Proteolysis by Sourdough Lactic Acid Bacteria: Effects on Wheat Flour Protein Fractions and Gliadin Peptides Involved in Human Cereal Intolerance

Fifty-five strains of lactic acid bacteria, previously isolated from sourdoughs from southern Italy, were used in this study (13). The species used were those commonly identified in sourdoughs: L. sanfranciscensis (1O, 2A, 5D, 5Q, 6G, 7A, 7H, 9F, 9M, 13A, 13R, 14C, 20C, 22E, and 22Z), Lactobacillus alimentarius (1A, 1B, 2B, 8D, 10α, 15A, 15F, 15M, 15R, 16B, 16I, and 16M), Lactobacillus brevis (1F, 1D, 6M, 10A, 14G, 17D, and 18B), Lactobacillus fermentum (6E, 18F, and 18I), Lactobacillus hilgardii (51B and 52B), Lactobacillus acidophilus (16A and 16α), Lactobacillus plantarum (17N, 18E, 19A, 20B, and 21B), Lactobacillus farciminis (I2), Lactobacillus fructivorans (DA106), Lactococcus lactis subsp. lactis (11M, 10γ, and 17C), Weissella confusa (14A) (11), and Leuconostoc citreum (10M, 11C, and 23B) (17).

The strains were routinely propagated for 24 h at 30 or 37°C in modified MRS broth (Oxoid, Basingstoke, Hampshire, England) with the addition of fresh yeast extract (5% [vol/vol]) and 28 mM maltose at a final pH of 5.6. When used for enzyme assays, sourdough fermentation, and subcellular fractionation, lactic acid bacterial cells were incubated until the late exponential phase of growth was reached (ca. 12 h).

Twelve-hour-old cells of lactic acid bacteria cultivated in modified MRS broth were harvested by centrifugation at 9,000 × g for 10 min at 4°C, washed twice with 20 mM sterile phosphate buffer (pH 7.0), and resuspended in the same buffer at a concentration of ca. 10⁹ CFU/ml.

A wheat flour hydrolysate was produced by stirring a suspension of wheat flour (30% [wt/vol] in 20 mM phosphate buffer, pH 7.0) for 1 h at 30°C. After incubation, the suspension was filtered (Whatman International Ltd. [Maidstone, England] no. 4), heated at 80°C for 5 min, centrifuged (10,000 × g for 15 min at 4°C), sterilized by filtration (0.45-μm-pore-size Millex-HA; Millipore S.A., Saint Quentin, France), and treated with 0.1% (wt/vol) protease from Bacillus licheniformis (ca. 3 U/mg) (Sigma Chemical Co., St. Louis, Mo.) having an optimum pH of 7.0. After 3 h of incubation at 30°C, the wheat flour hydrolysate treated with protease (WFHP) was heated at 80°C for 5 min to inactivate enzymes, sterilized again by filtration, and used as a substrate. The assay mixture, containing 0.8 ml of WFHP and 0.2 ml of the cellular suspension, was incubated at 30°C for 6 h, and the supernatant was recovered by centrifugation and used for electrophoresis. A preliminary assay conducted with undigested wheat flour hydrolysate and other non-cereal protein substrates gave unsatisfactory results. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted according to the Laemmli procedure (26); the gels contained 12.5% acrylamide (separation distance, 10 cm; gel thickness, 1.5 mm) and were stained with B10 Bio-Safe Coomassie blue (Bio-Rad Laboratories, Hercules, Calif.). Low-range SDS-PAGE molecular mass standards (Bio-Rad) were used. Three gels from each assay were analyzed for the protein band intensities in WFHP (piWFHP) and in WFHP treated with lactic acid bacteria (piWFHPL) with the Quantity One software package (Bio-Rad). The hydrolysis factors for individual protein bands were calculated as [(piWFHP − piWFHPL)/piWFHP] × 100.

The characteristics of the wheat flour used were as follows: moisture, 12.8%; protein (N × 5.70), 10.7% of dry matter (d.m.); fat, 1.8% of d.m.; ash, 0.6% of d.m.; and total soluble carbohydrates, 1.5% of d.m.

Two hundred grams of wheat flour, 70 ml of tap water, and 30 ml of cellular suspension containing 10⁹ CFU of each lactic acid bacterial strain/ml (final concentration in the dough, 5 × 10⁷ CFU/g) were used to produce 300 g of dough (dough yield: 150) with a continuous high-speed mixer (60 × g; dough mixing time, 5 min). Sourdough fermentations were carried out at 37°C for 4 and 8 h, which corresponded to the most typical times of fermentation used for Italian wheat sourdoughs (13, 36). All the sourdoughs started with selected lactic acid bacteria had pHs ranging from 4.3 to 4.6, depending on the time of incubation. A dough produced with 200 g of wheat flour and 100 g of tap water, without bacterial inoculum and containing 0.15 g of NaN₃ (wt/wt), was incubated under the same conditions and was used as the control. After incubation, the dough was acidified to pH 4.4 with a mixture of lactic and acetic acids in a molar ratio of 4:1, which corresponds to that usually found after sourdough fermentation (19). Under our experimental conditions, the total bacterial count of this unstarted dough varied from 2 × 10⁴ to 7 × 10⁴ CFU/g after incubation without appreciable variation in pH and dough rheology and taste. The chemical acidification was also carried out directly, without addition of NaN₃, during dough mixing or gradually during dough incubation.

Wheat flour protein fractions were extracted from sourdough and control samples following the method originally described by Osborne (32) and further modified by Weiss et al. (44). One gram of dough was diluted with 4 ml of 50 mM Tris-HCl (pH 8.8), held at 4°C for 1 h with vortexing at 15-min intervals, and centrifuged at 20,000 × g for 20 min. The supernatant contained albumins and globulins. In order to minimize cross contamination among albumins, globulins, and gliadins, the pellets were further extracted twice with 50 mM Tris-HCl (pH 8.8), and the supernatants were discarded. After being washed with distilled water to remove buffer ions, the pellets were diluted with 4 ml of ethanol (75% [vol/vol]), stirred at 25°C for 2 h, and centrifuged as described above. The supernatant contained gliadins. The extraction by ethanol was also repeated twice. Residual ethanol was eliminated by resuspending the pellets with distilled water and centrifugation. Finally, the pellets were diluted with 4 ml of a urea-dithiothreitol (DTT) mixture (6 M urea, 1% [vol/vol] Triton X-100, 0.5% [wt/vol] DTT, and 0.5% [vol/vol] 2-D Pharmalyte [pH 3 to 10]), held for 2 h at room temperature with occasional vortexing, and centrifuged. The supernatant contained glutenins. All extracts were stored at −80°C until they were used. To compare proteins with different solubilities, all the extracts were diluted with equal volumes of 6 M urea containing 1% (vol/vol) Triton X-100, 0.5% (wt/vol) DTT, and 0.5% (vol/vol) 2-D Pharmalyte (pH 3 to 10) before two-dimensional (2D) electrophoresis.

2D gel electrophoresis was performed using the immobiline-polyacrylamide system, essentially as described by Bjellqvist et al. (10). Aliquots (30 μl) were used for each electrophoretic run. Isoelectric focusing was carried out on immobiline strips providing a nonlinear 3-to-10 pH gradient (IPG strips; Amersham Pharmacia Biotech) by IPG-phore at 15°C. The voltage was as follows: 0 to 300 V for 1 h, 300 to 500 V for 3 h, 500 to 2,000 V for 4 h, and a constant 8,000 V for 4 h. After electrophoresis, the IPG strips were equilibrated for 12 min against 6 M urea, 30% (vol/vol) glycerol, 2% (wt/vol) SDS, 0.05 M Tris-HCl (pH 6.8), and 2% (wt/vol) dithioerythritol and for 5 min against 6 M urea, 30% (vol/vol) glycerol, 2% (wt/vol) SDS, 0.05 M Tris-HCl (pH 6.8), 2.5% (wt/vol) iodioacetamide, and 0.5% bromophenol blue. The second dimension was carried out in a Laemmli system (26) on 12% polyacrylamide gels (13 cm by 20 cm by 1.5 mm) at a constant current of 40 mA/gel and at 10°C for approximately 5 h until the dye front reached the bottom of the gel. The gels were calibrated with two molecular mass markers: comigration of the extracts with human serum proteins for a molecular mass range of 200 to 10 kDa and markers for two-dimensional electrophoresis (pI range, 7.6 to 3.8; molecular mass range, 17 to 89 kDa) from Sigma Chemical Co. The electrophoretic coordinates used for serum proteins were according to the method Bjellqvist et al. (10). The gels were silver stained as described by Hochstrasser et al. (25). The protein maps were scanned with an Image Scanner and analyzed with the Image Master 2D version 3.01 computer software (Amersham Pharmacia Biotech). Three gels were analyzed, and spot intensities of chemically acidified dough (siCAD) and sourdough (siSD) were normalized as reported by Bini et al. (9). In particular, the spot quantification for each gel was calculated as relative volume (percent volume); the relative volume was the volume of each spot divided by the total volume over the whole image. In this way, differences in color intensities among the gels were eliminated (2). The hydrolysis factor for individual proteins was expressed as [(siCAD − siSD)/siCAD] × 100. All the hydrolysis factors were calculated based on the average of the spot intensities of each of the three gels, and standard deviation was calculated. Only hydrolysis factors with statistical significances where the P value was <0.05 were reported (see Table 2).

The concentrations of free amino acids in the water extracts of chemically acidified dough and sourdoughs were determined. Ten grams of dough were diluted with 50 ml of distilled water, homogenized with a Classic Blender (PBI International, Milan, Italy), and incubated with stirred conditions (100 rpm) at 30°C for 30 min. After centrifugation at 12,000 × g for 15 min, the supernatant was freeze-dried. Twenty milligrams of extract was resuspended in 6 ml of distilled water and filtered through a membrane having a 500-Da cutoff. The permeate was previously derivatized in a 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate precolumn and then used for high-performance liquid chromatography analysis (AccQ-Tag method; Waters Associates, Milford, Mass.). The chromatographic separation was carried out on a Waters AccQ-Tag column at 37°C, and elution was at a flow rate of 1 ml/min with a ternary gradient composed of 50 mM acetate buffer, pH 5.0, containing phosphoric acid (A), acetonitrile (B), and water (C). A fluorescence detector was used at a 250-nm excitation wavelength and a 395-nm emission wavelength. Identification and quantification of amino acids were carried out by comparison with a standard mixture of amino acids (Sigma Chemical Co.).

For rheological analyses, the total weight of the dough was increased to 1.2 kg, keeping a constant dough yield of 150, and the time of incubation was varied from 1.5 to 4 h. NaCl at a concentration of 0.8% (wt/wt of dough), as normally used in Italian breadmaking (13), was also added to the dough. For rheological analyses, the chemical acidification of the dough was carried out directly only during mixing.

The maximum resistance (maximum height of the curve in extensographic units) and extensibility (length of the curve in millimeters) of sourdoughs fermented for 1.5 h were determined by the Brabender OHG (Duisburg, Germany) extensograph. The resistance to deformation (P; height of the curve in millimeters), the extensibility (L; length of the curve in millimeters), and the P/L ratio of sourdoughs fermented for 4 h were determined with the Chopin (Chopin, France) alveograph. The resistance to mixing was estimated by the Brabender farinograph on sourdoughs fermented for 4 h. The degree of softening of the dough, expressed in Brabender units, was defined as the difference between the starting and final resistance values during one run (20 min) in the farinograph.

Twelve-hour-old cells of lactic acid bacteria cultivated in modified MRS broth were used for subcellular fractionation by lysozyme treatment in 50 mM Tris-HCl buffer, pH 7.5, containing 24% (wt/vol) sucrose, as described by Gobbetti et al. (20, 21). The only modification was that spheroplasts resuspended in isotonic buffer were sonicated by four cycles (10 each) (Sony Prep model 150; Sanyo, Tokyo, Japan) to recover the cell cytoplasm. Two cellular fractions were used: cell wall and cytoplasm. Both fractions were dialyzed for 24 h at 4°C against 20 mM phosphate buffer, pH 7.0, and concentrated ca. 20-fold by freeze-drying (MOD E1PTB; Edwards, Milan, Italy). The protein profile of the cell wall fraction was checked by SDS-PAGE in five repeated assays, as described by De Angelis et al. (15).

Proteinase activity was measured in 20 mM phosphate buffer, pH 7.0, by the method of Twinning (41) with fluorescent casein (1.0% [wt/vol]) as the substrate. Peptidase activity was measured on Leu-p-nitroanilide by the method of Gobbetti et al. (20). Proteinase activity on fluorescent casein was mainly detected in the cell wall fraction, while as expected, peptidase activity was found only in the cytoplasmic preparation. Both cellular fractions showed activity towards the nonapeptide bradykinin (Sigma Chemical Co.), which was used to standardize the enzyme activities.

The 31-43 fragment of A-gliadin was chemically synthesized by the Neosystem Laboratoire (Strasbourg, France). The peptide has the following sequence: L-G-Q-Q-Q-P-F-P-P-Q-Q-P-Y. All of the enzyme preparations used in the assays showed ca. 80% hydrolysis on bradykinin substrate. The reaction mixture contained 160 μl of 20 mM phosphate buffer (pH 7.0), 75 μl of 4 mM fragment 31-43, 4 μl of NaN₃ (0.05% final concentration), and 100 μl of the enzyme preparation. The enzyme activity was stopped by addition of 0.1% (vol/vol) (final concentration) trifluoroacetic acid. Peptides were separated from the mixture by reversed-phase-fast performance liquid chromatography (RP-FPLC) using a PepRPC HR 5/5 column and FPLC equipment with a UV detector operating at 210 nm (Pharmacia Biotech). Elution was at a flow rate of 0.5 ml/min with a linear gradient (0 to 100%) of acetonitrile in 0.1% trifluoroacetic acid.

The 31-43 fragment of A-gliadin and the hydrolyzed mixtures produced by enzyme treatments were freeze-dried and used for agglutination tests.

Ethanol-extractable proteins (gliadins) from wheat flour (S. Pastore variety) were submitted to peptic-tryptic (PT) sequential digestion to produce the corresponding PT digest (4). After production, the PT digest was heated at 100°C for 30 min to inactivate enzymes. This peptide preparation was used directly for the agglutination test, or it was further digested with enzyme preparations from lactic acid bacteria before the agglutination test. A mixture containing 160 μl of 20 mM phosphate buffer (pH 7.0), 75 μl of PT digest (21 mg/ml), 4 μl of NaN₃ (0.05% final concentration), and 100 μl of enzyme preparation, which consisted of equal volumes of cell wall and cytoplasmic fractions, was incubated at 30°C for 24 h and then immediately assayed.

K 562 (S) subclone cells of human myelagenous leukemia origin from the European Collection of Cell Culture (Salisbury, United Kingdom) were used for the agglutination test (6). The cells were grown in RPMI medium (HyClone, Cramlington, United Kingdom) supplemented with 0.2 mM l-glutamine, 50 U of penicillin/ml, 50 mg of streptomycin/ml, and 10% (vol/vol) fetal calf serum (Flow Laboratories, Irvine, Scotland) at 37°C in a humidified atmosphere of 5% CO₂ in air for 96 h. After cultivation, the human cells were harvested by centrifugation at 900 × g for 5 min, washed twice with 0.1 M phosphate-buffered saline solution (Ca²⁺ and Mg²⁺ free; pH 7.4), and resuspended at a concentration of 10⁸/ml in the same buffer. Twenty-five microliters of this cell suspension was added to wells of a microtiter plate containing serial dilutions (0.218 to 7.0 g/liter) of PT digest, 4 mM 31-43 fragment of A-gliadin, or 4 mM mixtures of the 31-43 fragment hydrolyzed by lactobacillus enzyme preparations in phosphate-buffered saline. The total volume in the well was 100 μl, and the mixture was held for 30 min at room temperature. After incubation, a drop of the suspension was applied to a microscope slide to count clumped and single cells. Agglutination tests were carried out in triplicate, and photographs were taken with a Diaphot-TMD inverted microscope (Nikon Corp., Tokyo, Japan).

Fifty-five strains belonging to 12 species of lactic acid bacteria were preliminarily screened for proteolytic activity using the WFHP substrate, which contained a digest of albumin and globulin polypeptides with masses of 10.0 to 61.6 kDa (data not shown). Based on SDS-PAGE analysis, the strains were differentiated according to the hydrolysis of eight polypeptides. Strains belonging to the same species mainly grouped together, and all the strains fell into four main groups (Table 1). L. alimentarius, L. plantarum, L. farciminis, and L. lactis subsp. lactis strains had the same proteolysis profile as L. alimentarius 15M, which, compared to the WFHP substrate, completely degraded polypeptides 1, 5, and 7; gave rise to a new polypeptide band of ca. 53.4 kDa, probably by hydrolysis of polypeptide 1; and slightly hydrolyzed protein bands designated 2, 3, 4, and 6. L. brevis 14G did not hydrolyzed polypeptide 7, did not produce the ca. 53.4-kDa polypeptide, and had a lower activity on protein band 1. The other strains of L. brevis and those of L. citreum and L. fructivorans behaved similarly. All the strains of L. sanfranciscensis, including 7A, differed from L. brevis 14G mainly due to the presence of the new polypeptide. L. hilgardii 51B and L.acidophilus and W. confusa strains were characterized by a moderate activity on all the polypeptides considered.

L. alimentarius 15M, L. brevis 14G, L. sanfranciscensis 7A, and L.hilgardii 51B were used for further studies, since they were representative of the selected groups and are widely isolated from sourdoughs (19).

The selected lactobacilli were used to study proteolysis during sourdough fermentation. All the sourdoughs contained ca. 10⁹ CFU of each lactic acid bacterial strain/g and had final pH values ranging from 4.3 to 4.6, depending on the time of fermentation. In agreement with previous findings (21, 22), proteolysis at 4 h, expressed in terms of the free amino acid concentration, was 80 to 85% of that reached after 8 h. Results from sourdough fermented for 8 h were further described.

Wheat flour proteins were selectively extracted from sourdoughs and further analyzed by 2D electrophoresis. Figure 1 shows the albumin, globulin, gliadin, and glutenin fractions in the chemically acidified (pH 4.4) dough. In agreement with previous electrophoretic separations (35, 44), albumin and globulin polypeptides were distributed over the entire range of pIs (4.4. to 8.7) and molecular masses (15 to 100 kDa), while gliadins were located in well-defined regions: α, β, and γ fractions (28 to 50 kDa) formed a cluster in the alkaline zone of the gel (pIs, 6.5 to 8.5), and ω fractions (55 to 70 kDa) were confined to the pI area of 4.0 to 7.0. The high-molecular-mass glutenin subunits were positioned in the pI range of 5.1 to 5.4 with a mass of ca. 100 kDa, while low-molecular-mass subunits (35 to 50 kDa) exhibited pIs of 6.0 to 9.0 and partially overlapped the α-, β-, and γ-gliadins. The possibility of slight contamination among protein fractions during selective extraction cannot be excluded.

Since biological or chemical acidification caused a marked modification of the 2D polypeptide pattern with respect to the nonacidified dough (data not shown), the sourdoughs fermented by selected lactobacilli were compared to the chemically acidified dough. A total of 46 polypeptides belonging to albumin, globulin, and gliadin fractions were hydrolyzed by the selected lactic acid bacteria (Table 2). No hydrolysis of glutenins was detected under these conditions. L. alimentarius 15 M showed hydrolysis of 37 polypeptides; 24 of them were located in the region of the albumin and globulin fractions. Almost all the albumin and globulin polypeptides (22 of 24) were degraded by hydrolysis factors of ≥50%. Only 7 of the 13 gliadins were degraded to the same extent. Except for L. hilgardii 51B, the proteolytic activities of the lactobacilli affected polypeptides with a wide range of pIs and molecular masses. Hydrolysis was particularly intense towards protein spots with masses of less than 34 kDa. L. brevis 14G hydrolyzed 39 polypeptides, 24 of which were albumins and globulins. Compared to that of L. alimentarius 15M, the hydrolysis was more pronounced, since all of the albumin and globulin polypeptides and 12 of the 15 gliadins were degraded by hydrolysis factors of ≥50%. L. sanfranciscensis 7A showed proteolysis towards the largest number (41) of polypeptides, 24 albumins and globulins and 18 gliadins. L. hilgardii 51B showed slight proteolysis; only 14 polypeptides were degraded, 8 of them being albumins and globulins.

Proteolysis during sourdough fermentation was also estimated by the concentration of free amino acids (Table 3). The unstarted wheat flour dough incubated for 8 h at 37°C had a final pH of 5.6 and did not show an appreciable increase in total free amino acids with respect to the wheat flour. Also, the addition of the cell suspension to the dough did not increase the free amino acids (data not shown). Chemical acidification to pH 4.4 produced an increase in the total free amino acids (1,278 mg/kg), which reflected a nonspecific increase in individual amino acids. Fermentation by selected lactobacilli resulted in a markedly higher total concentration (1,767 to 2,020 mg/kg) with some differences in the amino acid patterns. Compared to the chemically acidified dough, all the lactobacilli caused a marked increase in Pro, Leu, and Phe concentrations, and L. alimentarius 15M, L. brevis 14G, and L.sanfranciscensis 7A also favored an increase in Asp and Glu. L. hilgardii 51B had the lowest capacity to liberate amino acids.

The sourdoughs used for proteolysis analyses were also evaluated for some rheological properties according to the method proposed by Martinez-Anaya et al. (31). Compared to the chemically acidified dough, the sourdough produced by L. brevis 14G had the lowest maximum resistance (560 versus 1,000 extensographic units) and the highest extensibility (97 versus 60 mm), as analyzed by the Brabender extensograph (Table 4). The sourdoughs started with L. alimentarius 15M and L. sanfranciscensis 7A behaved similarly, while that from L. hilgardii 51B had higher resistance and lower extensibility. These findings were confirmed by the P/L ratios of the Chopin alveograph, which decreased from 10.80 to 1.52 going from the chemically acidified dough to the L. brevis 14G dough. The greatest softening of the dough caused by the four lactobacilli was also demonstrated by the Brabender farinograph, which pointed out the hardness of the chemically acidified dough, characterized by a very low degree of softening (380 versus 430 to 500 Brabender units). An unacidified dough, having the same dough yield of 150, showed a degree of softening which approached that of the chemically acidified dough, as determined by the Brabender farinograph (data not shown).

The four selected lactobacilli were further used to hydrolyze the chemically synthesized 31-43 fragment of A-gliadin, which was found to be toxic to celiac patients by the most common in vitro systems (7, 34). A-gliadin is usually obtained as a sediment by ultracentrifugation (133,000 × g) of the acetic extract of wheat flour; its electrophoretic pattern in a dissociating buffer is identical to that of highly purified α-gliadin fractions. The hydrolysis of the peptide was assayed by using two bacterial cell fractions, cell wall and cytoplasm, which contained the greatest part of the proteinase and peptidase activity, respectively. The cell fractions were used in the enzyme assay at a concentration corresponding to 10⁹ CFU/ml. After 4 h of incubation, hydrolysis of the 31-34 peptide of A-gliadin by proteinases, expressed as percentage reduction of the peak area of the untreated substrate, was ca. 54 to 50% for L. alimentarius 15M and L. brevis 14G, 43% for L. sanfranciscenis 7A, and 35% for L. hilgardii 51B (Fig. 2b, c, d, and e). All the strains showed a common product of hydrolysis, which eluted after 18 min of the acetonitrile gradient; the most complex profile was for L. brevis 14G, which produced four main peptides. Compared to the cell wall, the activity of the cytoplasmic preparation was higher, always resulting in a percentage of hydrolysis greater than 50% (Fig. 2f, g, h, and i). The hydrolysis pattern was only partly similar to that found in the presence of the proteinase activity.

A PT digest of gliadins, obtained by simulating in vivo protein digestion, was used for the agglutination test on K 562 (S) cells. According to previous findings (6), no significant evidence of cell clustering was found when the undifferentiated K 562 (S) cells were not treated with the PT digest (Fig. 3a). On the contrary, the PT digest agglutinated 100% of the undifferentiated cells at the lowest concentration of 0.218 g/liter (Fig. 3b and Table 5). The agglutinated cells had a peculiar appearance, i.e., a tendency to form a continuous cell layer with high resistance to shearing and whirling forces. Before being used, the PT digest was further digested for 24 h with the cell wall and cytoplasmic preparations of selected lactic acid bacteria. When assayed alone, the enzyme preparations were ineffective in causing cell agglutination (data not shown). In the presence of the highest concentration of PT digest (7.0 g/liter), further hydrolysis by L. alimentarius 15M, L. brevis 14G, and L.sanfranciscensis 7A exerted a 50 to 60% inhibition of cell agglutination. L. hilgardii 51B had no effect (Table 5). At an intermediate concentration of 0.875 g/liter, the agglutination activity of the PT digest was completely inhibited by treatment with the L. alimentarius 15M and L. brevis 14G enzymes, while L. sanfranciscensis 7A gave complete inhibition only at 0.218 g/liter.

The agglutination test was also used to assay the activity of the 31-43 fragment of A-gliadin hydrolyzed by the enzyme preparations of lactobacilli. Compared to the whole fragment, all the peptide mixtures gave decreases of K 562 (S) cell agglutination which ranged from 40 to 85%.