Process of microbic biotechnology for completely degrading gluten in flours

Abstract

The present invention concerns the use of lactic acid bacteria selected and fungal enzymes for the gluten complete degradation from both bread and durum wheat, barley, rye and oat flour. In particular, the invention concerns the use of lactic acid bacteria selected and fungal enzymes for the gluten complete degradation (residual gluten concentration lower than 20 ppm) of cereal flours, which after detoxification can be used according to a standardized biotechnological protocol for the production of various gluten-free foods.

Claims

1. Mixture comprising or consisting of Lactobacillus sanfranciscensis DSM22063 and Lactobacillus plantarum DSM 22064 lactic acid bacteria, wherein the lactic acid bacteria each have an x-prolyl dipeptidyl aminopeptidase activity of between 17.5 U and 22 U, U being defined as the enzyme amount necessary to release 1 mol/min of amino acid.

2. Mixture according to claim 1, further comprising fungal proteases.

3. Mixture according to claim 2, wherein fungal proteases are selected from the group consisting of Aspergillus oryzae, Aspergillus niger proteases or mixtures thereof.

4. A mixture comprising: a) Lactobacillus sanfranciscensis DSM22063 and Lactobacillus plantarum DSM 22064 lactic acid bacteria; b) water; and c) flour; wherein the lactic acid bacteria each have an x-prolyl dipeptidyl aminopeptidase activity of between 17.5 U and 22 U, U being defined as the enzyme amount necessary to release 1 mol/min of amino acid; and wherein the flour is at least one gluten-containing flour selected from the group consisting of bread wheat, durum wheat, tender wheat, barley, rye and oat flours.

Description

(1) The present invention now will be described by an illustrative, but not limitative way according to preferred embodiments thereof, with particular reference to enclosed drawings.

(2) FIG. 1 shows N type aminopeptidase (PepN), dipeptidase (PepV) and tripeptidase (PepT) (a), and proline iminopeptidase (PepI), prolidase (PepQ), prolinase (PepR), dipeptidil-peptidase (PepX) (b) activities of Lactobacillus sanfranciscensis DPPMA12 (DSM22063) and Lactobacillus plantarum DPPMA125 (DSM22064), on Leu-p-NA, Leu-Leu, Leu-Leu-Leu and Pro-p-NA, Val-For-Gly and Gly-For-Wing synthetic substrates, respectively. Lactic acid bacteria used in the study of Rizzello et al. (Rizzello et al., 2007. Appl. Environ. Microbiol. 73:4499-4507) were employed as control: Lactobacillus alimentarius 15M, Lactobacillus brevis 14G, L. sanfranciscensis 7A, Lactobacillus hilgardii 51B and L. sanfranciscensis LS3, LS10, LS19, LS23, LS38 and LS47. The enzymatic activity was expressed as activity unit (U), i.e. enzyme amount necessary to release 1 mol/min of p-nitroanilide or 1 mol/min of amino acid for the activities on substrates different from p-nitroanilide.

(3) FIG. 2 shows bi-dimensional electrophoretic profiles of various durum wheat varieties (Svevo and Duilio) before and after treatment with selected lactic acid bacteria and fungal proteases.

(4) FIG. 3 shows the protein composition of wheat flour before and after the hydrolysis process by selected lactic acid bacteria and fungal proteases.

(5) FIG. 4 shows aminopeptidase (A), proline iminopeptidase (B) and prolyl-dipeptidil aminopeptidase (C) activities of lactic acid bacteria used according to WO2008/010252 (Lactobacillus sanfranciscensis LS40, LS13, LS44, LS35, LS14, LS11, LS18, LS4, LS15 and LS41) and the present invention [L. sanfranciscensis DPPMA12 (DSM22063) and Lactobacillus plantarum DPPMA125 (DSM22064)]. The acronyms 15M, 14G, 7A, 51B, LS3, LS10, LS19 LS23, LS38 and LS47 stand for biotypes as used according to Rizzello et al., 2007, publication.

(6) FIG. 5 shows the residual gluten concentration (ppm) in fermented doughs from Lactobacillus sanfranciscensis LS40, LS13, LS44, LS35, LS14, LS11, LS18, LS4, LS15 and LS41 (WO2008/010252) and from L. sanfranciscensis DPPMA12 (DSM22063) and Lactobacillus plantarum DPPMA125 (DSM22064) for 12 h at 37 C.

(7) FIG. 6 shows the concentration of total free amino acids (mg/kg) in fermented dough using different combinations of lactic bacteria according to WO2008/010252 (dough 1, 2, 3, 4 and 5) and wheat flour dough fermented with two lactic acid bacteria (DDPPMA12 and DPPMA125) of the present invention.

(8) FIG. 7 shows the Principal Component Analysis (PCA) of the data obtained from sensory analysis of breads (1, 2, 4 and 5) according to WO2008/010252 and bread (DPPMA12 and DPPMA125) obtained using detoxified wheat flour according to the invention.

EXAMPLE 1

Peptidase Activity of Selected Lactic Bacteria

(9) L. sanfranciscensis DPPMA12 and L. plantarum DPPMA125 from the Culture Collection of the Dipartimento di Protezione delle Piante and Microbiologia Applicata dell'Universita degli Studi di Bari, previously isolated from sourdoughs, were propagated at 30 C. for 24 h in modified MRS (mMRS), containing, in addition to usual ingredients, 5% maltose and 10% yeast waterfinal pH 5.6. As control for peptidase activities of lactic bacteria used in the recent publication by Rizzello et al. (Rizzello et al., 2007. Appl. Environ. Microbiol. 73:4499-4507) were used: Lactobacillus alimentarius 15M, Lactobacillus brevis 14G, L. sanfranciscensis 7A, Lactobacillus hilgardii 51B and L. sanfranciscensis LS3, LS10, LS19, LS23, LS38 and LS47.

(10) Cells cultivated for 24 h, harvested by centrifugation (10000 rpm, 4 C.), washed twice in phosphate buffer 50 mM, pH 7.0 and re-suspended in same buffer at 2.5 (A.sub.620 nm) optical density, corresponding to 10.sup.8 cfu/ml, were used for enzyme assays. Type N (PepN) aminopeptidase and proline iminopeptidase (PepI) activities, were determined using Leu-p-NA and Pro-p-NA synthetic substrates, respectively. The reaction mixture consisted of: 0.9 ml of K-phosphate buffer 50 mM, pH 7.0 containing dissolved synthetic substrate (final concentration 2 mM) and 100 l of cellular suspension. The enzymatic activity, expressed as activity unit (U), corresponds to enzyme amount necessary to release 1 mol/min of p-nitroanilide (Gobbetti et al., 1996. The proteolytic system of Lactobacillus sanfranciscensis CB1: purification and characterization of a proteinase, a dipeptidase, and an aminopeptidase. Appl. Environ. Microbiol. 62: 3220-3226). Prolidase (PepQ), prolinase (PepR) and dipeptidil-peptidase (PepX) were determined as described by Cagno and co-workers, (Di Cagno et al., 2004. Sour dough bread made from wheat and nontoxic flours and starter with selected lactobacilli is tolerated in celiac sprue patients, Appl. Environ. Microbiol. 70: 1088-1096) on, respectively, Val-Pro, Pro-Gly and Gly-Pro-Ala. Dipeptidase (PepV) and tripeptidase (PepT) were determined according to Cd-ninidrine method (Gobbetti et al., 1999. Study of the effects of temperature, pH, NaCl, and aw on the proteolytic and lipolytic activities of cheese-related lactic bacteria by quadratic response surface methodology, Enzyme Microbial Technol 25: 795-809) using, respectively, Leu-Leu and Leu-Leu-Leu. One activity unit (U) is defined as the enzyme amount necessary to release 1 mol/min of amino acid.

(11) For comparative purposes, the test was repeated also for the lactic bacteria described in WO2008/010252 (L. sanfranciscensis LS40, LS13, LS44, LS35, LS14, LS11, LS18, LS4, LS15 and LS41).

EXAMPLE 2

Protein Extraction from Wheat Flour and Electrophoretic Analysis

(12) Proteins were extracted from wheat flour according to method described by Weiss et al. (Weiss et al., 1993. Electrophoretic characterization of wheat grain allergens from different cultivars involved in bakers asthma. Electrophoresis. 14:805-816). Bi-dimensional electrophoretic analysis of ca. 30 g of extracted fraction protein was carried out according to immobiline-polyacrilamide method (De Angelis et al., 2005. Biochim. Biophys. Acta. 1762:80-93). Four gels for each independent fermentation were analyzed and data were normalized according to procedure as proposed by Bini et al. (Bini et al., 1997. Protein expression profiles in human breast ductal carcinoma and histologically normal tissue. Electrophoresis. 18:2831-2841).

EXAMPLE 3

Immunological and Mass Spectrometry MALDI-TOF Analyses

(13) Immunological analyses were carried out by using of R5 antibody, and sandwich and competitive ELISA test (Transia Plate, Diffchamb) (Valdez et al., 2003. Innovative approach to low-level gluten determination in foods using sandwich enzyme-linked immunosorbent assay protocol. Eur. J. Gastroenterol. Hepatol. 15:465-474). MALDI-TOF spectrometry analysis was carried using Voyager-De Pro-workstation (PerSeptive Biosystems United Kingdom) according to method reported by Hernando et al. (Hernando et al., 2003. New strategy for the determination of gliadin in maize or rice-based foods matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fractionation of gliadin from maize or rice-prolamins by acid treatment. J. Mass Spectrom. 38:862-871).

(14) Protein concentration was determined according to Bradford method (Bradford, 1976. A rapid and sensitive method for the quantification of microgram quanties of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254). Organic nitrogen concentration was determined according to Kjeldahl method. Free amino acid concentration was determined using an amino acid analyzer (Biochrom Ltd., Cambridge Science Park, United Kingdom) (Di Cagno et al., 2004. Appl. Environ. Microbiol. 70:1088-1096).

(15) For comparative purposes, free amino acid concentration was determined also in dough obtained using lattobacilli described in WO2008/010252 (L. sanfranciscensis LS40, LS13, LS44, LS35, LS14, LS11, LS18, LS4, LS15 and LS41) after fermentation for 24 hours at 30 C. according to procedures as indicated in the protocol reported in FIG. 8 of said document.

EXAMPLE 4

Manufacture of Leavened Baked Goods Using Detoxified Wheat Flour

(16) Cultures of two selected lactic acid bacteria were propagated in culture medium, washed and re-suspended in water as previously described. Wheat flour was mixed at 30% with water (70%) containing the mixture of said two lactic acid bacteria at a cell density of ca. 10.sup.8 cfu/g, and fungal enzymes, each at 400 ppm concentration, were added. Fermentation was carried out for 12 h at 37 C. After fermentation, directly to liquid dough native maize (10%), rice flour (10%), egg (5%), sugar (3%), butter (1%) and baker's yeast (1.5%) were added. The concentrations are based on the total dough weight. After kneading, the fermentation is allowed for 1.5 h at 30 C., before the baking of leavened dough for 50 minutes at 250 C.

(17) The process can further comprise a drying step of liquid wheat flour dough. Different ingredients were also used for the production of gluten-free bread.

(18) For comparison purposes also breads 1, 2, 4 and 5 were produced according to the protocol of Patent Application WO2008/010252. Sensory analysis of breads obtained according to the invention and known art, respectively, was carried out, particularly the following descriptors were considered: elasticity, acid fragrance, acid taste, sweetness, dryness and fragrance. Each descriptor was evaluated according to a score scale from 0 to 100. The results of sensory analysis were processed by Principal Component Analysis. Moreover, the breads were analyzed for specific volume, crumb structure, stiffness and fibre content according to standard methods of the American Association of Cereal Chemistry (AACC).

EXAMPLE 5

Administration to Celiac Patients of Leavened Baked Goods Made of Detoxified Wheat Flour

(19) Baked goods based on detoxified wheat flour, obtained as previously described, were administered to 5 celiac patients. Opportunely the celiac pathology diagnosis has been acquired according to criteria as proposed by the European Society for Pediatric Gastroenterology, Hepatology and Nutrition. Patient average age was ca. 15 years. Celiac patients were under remission conditions since at least two years and subjected to a controlled gluten-free diet. All patients at recruitment showed serological pathology negative indicators, as well as negative histochemical assays. Each patient, during a 60 day period, daily consumed baked goods which contained detoxified wheat flour corresponding to 10 g of native gluten equivalent. Immunochemical and histological assays were carried out at the Dipartimento di Pediatria e Gastroeneterologia dell'Universita degli Studi di Napoli, Federico II. Patient recruitment occurred with informed consent of the parents to which the experimental schedule, previously approved by the Ethical Committee of the University of Naples, has been subjected.

(20) Results

(21) (1) Peptidase Activity of Selected Lactic Acid Bacteria

(22) Peptidase activity was assayed on synthetic substrates relatively specific for peptidase activities which are important for the degradation of gluten derived oligopeptides (FIG. 1). It is possible to observe that L. sanfranciscensis DPPMA12 and L. plantarum DPPMA125 display all the considered enzymatic activities. With the exception of tripeptidase (PepT) type activity, two selected lactic acid bacteria, and particularly L. plantarum DPPMA125, display values for other peptidase activities significantly (P<0.05) higher than biotypes used in the study by Rizzello et al. (Rizzello et al., 2007. Appl. Environ. Microbiol. 73:4499-4507). Significant differences, particularly for PepI, PepQ, PepR and PepX activities, with the presence of proline residues at various bond positions, were detected. Glutenin and in particular gliadins, contain a very high and unusual percentage (45-60%) of glutamine and proline residues. This last iminoacid, consequently, particularly occurs in toxic epitopes, resulting from wheat flour, and responsible of celiac pathology. To provide for microorganisms suitable for high degradation of bond where proline is involved in, this enzyme activity is certainly a pre-requirement for intense gluten degradation and rapid hydrolysis process. The availability of a large Culture Collection to be screened and the large number of assayed enzymatic activities represent, therefore, the requirement to get not commonly available selected strains. Peptidase activity of selected lactic bacteria is enhanced by the complementary use of fungal proteases routinely used in bread-making processes. Such enzymes are employed in the bread-making industry in order to modify the protein concentration and, therefore, the flour strength, depending on baked goods they are designed to.

(23) FIG. 4 shows a comparison of peptidase activities for known art lactic acid bacteria and two lactic acid bacteria (DPPMA12 and DPPMA125) according to the invention, respectively, and it is apparent that the latter display markedly higher aminopeptidase, proline iminopeptidase and prolyl-dipeptidil aminopeptidase activities.

(24) (2) Characterization of Hydrolised Flour

(25) After fermentation for 12 h at 37 C., protein fractions were selectively extracted and subjected to complementary analytical assays. As it is apparent using bi-dimensional electrophoretic analysis (FIG. 2), at the end of fermentation process no traces of gliadins from Svevo and Duilio durum wheat variety flours are detectable. Similar results were found in the glutenin fraction for bread commercially available 00 type wheat, other durum wheat tested varieties (Arcangelo, Ciccio, Colosseo, Gargano and Simeto) and barley, rye and oat flours. Wheat flour proteins, extracted with 60% ethanol, were analyzed with MALDI-TOF MS technique. Peaks corresponding to gliadin European standard completely disappeared after fermentation for 12 h at 37 C. Only some peaks with molecular mass lower than 8 kDa were detected by spectrometry analysis. Immunological analyses carried out using R5 antibodies and ELISA assays confirmed that no gliadin traces were detectable in fermented sample. According to same method the residual gluten concentration determined in bread commercially available type 00 wheat, durum wheat variety and barley, rye and oat flours was, in all the cases, lower than 20 ppm. The method used for these determinations is an AIC (Associazione Italiana Celiachia), WHO and FAO official method. With respect to literature reported data (Rizzello et al., 2007. Appl. Environ. Microbiol. 73:4499-4507), the hydrolysis process is carried out with the same effectiveness (residual gluten <20 ppm) but in a markedly shorter times (12 versus 48 h). This process high rate results, on the one hand, from the use of higher concentration of each of fungal proteolytic enzymes (400 ppm) and, on the other hand, mainly from higher peptidase activity of selected lactic bacteria biotypes. Again in comparison to said literature reference which only considered bread wheat flour with low initial gluten concentration, the present invention shows the protocol effectiveness also on various durum wheat varieties, and barley, rye and oat flours having also initial elevated protein concentrations.

(26) In FIG. 3 organic nitrogen content of bread commercially available type OO wheat flour before and after the fermentation process is reported. Hydrolysed flour almost totally consists of a mixture of low molecular weight peptides and amino acids. Only an amount lower than 20% of initial glutenins is yet present in hydrolysed flour. Amino acid concentration in hydrolysed flour is about 15000 mg/kg compared to <1.000 mg/kg occurring in wheat flour. Higher bioavailability of free amino acids makes this hydrolysed wheat flour a raw material with high nutritional content, retaining at the same time other cereal nutritional characteristics in terms of mineral salts, vitamins and fibres. Where used as an ingredient for the production of gluten-free foods, the hydrolysed wheat flour would cover nutritional imbalances resulting from gluten-free diet (Grehn et al., 2001. Scand J Nutr 45: 178-182; Mariani et al., 1998. J Pediart Gastroenterol Nut 27: 519-523; Thompson et al., 2005. J. Human. Nutr. Diet. 18:163-169).

(27) Comparative test with known art lactic acid bacteria shows that the amino acid concentration in the dough is remarkably lower and equal to about 2000 mg/kg after highest hydrolysis condition (FIG. 6). This confirms the different hydrolysis degree of wheat proteins. Moreover, since released amino acids are precursors of volatile compounds which are generated during baking process and are responsible for baked goods taste, remarkably higher free amino acid concentration, as in the case of the present invention, indicates higher synthesis of volatile compounds and, therefore, a better taste of the products according to the invention.

(28) (3) Production of Leavened Baked Goods Using Detoxified Wheat Flour

(29) An application example of biotechnological protocol for the manufacture of leavened baked goods based on detoxified wheat flour was reported above. In addition to the manufacture of baked goods, the protocol was standardized and optimized also for gluten-free bread production with previously described ingredients. In addition to the possible direct use of detoxified wheat flour, a treatment thereof using spry-drier and subsequent use as dried matter is possible. This further technological possibility allows an easy conservation of the raw material over the time, without any alteration of wheat flour nutritional characteristics.

(30) FIG. 7 shows the best sensory properties of the bread according to the present invention (DPPMA12+DPPMA125) compared to known art breads. Moreover, table 1 shows that the bread according to the present invention is characterized by higher specific volume, more crumb bubbles, lower stiffness, and higher fibre content compared to known art bread. These differences result from the presence of wheat flour that although detoxified is suitable to favour better rheological and chemical characteristics.

(31) TABLE-US-00001 TABLE 1 Rheological and Bread Bread chemical parameters Starter 1* (DPPMA12 + DPPMA125) Specific volume 1.35 0.04 1.48 0.07 (cm3/g) Crumb bubbles (%) 39.2 0.34 42.3 0.47 Stiffness (n) 16.62 0.27 14.75 0.21 Fibre content (%) 1.5 0.44 2.0 0.52 *Lactobacillus sanfranciscensis DSM18426, DSM18427, Lactobacillus plantarum DSM18430

(32) (4) Administration to Celiac Patients of Baked Goods Made of Detoxified Wheat Flour

(33) Baked goods prepared according to previously described biotechnological protocol have been administered daily to celiac patients corresponding to a dose equivalent to 10 g of native gluten. In Table 2 immunochemical and histological indices of in remission celiac patients subjected to consumption (10 g of gluten equivalent to the day for 60 gg) based on detoxified wheat flour are reported.

(34) TABLE-US-00002 TABLE 2 Immunochemistry Anti-tTG CD3 CD25 Marsh Grade T.sub.0 T.sub.60 T.sub.0 T.sub.60 T.sub.0 T.sub.60 T.sub.0 T.sub.60 T.sub.0 T.sub.60 F.I. 1.6 1 39 38 6 5 5.6 8.6 0 0 I.C. 1.9 1.1 3.7 11 11 9 0.9 3.8 0 0 R.R. 0.3 0.3 53 56 3 4 11.5 17.8 1 1 I.I. 0.5 0.3 31 36 21 21 8.4 12.8 0 0

(35) As it is possible to observe, all the patients at recruitment time (T.sub.0) display normal serological and histological values (Marshes Grade). After each daily assumption of 10 g gluten equivalent, over a 60 day period (T.sub.60) none of biochemical and immunohistochemical values was different compared to initial value. In particular, it is to be observed that Marsh Grade, who represents the integrity condition and functionality of the intestinal mucosa, as detected based on bio-optical sample, is absolutely identical to initial value. No patient developed atrophy of intestinal villas during the challenge. Only one out 5 recruited patients interrupted the test due to personal reasons and not depending on eventual pathological condition. On the base of acquired results based on most careful in vivo clinical analyses, it is possible to state that the detoxified wheat flour was tolerated by all the patients. In conclusion, detoxified wheat flour can be used for the preparation of gluten-free foods.