METHOD FOR DEGRADATION OF GLIADIN TO OBTAIN GLUTEN-FREE FLOUR

Abstract

The present invention relates to a method for the degradation of gliadin in flour for bread making, by means of a step of mixing the flour with water, at least one step of performing enzymatic hydrolysis, at least one step of fermenting using microorganisms in controlled-pH conditions, and a step of drying to obtain a gliadin-free flour.

Claims

1. A process for degradation of gliadin in flour for bread making, characterized in that it comprises: a step of mixing the flour with water; at least one step of enzymatic hydrolysis to hydrolize gliadin from the mixture obtained in the mixing step; at least one step of fermenting the mixture obtained in the enzymatic hydrolysis step by using at least one microorganism under controlled-pH conditions; wherein the microorganism is selected from Bacillus amyloliquefacients, Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus reuteri, Lactobacillus helveticus or mixtures thereof; and, a step of drying to obtain a gliadin-free flour.

2. The process for degradation of gliadin in flour for bread making according to claim 1, further characterized in that fungal enzymes are used in the enzymatic hydrolysis step.

3. The process for degradation of gliadin in flour for bread making according to claim 2, further characterized in that enzymes from Aspergillus niger and/or Aspergillus oryzae are used.

4. The process for degradation of gliadin in flour for bread making according to claim 3, further characterized in that the enzymes from Aspergillus niger and Aspergillus oryzae are present in a 1:1 ratio.

5. The process for degradation of gliadin in flour for bread making according to claim 2, further characterized in that the enzymes are added to the mixture in a proportion from between 0.01 and 0.1% by weight relative to the suspension.

6. The process for degradation of gliadin in flour for bread making according to claim 1, further characterized in that the enzymatic hydrolysis step is carried out at a temperature from between 35 and 37 C.

7. The process for degradation of gliadin in flour for bread making according to claim 1, further characterized in that the enzymatic hydrolysis step is carried out under constant stirring.

8. The process for degradation of gliadin in flour for bread making according to claim 1, further characterized in that hydrolysis time is from between 4 and 8 hours.

9. The process for degradation of gliadin in flour for bread making according to claim 1, further characterized in that after the hydrolysis step is completed, a pH adjustment is carried out.

10. The process for degradation of gliadin in flour for bread making according to claim 9, further characterized in that the pH is adjusted to between 6.5 and 8.

11. The process for degradation of gliadin in flour for bread making according to claim 9, further characterized in that the pH adjustment is carried out by adding a base.

12. The process for degradation of gliadin in flour for bread making according to claim 11, further characterized in that the base is K.sub.2CO.sub.3.

13. (canceled)

14. The process for degradation of gliadin in flour for bread making according to claim 1, further characterized in that the microorganism used is added to the mixture in an amount from between 10.sup.3 and 10.sup.9 CFU/g of flour.

15. The process for degradation of gliadin in flour for bread making according to claim 1, further characterized in that the fermenting step is carried out at a temperature of from 36 to 38 C.

16. The process for degradation of gliadin in flour for bread making according to claim 1, further characterized in that the fermenting step is carried out for a period of time from between 4 and 8 hours.

17. The process for degradation of gliadin in flour for bread making according to claim 1, further characterized in that after the fermenting step is completed, a second pH adjustment is carried out.

18. The process for degradation of gliadin in flour for bread making according to claim 17, further characterized in that the pH is adjusted to between 6.5 and 8.

19. The process for degradation of gliadin in flour for bread making according to claim 17, further characterized in that the second pH adjustment is carried out by adding a base.

20. The process for degradation of gliadin in flour for bread making according to claim 19, further characterized in that the base is K.sub.2CO.sub.3.

21. The process for degradation of gliadin in flour for bread making according to claim 1, further characterized in that it comprises a second enzymatic hydrolysis step.

22. The process for degradation of gliadin in flour for bread making according to claim 21, further characterized in that fungal enzymes are used in the second enzymatic hydrolysis step.

23. The process for degradation of gliadin in flour for bread making according to claim 22, further characterized in that enzymes from Aspergillus niger and/or Aspergillus oryzae are used in the second enzymatic hydrolysis step.

24. The process for degradation of gliadin in flour for bread making according to claim 23, further characterized in that the enzymes from Aspergillus niger and Aspergillus oryzae are present in a 1:1 ratio.

25. The process for degradation of gliadin in flour for bread making according to claim 22, further characterized in that the enzymes are added to the mixture in a proportion from between 0.01 and 0.1% by weight relative to the suspension.

26. The process for degradation of gliadin in flour for bread making according to claim 21, further characterized in that the second enzymatic hydrolysis step is carried out at a temperature from between 35 and 37 C.

27. The process for degradation of gliadin in flour for bread making according to claim 21, further characterized in that the second enzymatic hydrolysis step is carried out under constant stirring.

28. The process for degradation of gliadin in flour for bread making according to claim 21, further characterized in that a second enzymatic hydrolysis time is of from 2 to 4 hours.

29. The process for degradation of gliadin in flour for bread making according to claim 1, further characterized in that it comprises a second fermenting step.

30. The process for degradation of gliadin in flour for bread making according to claim 29, further characterized in that the second fermenting step is carried out by using at least one microorganism under controlled-pH conditions.

31. The process for degradation of gliadin in flour for bread making according to claim 30, further characterized in that, in the second fermenting step, the microorganism is selected from Bacillus amyloliquefaciens, Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus reuterii, Lactobacillus helveticus microorganisms or mixtures thereof.

32. The process for degradation of gliadin in flour for bread making according to claim 31, further characterized in that a mixture of Bacillus amyloliquefaciens and at least two lactobacilli selected from: Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus reuterii, and Lactobacillus helveticus is used.

33. The process for degradation of gliadin in flour for bread making according to claim 32, further characterized in that the microorganisms are present in a 1:1:1 ratio.

34. The process for the degradation of gliadin flour for bread making according to claim 32, further characterized in that the microorganism is added to the mixture in an amount from between 10.sup.3 and 10.sup.9 CFU/g of flour.

35. The process for degradation of gliadin in flour for bread making according to claim 21, further characterized in that the second fermenting step is carried out at a temperature from between 35 and 37 C.

36. The process for degradation of gliadin in flour for bread making according to claim 32, further characterized in that the second fermenting step is carried out under constant stirring.

37. The process for degradation of gliadin in flour for bread making according to claim 32, further characterized in that the second fermenting step is carried out for a period of time from between 4 and 12 hours.

38. The process for degradation of gliadin in flour for bread making according to claim 1, further characterized in that the drying step is a spray drying process.

39. The process for degradation of gliadin in flour for bread making according to claim 38, further characterized in that inlet air is at a temperature from between 150 and 180 C. and outlet air is at a temperature from between 70 and 90 C. in the spray drying.

Description

DESCRIPTION OF DRAWINGS

[0022] FIG. 1 shows a chromatogram of flour treated by means of a 5-hour proteolysis and lactobacillus fermentation.

[0023] FIG. 2 shows a chromatogram of flour treated by means of a 10-hour proteolysis and lactobacillus fermentation.

[0024] FIG. 3 shows a chromatogram of flour treated by means of fermentation by L. johnsonii for 24 hours.

[0025] FIG. 4 shows a chromatogram of flour treated by means of a proteolysis using HT Proteolytic 200 for 10 hours.

[0026] FIG. 5 shows a chromatogram of flour treated by means of a proteolysis using Harizyme G for 10 hours.

[0027] FIG. 6 shows a chromatogram of flour treated by means of a 5-hour proteolysis, lactobacillus fermentation, and a proteolysis using protease HT Proteolitic 200 for 5 hours.

[0028] FIG. 7 shows a concentration of gliadin in flour fermented by L. sanfranciscensis.

[0029] FIG. 8 shows a concentration of gliadin in flour fermented by L. brevis.

[0030] FIG. 9 shows a concentration of gliadin in flour fermented by Bacillus amyloliquefaciens.

[0031] FIG. 10 shows a concentration of L-tyrosine during an enzymatic proteolysis using protease N-1000.

[0032] FIG. 11 shows a concentration of L-tyrosine during an enzymatic proteolysis using protease HT Proteolitic 200.

[0033] FIG. 12 shows a liquid chromatogram of the flour treated by using a process comprising two enzymatic hydrolysis steps and two fermenting steps.

DETAILED DESCRIPTION OF THE INVENTION

[0034] As mentioned in the background, the degradation of gluten fractions, especially gliadins, is highly important for making products suitable for celiac disease patients. To solve this problem, it has been found that by means of a process consisting of at least one step of enzymatic hydrolysis and at least one step of fermentation using lactobacilli, the total degradation of gliadin contained in baking flours is achieved.

[0035] It has been observed that the acidification process of the medium that takes place when treating flours by fermentation and enzymatic hydrolysis negatively affects the degree of gliadin degradation in the flour because the action of microorganisms and enzymes is inhibited. This is why in order to obtain a flour with optimally degraded gliadins, not only must bacterial consortia and reaction conditions be carefully selected, but also each of these processes must be carried out in a specific order that optimizes the microorganism activity and that avoids the enzymatic inhibition.

[0036] Thus, the process for degradation of gliadin in flour for bread making of the present invention solves the problems and disadvantages mentioned in the state of the art.

[0037] In accordance with the foregoing, the present invention relates to a process for degradation of gliadin in flour for bread making comprising: a step of mixing the flour with water; at least one step of enzymatic hydrolysis to hydrolize gliadin from the mixture obtained in the mixing step; at least one step of fermenting the mixture obtained in the enzymatic hydrolysis step by using at least one microorganism under controlled-pH conditions; and, a step of drying to obtain a gliadin-free flour.

[0038] The flours for bread making are all of those having the suitable properties for making bread goods. They are characterized by having a high gluten content, which allows said flour to have a good water absorption capacity, cohesiveness, viscosity, and elasticity. Some examples of flours for bread making are flours derived from wheat, rye, barley, oatmeal, and triticale.

[0039] The first step of said process comprises mixing flour for bread making with water.

[0040] In a preferred embodiment of the present invention, fungal enzymes are preferably used in the enzymatic hydrolysis step. More preferably, enzymes from Aspergillus niger and/or Aspergillus oryzae. Said enzymes are preferably present in a 1:1 ratio and are added to the mixture in a proportion from between 0.01 and 0.1% by weight relative to the suspension. The enzymatic hydrolysis step is preferably carried out at a temperature from between 35 and 37 C., under constant stirring. The hydrolysis time is preferably from between 4 and 8 hours. In this first step, a degradation of about 88% gliadin present in the initial flour is achieved.

[0041] As previously stated, the medium tends to be acidified by the enzymatic hydrolysis, and it is known that the enzyme activity tends to decrease at an acidic pH. That is why that in an optional embodiment of the present invention, after the hydrolysis step is completed, a pH adjustment is carried out. Preferably, a pH between 6.5 and 8 is sought. To carry out said pH adjustment, a base, preferably K.sub.2CO.sub.3, is added in an amount sufficient to reach the pH required.

[0042] During the hydrolysis step described, the hydrolysis of gliadin and the breakdown of target peptides present in the gluten molecules are achieved. Peptides and amino acids generated by the enzymatic hydrolysis process can be degraded efficiently by some LABs. That is why a subsequent step of the process for degradation of gliadin is a step of fermenting the mixture obtained in the enzymatic hydrolysis step by using at least one LAB under controlled-pH conditions.

[0043] As to the step of fermenting, in a preferred embodiment of the invention, it is carried out by using Bacillus amyloliquefaciens, Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus reuterii, Lactobacillus helveticus or mixtures thereof as the microorganisms. The microorganism used is preferably added to the mixture in an amount from between 10.sup.3 and 10.sup.9 CFU/g of flour. Preferably, the step of fermenting is carried out at a temperature of from 36 to 38 C. and for a period from between 4 and 8 hours. In the mentioned step of fermenting, up to an additional 10% of initial gliadins can be degraded, with the remaining flour having about 2% of gliadins.

[0044] As in the hydrolysis step, during the step of fermenting, a considerable decrease in pH is observed, thus in an optional embodiment of the present invention, a second pH adjustment is carried out. Preferably a pH between 6.5 and 8 is sought. To carry out said pH adjustment, a base, preferably K.sub.2CO.sub.3, is added in an amount sufficient to reach the pH required.

[0045] To degrade the remaining 2% of gliadins, a second hydrolysis step may be implemented. Thus, in a further preferred embodiment of the present invention, a second enzymatic hydrolysis step is carried out by using preferably fungal enzymes. More preferably enzymes from Aspergillus niger and/or Aspergillus oryzae. Said enzymes are preferably present in a 1:1 ratio and are added to the mixture in a proportion from between 0.02 and 0.08% by weight relative to the suspension. The second enzymatic hydrolysis step is preferably carried out at a temperature from between 35 and 37 C., under constant stirring. The hydrolysis time is preferably from 2 to 4 hours.

[0046] Additionally, after the second hydrolysis step, a second fermenting step can be carried out to achieve a maximum possible degradation of gliadins. Accordingly, in a preferred embodiment of the present invention, a second step of fermenting the mixture obtained in the second enzymatic hydrolysis step is carried out by using at least one microorganism under controlled-pH conditions. Preferably, the second fermenting step is carried out by using Bacillus amyloliquefaciens, Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus reuterii, Lactobacillus helveticus or mixtures thereof as the microorganisms. Preferably, a mixture of Bacillus amyloliquefaciens and at least two lactobacilli selected from: Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus reuterii, and Lactobacillus helveticus is used. Preferably, the microorganisms are present in a 1:1:1 ratio. Preferably, the microorganisms are added to the mixture in an amount from between 10.sup.3 and 10.sup.9 CFU/g of flour. Preferably, the second fermenting step is carried out at a temperature from between 35 and 37 C., under constant stirring and for a period of time from between 4 and 12 hours.

[0047] Finally, to obtain a final product that can be used in the production of bakery products, the process includes an additional step of drying to obtain a gliadin-free flour.

[0048] In a preferred embodiment, the drying step is a spray drying process.

[0049] Preferably, in the spray drying, inlet air is at a temperature from between 150 and 180 C. and outlet air is at a temperature from between 70 and 90 C.

[0050] The present invention will be better understood from the following examples, which are presented for illustrative purposes only to allow a full understanding of the preferred embodiments of the present invention, without implying that there are no other non-illustrated embodiments that can be put into practice based on the detailed description set forth above.

Example 1

[0051] Several studies were conducted to observe the gliadin degradation by various methods.

[0052] In the first study, a process consisting of a proteolysis step and a fermentation step was carried out. For this study, a flour-in-water suspension was made, and said suspension was kept under constant stirring at a temperature of 37 C. For the proteolysis step, 1 g enzyme HT Proteolytic per kg of flour was added and left to hydrolize for 5 hours. Subsequently, 10.sup.9 CFU of the lactobacilli Lactobacillus brevis and Lactobacillus sanfranciscensis were added for the fermentation step, which lasted 9 hours.

[0053] In the second study, the previous assay was repeated, but the enzymatic proteolysis time was changed from 5 to 10 hours.

[0054] In the third study, a fermentation process was carried out, for which a flour-in-water suspension was made, and 10.sup.9 CFU of L. johnsonii were added to said suspension. The suspension was kept under constant stirring at a temperature of 37 C. for 24 hours.

[0055] The fourth study consisted of a proteolysis process, for which a suspension of 33.3 g flour and 130 ml water was made, and 1 g enzyme HT Proteolitic 200 per kg of flour was added to said suspension. Said suspension was kept under constant stirring at 37 C. for 10 hours.

[0056] In the fifth study, a proteolysis process was carried out, for which a suspension of 33.3 g flour and 130 ml water was made, and 1.2 g enzyme Harizyme G per kg of flour were added to said suspension. The suspension was kept under constant stirring at 37 C. for 10 hours.

[0057] At the end of each experiment, the flour was analyzed by liquid chromatography. The results can be seen in Table 1. In each of FIGS. 1 to 5, a liquid chromatogram of the flour Hoja de Plata is indicated by a letter A, and a chromatogram of the flour treated by each of the methods of the present study is indicated by the letter B. Also, the position of the chromatogram that shows the analyzed gliadin content is indicated by a circle in each figure.

TABLE-US-00001 TABLE 1 ExperimentResult Result Chromatogram Proteolysis for 5 h and A considerable decrease of gliadin FIG. 1 lactobacillus fermentation fractions was observed although not completely degraded Proteolysis for 10 h and A considerable gliadin degradation FIG. 2 lactobacillus fermentation was observed, however, only 20% reduction compared to the previous proteolysis treatment Fermentation by L. A decrease of gliadin fractions was FIG. 3 johnsonii for 24 h observed, however, an increase in higher molecular weight peptides was detected Proteolysis using HT A considerable gliadin decrease FIG. 4 Proteolitic 200 for 10 h was observed as well as a decrease in higher molecular weight peptides detected in previous experiments Proteolysis using A profile very similar to that of the FIG. 5 Harizyme G for 10 h standard flour Hoja de Plata was observed, which indicates that gliadin degradation was minimal

[0058] From the results shown in Table 1 and their respective Figures, it is concluded that the most efficient gluten degradation processes are those wherein a proteolysis was carried out followed by a step of lactobacillus fermentation.

Example 2

[0059] An assay was carried out to observe gliadin degradation by a process comprising a 5-h proteolysis step using enzyme N1000, a fermentation step using lactobacilli and an additional proteolysis step using HT Proteolitic 200.

[0060] For this assay, a first experiment was carried out wherein a flour-in-water suspension was made, and 0.4 g enzyme N-1000 per kg of flour was added to said suspension. The suspension was kept under constant stirring for 5 hours at a temperature of 37 C. Subsequently, 10.sup.9 CFU Lactobacillus brevis and Lactobacillus sanfranciscensis were added and kept under constant stirring for 9 hours at a temperature of 37 C. Finally, 1 g enzyme HT Proteolitic 200 per kg of flour was added and the suspension was kept under constant stirring for 5 hours at a temperature of 37 C.

[0061] The experiment was repeated but changing only the second proteolysis time to 10 hours.

[0062] From said experiment, the chromatogram of FIG. 6 was obtained wherein a chromatogram of the flour Hoja de Plata at different concentrations is indicated by the letter A and a chromatogram of the flour treated by the present method is indicated with the letter B. In the same figure, the position of the chromatogram that shows the gliadin content of the analized flour is indicated by a circle.

[0063] From FIG. 6, it can be seen that after all the process steps were carried out, it was possible to degrade gliadin to about 75 ppm. FIG. 6 also shows that the results of the first experiment were very similar to those of the second experiment.

Example 3

[0064] Three fermentation processes were carried out. In each process, a flour-in-water suspension was made, and 10.sup.9 CFU of a microorganism were added to said suspension. In the first experiment, the microorganism was L. brevis; in the second experiment, the microorganism selected was L. sanfranciscensis; and in the third experiment it was B. amyloliquefaciens. Each fermentation lasted 24 hours at 37 C. under constant stirring. Subsequently, the gliadin degraded was quantified by liquid chromatography. The results can be seen in FIGS. 7 for L. sanfranciscensis, 8 for L. brevis, and 9 for B. amyloliquefaciens. Wherein A lines indicate gliadin standards and B line indicates the gliadin concentration in flour fermented by each of the microorganisms.

[0065] As can be seen, the best microorganism for degrading gliadin is B. amyloliquefaciens.

Example 4

[0066] A study was conducted to determine the operating time required to carry out enzymatic hydrolysis. For this study, the tyrosine release was quantified during two wheat gluten hydrolysis processes, each with a different protease. The purpose of this experiment was to determine the hydrolysis time required for tyrosine to reach steady state, that is, when its stops being generated.

[0067] For these experiments, the tyrosine content in flour suspensions was quantified in the hydrolysis process every 15 to 30 minutes. For the first experiment, a suspension of 4.3 g flour in 130 ml water was used, and 0.4 g protease N-1000 was added to said suspension. For the second experiment, a suspension of 4.3 g gluten in 130 ml water was used, and 1 g protease Proteolitic 200 HT was added to said suspension.

[0068] The results obtained can be seen in FIG. 10 for protease N-1000 and in FIG. 11 for protease Proteolitic 200 HT, respectively. In these Figures, the tyrosine concentration is shown over time and it can be clearly seen that on average, the tyrosine concentration in the suspensions reaches the steady state between 4 and 8 hours of hydrolysis.

Example 5

[0069] An assay was carried out under conditions equivalent to those mentioned in the previous examples to determine the gliadin concentration present in the flour after being treated by the gliadin degradation process of the present invention.

[0070] For this study, a flour-in-water suspension was made, it was first treated by means of an enzymatic hydrolysis step using enzymes ENZECO PROTEASE FNP and ENZECO FUNGAL PROTEASE, then by a fermentation step using Bacillus amyloliquefaciens, followed by another enzymatic hydrolysis step using proteases ENZECO PROTEASE FNP and ENZECO FUNGAL PROTEASE, and finally by a second fermentation using Bacillus amyloliquefaciens in combination with L. brevis and L. delbrueckii. All of the steps were carried out at 37 C. under constant stirring.

[0071] Hydrolized flour samples were obtained by the quartering method. For each replica, 200 mg of sample were taken and placed in Falcon tubes with a glass bead to improve extraction.

[0072] Protein extraction was carried out by performing three washes with a 70% ethanolic solution. Each wash was performed by adding 3 ml ethanolic solution to sample and glass bead-containing tubes and they were stirred for one hour in a 70-RPM rotary incubator. After centrifuging the sample, it was re-suspended with solvent using a vortex. The soluble part in the solvent was decanted after centrifuging the tube for 10 minutes at 2500 RPM, and finally the extract was placed in another Falcon tube wherein the three washes were combined.

[0073] 3 ml extract from each sample were filtered through a 0.22-m membrane to analyze the gliadin concentration by HPLC.

[0074] The sample extractions were passed through an HPLC equipment at a temperature of 50 C. using two mobile phases, the first named solution A which consisted of a mixture of 99% water and 1% water with 0.001% acid trifluoroacetic. The mobile phase flow was 0.6 ml/min with a gradient of 27% B to 50% B for the first 20 minutes and finally 50% B to 75% B for up to 30 minutes, after the injection of a sample, a wash with phase A was performed for 10 minutes. 100 m sample or phase A were injected during the analysis and washed respectively. Peptide signals present at 210 nm were detected by using a UV detector, which can be seen in FIG. 12, wherein a liquid chromatogram of a standard flour is indicated by the letter A and a liquid chromatogram of a flour treated by means of the disclosed method is indicated by the letter B.

[0075] From FIG. 12, it can be seen that the fractions corresponding to the different gliadins (RF between 12 and 17 minutes) apparently disappear in the samples having flour treated by the method of the present invention.

[0076] In accordance with the foregoing, it shall be apparent to a person skilled in the art that the preferred embodiment of the process for degradation of gliadin in flour for bread making illustrated above is set forth for illustrative purposes only but not limited to the present invention, since a person skilled in the art can make numerous variations thereto, provided they are designed according to the principles of the present invention.

[0077] Accordingly, the present invention includes all of the embodiments that a person skilled in the art can pose from the concepts contained in the present specification, in accordance with the following claims.