRS media, which was incubated at 37 C. and stirring at 50 to 150 rpm over 10 to 16 hours. Each flask was sampled to measure medium absorbance (turbidity).

(38) In order to remove the remaining media and concentrate the microorganisms, the culture was placed in ethanol sanitized 250 mL centrifuge vials, and was then centrifuged at 2000 to 5000 rpm for 20 minutes.

(39) The pellet obtained was resuspended in sterile saline (sodium chloride, 9 g/L). Once the microorganisms were re-suspended and the volume required adjusted, the inoculum to be microencapsulated was prepared also in aseptic conditions by mixing the required amounts of each strain and getting to the final volume.

Example 2

Micro-encapsulation of the Bacterial Consortium

(40) Lactic serum isolated protein with 90% protein due to its barrier properties to oxygen, maltodextrin with a dextrose equivalent of 10 and Arabic gum were selected as encapsulating agents in different ratios.

(41) To support reactivation of the bacteria in the consortium, the addition of three types of prebiotic agents was analyzed: polidextrose, inulin and maguey honey. Also, trehalose was used as a specific nutritional ingredient for the bacteria forming the consortium.

(42) Encapsulating agents were dissolved in water at the proper ratios and to have 20% to 30% of dissolved solids. Prebiotics and trehalose were added, and this dispersion was inoculated with such amount of bacterial consortium so as to have 10.sup.10 CFU/g powder microencapsulated.

(43) The previous mixture was dried by a spray-dryer, at an inlet temperature of 150 C. and an outlet one of 80 C., with a feed rate of 22 mL/min.

(44) As a result, the powder microencapsulated microbial consortium was obtained.

Example 3

Prebiotic Ratios Determination Favoring Lactic-Acid Bacteria (BAL) Development

(45) As mentioned above, the addition of polidextrose (P), inulin (I) and maguey honey (M) to the microencapsulated consortium was assessed. To that end, a single centroid mixture design was used in the experiment, as shown in Table 1:

(46) TABLE-US-00001 TABLE 1 Prebiotic formulations assessed for microencapsulation of the microbial consortium Prebiotic (g) Treatment P I M 1 0.76 2.00 0.23 2 4.50 0 0 3 0 0 1.40 4 0 3.00 0 5 0 1.50 0.70 6 2.25 0 0.70 7 3.00 0.51 0.23 8 1.48 0.90 0.46 9 2.25 1.50 0 10 0.76 0.51 0.93 11 1.48 0.90 0.46

(47) Each mixture was added to the corresponding LAB dispersion and wall material and dried by spraying, as described above.

(48) Once the microencapsulated was obtained, 1 g of the powder was taken and rehydrated in sterile water to 30 C. with stirring. An aliquot of 1 ml was taken and serial dilutions were made, and plate seeding to determine the most probable number. The results of bacterial survival are shown in Table 2.

(49) TABLE-US-00002 TABLE 2 Survival percentage of LAB depending on prebiotic combination Treatment % LAB Survival 1 84 2 60 3 98 4 70 5 75 6 80 7 72 8 78 9 60 10 80 11 85

(50) According to the above, it can be seen that the greatest survival (98%) was reached when the prebiotic was maguey honey.

Example 4

Sourdoughs Preparation

(51) White wheat flour commercially available, suitable for manual baking was used, as well as the microencapsulated bacterial consortium obtained according to Example 2, with a concentration of 10.sup.10 CFU/g, and in addition to encapsulating agents and trehalose, further containing maguey honey as a prebiotic.

(52) As proteolytic enzymes, one of bacterial origin (HT proteolitic 200, Enmex S. A. de C. V., Mexico) and another one of fungal origin (Harizyme G, Enmex S. A. de C. V., Mexico) were used.

(53) For sourdoughs preparation, the dough was prepared with an MY (mass yielding) between 150-160 from wheat flour, mixing 400 g flour, 150 mL of water. 0.08 g of proteolytic enzyme of bacterial origin (HT proteolitic 200, Enmex S. A. de C. V., Mexico) and 0.12 g of proteolytic enzyme of fungal origin (Harizyme G, Enmex S. A. de C. V., Mexico) were added, both previously dissolved in the kneading water. Subsequently, the microbial consortium micro-capsules of the present invention were added, suspended in water, in such a way to achieve 10.sup.10 CFU/g.

(54) It was mixed for 8 min in a standard blender and then subjected to fermentation for 31 h at 35 C. and a relative humidity of 76%. pH during fermentation and the intensity of gluten degradation at the end of the fermentation were monitored by electrophoretic analysis (Western Blot test). The results are shown in Table 3.

(55) TABLE-US-00003 TABLE 3 Kinetics of degradation of inoculated sourdoughs with the microencapsulated bacterial consortium t1 t2 t3 t4 t5 t6 t7 Type of Pow- Pow- Powder Powder Powder Powder Powder inoculum der der Time (h) 3 6 7 11 23 27 31 Gliadines 4.1 3.0 <5.0 <3.0 4.1 <3.0 <3.0 (ppm) Hordeines NA NA NA NA NA NA NA (ppm) Secalines NA NA NA NA NA NA NA (ppm) Areninas NA NA NA NA NA NA NA (ppm)

(56) According to the data shown in the table, it is possible to see an intensive degradation of the gluten gliadin fraction by the effect of the microencapsulated bacterial consortium of the present invention, in combination with the proteolytic enzymes, within the first 3 hours of fermentation.

Example 5

Elaboration of Astorga Cupcake-Type Sweet Bread with Sourdoughs by Lactic-Acid Fermentation

(57) Cupcake-type, gluten-free, sweet bread was made using the sourdough obtained according to Example 4. To this end, butter, margarine and sugar glass were beat using the mixer palette, first at low speed until incorporation, and then increasing the speed and mixing for 20 more minutes to incorporate the largest amount of air into the mixture.

(58) Three egg yolks were added little by little, until its complete incorporation, and subsequently milk, flavoring (orange, lime and chocolate) and potassium sorbate were added.

(59) The fermented sourdough according to Example 4, maize starch, nixtamalized flour, baking soda and baking powder, pre-mixed in a plastic bag were incorporated to the previous mixture. Water was added and it was mixed at high speed for 8 minutes.

(60) Separately, the egg whites were beat until stiff and then were blended in with the rest of the dough, at medium speed so as not to break the foam.

(61) The fluid mass was deposited in cupcake paper cups in oven baking trays and it was baked for 10 minutes at 200 C.

Example 6

Astorga Cupcakes Assessment by Consumers

(62) In order to determine the acceptability of the cupcakes with orange, lime and chocolate flavor, prepared according to Example 5 (using the sourdough obtained with the microencapsulated microbial consortium, in combination with proteolytic enzymes, of the present invention), compared to natural cupcakes made with traditional dough, an assessment was carried out among consumers.

(63) To this end, a test was carried out with 70 consumers of Astorga cupcakes from the Universidad Iberoamericana, Mexico City campus. The participants, 50% men and 50% women, aged from 15 to over 45, were consumers of commercial sweet bread consuming at least every 15 days. The significance and confidence level of the test is 0.05% and 95%, respectively. The results are shown in Table 4.

(64) TABLE-US-00004 TABLE 4 Test Results with consumers of cupcakes made from fermented sourdough with microencapsulated bacterial consortium in combination with proteases. General acceptance Appearance Texture Flavor Range: 1-5 (1 least Range: 1-5 pleasant, 5 most Cupcake (1 least pleasant, 5 most pleasant) pleasant) Natural.sub.a 4.01.sub.a 3.87.sub.a 4.04.sub.a 7.96.sub.a Good Good Good Orange flavor.sub.b 3.9.sub.ab 3.99.sub.ab 4.00.sub.ab 8.1.sub.ab Good Good Good Chocolate 3.77.sub.ac 3.89.sub.ac 3.76.sub.ac 7.64.sub.ac flavor.sub.c Almost good Good Almost good Lime flavor.sub.d 3.41.sub.d 3.56.sub.d 3.26.sub.d 6.81.sub.d Between Between Regular regular and good and good regular

(65) The results in Table 4 show that there is not a significant difference in 95% (0.05) with respect to the natural cupcake in all the assessed attributes (appearance, texture and flavor) and in general acceptance of cupcakes with orange flavor and chocolate flavor.

(66) On the contrary, the lime cupcake does present significant difference at 95% with respect to the natural cupcake in all the assessed attributes, in favor of the natural cupcake.

(67) In accordance with the above-described, it will be observed that the microencapsulated bacterial consortium for gluten degradation in sourdoughs and the process of making the same, as well as the baked products obtained from such sourdoughs, have been devised to have gluten-free products, that might be suitable for consumption by celiac patients, and it will be apparent to any expert in the art that the described embodiments of the microencapsulated consortium for gluten degradation in sourdoughs and the process of making thereof as described, are only illustrative and non-limiting of the present invention, since various changes of consideration are possible in their details without departing from the scope of the invention.

(68) Therefore, the present invention should not be considered restricted except for what is demanded by the prior art and by the scope of the appended claims.