Medical nutrition therapy: use of sourdough lactic acid bacteria as a cell factory for delivering functional biomolecules and food ingredients in gluten free bread

Celiac disease (CD) is an immune-mediated disease, triggered in genetically susceptible individuals by ingesting gluten from wheat, rye, barley, and other closely related cereal grains. Currently, the estimated prevalence of CD is around 1 % of the population in the western world and medical nutritional therapy (MNT) is the only accepted treatment for celiac disease. To date, the replacement of gluten in bread presents a significant technological challenge for the cereal scientist due to the low baking performance of gluten free products (GF). The increasing demand by the consumer for high quality gluten-free (GF) bread, clean labels and natural products is rising. Sourdough has been used since ancient times for the production of rye and wheat bread, its universal usage can be attributed to the improved quality, nutritional properties and shelf life of sourdough based breads. Consequently, the exploitation of sourdough for the production of GF breads appears tempting. This review will highlight how sourdough LAB can be an efficient cell factory for delivering functional biomolecules and food ingredients to enhance the quality of gluten free bread.     

Rope-producing strains of Bacillus spp. from wheat bread and strategy for their control by lactic acid bacteria

Two types of white wheat bread (high- and low-type loaves) were investigated for rope spoilage. Thirty of the 56 breads tested developed rope spoilage within 5 days; the high-type loaves were affected by rope spoilage more than the low-type loaves. Sixty-one Bacillus strains were isolated from ropy breads and were characterized on the basis of their phenotypic and genotypic traits. All of the isolates were identified as Bacillus subtilis by biochemical tests, but molecular assays (randomly amplified polymorphic DNA PCR assay, denaturing gradient gel electrophoresis analysis, and sequencing of the V3 region of 16S ribosomal DNA) revealed greater Bacillus species variety in ropy breads. In fact, besides strains of B. subtilis, Bacillus licheniformis, Bacillus cereus, and isolates of Bacillus clausii and Bacillus firmus were also identified. All of the ropy Bacillus isolates exhibited amylase activity, whereas only 32.4% of these isolates were able to produce ropiness in bread slices after treatment at 96 degrees C for 10 min. Strains of lactic acid bacteria previously isolated from sourdough were first selected for antirope activity on bread slices and then used as starters for bread-making experiments. Prevention of growth of approximately 10(4) rope-producing B. subtilis G1 spores per cm(2) on bread slices for more than 15 days was observed when heat-treated cultures of Lactobacillus plantarum E5 and Leuconostoc mesenteroides A27 were added. Growth of B. subtilis G1 occurred after 7 days in breads started with Saccharomyces cerevisiae T22, L. plantarum E5, and L. mesenteroides A27.     

Improvement of the quality of wheat bread by addition of glycoside hydrolase family 10 xylanases

Although many xylanases are widely used in the baking industry, only one glycoside hydrolase family 10 (GH 10) xylanase has previously been reported to be effective in baking. In this study, we compared the effectiveness of two GH 10 xylanases, psychrophilic XynA from Glaciecola mesophila and mesophilic EX1 from Trichoderma pseudokoningii, in bread making. The optimal dosages needed to improve wheat flour dough and bread quality were 270-U/kg flour for EX1 and 0.9-U/kg flour for XynA. At their optimal dosage, both XynA and EX1 had significant dough-softening ability, resulting in a 50% reduction in Brabender units. XynA was more effective than EX1 in reducing the time to reach maximum consistency. XynA and EX1 showed similar effects in improving the bread volume (~30% increase). EX1 was more effective in reducing the initial crumb firmness. Although both enzymes exhibited similar anti-staling effects on the bread, based on a decrease in the bread firmness, XynA had a greater effect on reducing the firming rate, and EX1 showed an enhanced reduction in the initial firmness. These results show that these two GH 10 xylanases have unique advantages in improving dough and bread quality and indicate their potential in bread making.     

Mutations affecting the activity of the cyclodextrin glucanotransferase of Paenibacillus pabuli US132: insights into the low hydrolytic activity of cyclodextrin glucanotransferases

The cyclodextrin glucanotransferase from Paenibacillus pabuli US132 (US132 CGTase) was engineered using a rational approach in an attempt to provide it with anti-staling properties comparable to those of the commercial maltogenic amylase (Novamyl). The study aimed to concurrently decrease the cyclization activity and increase the hydrolytic activity of US132 CGTase. A five-residue loop (PAGFS) was inserted, alone or with the substitution of essential residues for cyclization (G180, L194 and Y195), mimicking the case of Novamyl. The findings indicate that, unlike the case of the CGTase of Thermoanerobacterium thermosulfurigenes strain EM1 whose initial high hydrolytic activity was exceptional, these mutations completely abolished the cyclization and hydrolytic activities of the US132 CGTase. This suggests that those mutations are not able to convert conventional CGTases, whose hydrolytic activities are very weak, into hydrolases. Accordingly, and for the first time, a structural barrier at subsite ?3 was advanced as an influential factor which might explain the low hydrolytic activity of conventional CGTases.     

SENSORY-SPECIFIC SATIETY FOR SELECTED BREAD PRODUCTS1

Subjects rated the intensities of 42 different attributes for 26 different bread products. Principal component analysis was used to categorize the breads based on their attribute ratings. The first two principal components were interpreted as a richness dimension and a nutrition/fiber dimension. The influence of these dimensions on sensory-specific satiety in bread products was investigated. Subjects tasted and rated their liking of small portions of a set of bread products representative of the extremes of the richness dimension and the nutrition/fiber dimension before and after eating a 400 calorie serving of one of the breads. Measurements of hunger were taken before and after the meal. Bread appeared resistant to sensory-specific satiety. The richness of the breads did not affect the amount of sensory-specific satiety. The high nutrition/fiber breads decreased hunger and tended to decrease liking more than the low nutrition/fiber breads.     

Stability of sterigmatocystin during the bread making process and its occurrence in bread from the Latvian market

Sterigmatocystin (STC) is a carcinogenic and mutagenic mycotoxin produced by fungi of many Aspergillus species. The aim of this research was to test the stability of STC during the bread making process and to check bread samples from the Latvian market for STC contamination, using a previously developed electrospray positive ionisation (ESI⁺) liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Wheat grain naturally contaminated with STC was used for bread baking. STC was found to be stable during the bread-making process. In the food survey 17% of the analysed breads were positive for STC, with concentration levels of 2-7 μg kg^-1. One out of six rye bread samples, one out of nine rye-wheat bread samples and three out of 14 wheat bread samples were contaminated with STC. Four out of five contaminated samples contained whole grains as the main ingredient. We conclude that whole grain bread may be a possible source of STC, although even STC-positive bread samples identified in this study contained quite low toxin levels.     

Glutamine deamidation by cereal-associated lactic acid bacteria

AIMS: It was the aim of our work to investigate glutamine deamidation by lactic acid bacteria isolated from cereal fermentations and to elucidate the ecological and technological relevance in baking of the conversion of glutamine to glutamate. METHODS AND RESULTS: Lactobacillus sanfranciscensis and Lact. reuteri were found to display glutaminase activity. The addition of glutamine to modified Man, Rogosa and Sharp medium increased the cell yields of Lact. sanfranciscensis, as well as the production of lactic and acetic acid. The final pH; however, was increased in the glutamine-containing medium. The addition of 47 mmol kg(-1) glutamate to chemically acidified doughs significantly changed the bread flavour. In sourdoughs with enhanced proteolytic activity, strain-dependent production of 27-120 mmol glutamate per kilogram sourdough was observed. CONCLUSIONS: Lactobacillus sanfranciscensis and Lact. reuteri converted glutamine into glutamate; this conversion improves the acid tolerance of lactobacilli and significantly influences wheat bread flavour. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper illustrates the complex interaction of sourdough-lactobacilli with their environment: the flour provides substrates for metabolic activities that enable the lactobacilli to reach higher cell counts, and the produced metabolite may be one of the reasons why the flavour of fermented breads is different to the flavour of chemically acidified breads.     

In-vitro selection of highly stabilized protein variants with optimized surface

Thermostable proteins are of prime importance in protein science, but it has remained difficult to develop general strategies for stabilizing a protein. Site-directed mutagenesis based on comparisons with thermophilic homologs is rarely successful because the sequence differences are too numerous and dominated by neutral mutations. Here we used a method of directed evolution to increase the stability of a mesophilic protein, the cold shock protein Bs-CspB from Bacillus subtilis. It differs from its thermophilic counterpart Bc-Csp from Bacillus caldolyticus at 12 surface-exposed positions. To elucidate the stabilizing potential of exposed amino acid residues, six of these variant positions were randomized by saturation mutagenesis, the corresponding library of sequences was inserted into the gene-3-protein of the filamentous phage fd, and stabilized variants were selected by the Proside technique. Proside links the increased protease resistance of stabilized protein variants with the infectivity of the phage. Many strongly stabilized variants of Bs-CspB were identified in two selections, one in the presence of a denaturant and the other at elevated temperature. Several of them are significantly more stable than the naturally thermostable homolog Bc-Csp, and the best variant reaches Tm-Csp (the homolog from the hyperthermophile Thermotoga maritima) in stability. Remarkably, this variant differs from Tm-Csp at five and from Bc-Csp at all six randomized positions. This indicates that proteins can be strongly stabilized by many different sets of surface mutations, and Proside selects them efficiently from large libraries. The course of the selection could be directed by the conditions. In an ionic denaturant non-polar surface interactions were optimized, whereas at elevated temperature variants with improved electrostatics were selected, pointing to two different strategies for stabilization at protein surfaces.     

Characterization of a xylanase from the newly isolated thermophilic Thermomyces lanuginosus CAU44 and its application in bread making

AIMS: A xylanase from the newly isolated thermophilic fungus, Thermomyces lanuginosus CAU44, was characterized and evaluated for its suitability in bread making. METHODS AND RESULTS: Xylanase was purified 3.5-fold to homogeneity with a recovery yield of 32.8%. It appeared as a single protein band on SDS-PAGE gel with a molecular mass of c. 25.6 kDa. The purified xylanase had an optimum pH of 6.2, and it was stable over pH 5.6-10.3. The optimal temperature of xylanase was 75 degrees C and it was stable up to 65 degrees C at pH 6.2. Study was further carried out to investigate the effect of the purified xylanase on the properties of wheat bread and its staling during storage. CONCLUSIONS: The purified xylanase from T. lanuginosus CAU44 was stable up to 65 degrees C and had a broad pH range. The presence of thermostable xylanase during bread making led to an improvement of the specific bread volume and better crumb texture. Besides, addition of xylanase provided an anti-staling effect. SIGNIFICANCE AND IMPACT OF THE STUDY: The xylanase from the newly isolated Thermomyces lanuginosus CAU44 shows great promise as a processing aid in the bread-making industry.     

Cloning and Characterization of a Weissella confusa Dextransucrase and Its Application in High Fibre Baking

Wheat bran offers health benefits as a baking ingredient, but is detrimental to bread textural quality. Dextran production by microbial fermentation improves sourdough bread volume and freshness, but extensive acid production during fermentation may negate this effect. Enzymatic production of dextran in wheat bran was tested to determine if dextran-containing bran could be used in baking without disrupting bread texture. The Weissella confusa VTT E-90392 dextransucrase gene was sequenced and His-tagged dextransucrase Wc392-rDSR was produced in Lactococcus lactis. Purified enzyme was characterized using ¹⁴C-sucrose radioisotope and reducing value-based assays, the former yielding K _m and V _max values of 14.7 mM and 8.2 μmol/(mg∙min), respectively, at the pH optimum of 5.4. The structure and size of in vitro dextran product was similar to dextran produced in vivo. Dextran (8.1% dry weight) was produced in wheat bran in 6 h using Wc392-rDSR. Bran with and without dextran was used in wheat baking at 20% supplementation level. Dextran presence improved bread softness and neutralized bran-induced volume loss, clearly demonstrating the potential of using dextransucrases in bran bioprocessing for use in baking.     

X-ray structure of Novamyl, the five-domain "maltogenic" alpha-amylase from Bacillus stearothermophilus: maltose and acarbose complexes at 1.7A resolution.

The three-dimensional structure of the Bacillus stearothermophilus "maltogenic" alpha-amylase, Novamyl, has been determined by X-ray crystallography at a resolution of 1.7 A. Unlike conventional alpha-amylases from glycoside hydrolase family 13, Novamyl exhibits the five-domain structure more usually associated with cyclodextrin glycosyltransferase. Complexes of the enzyme with both maltose and the inhibitor acarbose have been characterized. In the maltose complex, two molecules of maltose are found in the -1 to -2 and +2 to +3 subsites of the active site, with two more on the C and E domains. The C-domain maltose occupies a position identical to one previously observed in the Bacillus circulans CGTase structure [Lawson, C. L., et al. (1994) J. Mol. Biol. 236, 590-600], suggesting that the C-domain plays a genuine biological role in saccharide binding. In the acarbose-maltose complex, the tetrasaccharide inhibitor acarbose is found as an extended hexasaccharide species, bound in the -3 to +3 subsites. The transition state mimicking pseudosaccharide is bound in the -1 subsite of the enzyme in a 2H3 half-chair conformation, as expected. The active site of Novamyl lies in an open gully, fully consistent with its ability to perform internal cleavage via an endo as opposed to an exo activity.     

Application of novel starter cultures for sourdough bread production

Sourdough application has been extensively increased in the last years due to the consumers demand for food consumption without the addition of chemical preservatives. Several starter cultures have been applied in sourdough bread making targeting the increase of bread self-life and the improvement of sensorial character. More specific, Lactobacillus acidophilus and Lactobacillus sakei as single and mixed cultures were used for sourdough bread making. Various sourdough breads were produced with the addition of sourdough perviously prepared with 10% w/w L. acidophilus, 10% w/w L. sakei and 5% w/w L. acidophilus and 5% w/w L. sakei at the same time. Various chemical parameters were determined such as lactic acid, total titratable acidity and pH. The results revealed that the produced sourdough bread made with sourdough containing the mixed culture was preserved for more days (12 days) than all the other breads produced in the frame of this study, since it contained lactic acid in higher concentrations. The respective total titratable acidity varied between 10.5 and 11 ml NaOH N/10. The same sourdough bread had a firmer texture, better aroma, flavor and overall quality compared to other sourdough breads examined in this study, as shown by sensory evaluation tests and results obtained through SPME GC–MS analysis, which revealed significant differences among the different bread types.     

Protein engineering of alpha-amylase for low pH performance.

Industrial-scale starch liquefaction is currently constrained to operating at pH 6.0 and above, as the enzyme used in the process, Bacillus licheniformis alpha-amylase, is unstable at lower pH under the conditions used. There is a need to develop an enzyme that can operate at lower pH. Recent progress has been made in engineering the B. licheniformis enzyme for improved industrial performance. The availability of crystal structures and subsequent analysis of improved variants, in a structural context, is revealing common factors and a rationale to make further improvements.     

Production of sour maize bread using starter-cultures

Eight lactic acid bacteria were isolated from fermenting maize meal. They were identified as Lactobacillus brevis, L. casei, L. fermentum, Pediococcus acidilacti, P. pentosaceus, Lactobacillus spp. I and Pediococcus spp. I and II. L. brevis and Lactobacillus spp. I isolated from the spontaneously fermented maize meal together with L. brevis isolated from rye sour dough and L. plantarum from ogi, a fermented maize gruel, were selected as starter organisms. There was a decrease in the final pH from 4.9 to 3.8 and an increase in the acid equivalent and temperature of the spontaneously-generated sour maize meal at the end of 24h fermentation. There was a decrease in the pH and moisture content of the sour maize breads relative to the conventional wheat bread. An improvement in the shelf-life of the bread samples was also obtained. Crude protein values of the sour maize breads were between 4.36% and 8.87%, while crude fat contents ranged between 3.66% to 7.67%. The ash contents increased from 2.29% to 2.54% while total carbohydrate values were between 46.31% and 65.3%. Calcium, phosphorus and potassium contents ranged from 0.015, 0.26 and 0.018% to 0.036, 0.47 and 0.036% respectively. Physical examination of the bread samples showed that all were cracked and relatively hard. Weight, height and volume ranged from 316 to 380g; 4.2 to 5.2cm and 200 to 320cm3 respectively. Statistical analysis of the sensory attributes revealed a consumer acceptance of the sour maize breads, although ranking test showed preference for the baker's yeast leavened bread that served as a control.     

Production of sour maize bread using starter-cultures

Eight lactic acid bacteria were isolated from fermenting maize meal. They were identified as Lactobacillus brevis, L. casei, L. fermentum, Pediococcus acidilacti, P. pentosaceus, Lactobacillus spp. I and Pediococcus spp. I and II. L. brevis and Lactobacillus spp. I isolated from the spontaneously fermented maize meal together with L. brevis isolated from rye sour dough and L. plantarum from ogi, a fermented maize gruel, were selected as starter organisms. There was a decrease in the final pH from 4.9 to 3.8 and an increase in the acid equivalent and temperature of the spontaneously-generated sour maize meal at the end of 24h fermentation. There was a decrease in the pH and moisture content of the sour maize breads relative to the conventional wheat bread. An improvement in the shelf-life of the bread samples was also obtained. Crude protein values of the sour maize breads were between 4.36% and 8.87%, while crude fat contents ranged between 3.66% to 7.67%. The ash contents increased from 2.29% to 2.54% while total carbohydrate values were between 46.31% and 65.3%. Calcium, phosphorus and potassium contents ranged from 0.015, 0.26 and 0.018% to 0.036, 0.47 and 0.036% respectively. Physical examination of the bread samples showed that all were cracked and relatively hard. Weight, height and volume ranged from 316 to 380g; 4.2 to 5.2cm and 200 to 320cm3 respectively. Statistical analysis of the sensory attributes revealed a consumer acceptance of the sour maize breads, although ranking test showed preference for the baker's yeast leavened bread that served as a control.     

A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display

There is growing interest in the fast and robust engineering of protein pH-sensitivity that aims to reduce binding at acidic pH, compared to neutral pH. Here, we describe a novel strategy for the incorporation of pH-sensitive antigen binding functions into antibody variable domains using combinatorial histidine scanning libraries and yeast surface display. The strategy allows simultaneous screening for both, high affinity binding at pH 7.4 and pH-sensitivity, and excludes conventional negative selection steps. As proof of concept, we applied this strategy to incorporate pH-dependent antigen binding into the complementary-determining regions of adalimumab. After 3 consecutive rounds of separate heavy and light chain library screening, pH-sensitive variants could be isolated. Heavy and light chain mutations were combined, resulting in 3 full-length antibody variants that revealed sharp, reversible pH-dependent binding profiles. Dissociation rate constants at pH 6.0 increased 230- to 780-fold, while high affinity binding at pH 7.4 in the sub-nanomolar range was retained. Furthermore, binding to huFcRn and thermal stability were not affected by histidine substitutions. Overall, this study emphasizes a generalizable strategy for engineering pH-switch functions potentially applicable to a variety of antibodies and further proteins-based therapeutics.     

A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display

There is growing interest in the fast and robust engineering of protein pH-sensitivity that aims to reduce binding at acidic pH, compared to neutral pH. Here, we describe a novel strategy for the incorporation of pH-sensitive antigen binding functions into antibody variable domains using combinatorial histidine scanning libraries and yeast surface display. The strategy allows simultaneous screening for both, high affinity binding at pH 7.4 and pH-sensitivity, and excludes conventional negative selection steps. As proof of concept, we applied this strategy to incorporate pH-dependent antigen binding into the complementary-determining regions of adalimumab. After 3 consecutive rounds of separate heavy and light chain library screening, pH-sensitive variants could be isolated. Heavy and light chain mutations were combined, resulting in 3 full-length antibody variants that revealed sharp, reversible pH-dependent binding profiles. Dissociation rate constants at pH 6.0 increased 230- to 780-fold, while high affinity binding at pH 7.4 in the sub-nanomolar range was retained. Furthermore, binding to huFcRn and thermal stability were not affected by histidine substitutions. Overall, this study emphasizes a generalizable strategy for engineering pH-switch functions potentially applicable to a variety of antibodies and further proteins-based therapeutics.     

Application of Bifidobacterium strains to the breadmaking process

The possible use of bifidobacterial strains from different origin (adult and infant humans, and chicken) as novel starter cultures for breadmaking was evaluated. Fermentative parameters of doughs (pH, volume, total titrable acidity [TTA], lactic and acetic acids production and rheofermentative parameters) and technological parameters of breads (specific volume, bread shape and crumb hardness) were analyzed. Human bifidobacterial strains could replace Lactobacillus strains, commercialized for breadmaking, as they yielded breads with similar characteristics but with the advantage of having softer crumbs. Important differences between the behavior of chicken bifidobacterial strains and human bifidobacterial strains were found when comparing bread TTA, bread shape and bread volume. Breads made with chicken strains showed significantly lower (p < 0.05) specific bread volume than those made with human strains, while showing similar values of TTA. The effects observed when using bifidobacterial strains from different origin as novel starter cultures for breadmaking seemed to depend on the strain and its origin.     

Rope-Producing Strains of Bacillus spp. from Wheat Bread and Strategy for Their Control by Lactic Acid Bacteria

Two types of white wheat bread (high- and low-type loaves) were investigated for rope spoilage. Thirty of the 56 breads tested developed rope spoilage within 5 days; the high-type loaves were affected by rope spoilage more than the low-type loaves. Sixty-one Bacillus strains were isolated from ropy breads and were characterized on the basis of their phenotypic and genotypic traits. All of the isolates were identified as Bacillus subtilis by biochemical tests, but molecular assays (randomly amplified polymorphic DNA PCR assay, denaturing gradient gel electrophoresis analysis, and sequencing of the V3 region of 16S ribosomal DNA) revealed greater Bacillus species variety in ropy breads. In fact, besides strains of B. subtilis, Bacillus licheniformis, Bacillus cereus, and isolates of Bacillus clausii and Bacillus firmus were also identified. All of the ropy Bacillus isolates exhibited amylase activity, whereas only 32.4% of these isolates were able to produce ropiness in bread slices after treatment at 96°C for 10 min. Strains of lactic acid bacteria previously isolated from sourdough were first selected for antirope activity on bread slices and then used as starters for bread-making experiments. Prevention of growth of approximately 10⁴ rope-producing B. subtilis G1 spores per cm² on bread slices for more than 15 days was observed when heat-treated cultures of Lactobacillus plantarum E5 and Leuconostoc mesenteroides A27 were added. Growth of B. subtilis G1 occurred after 7 days in breads started with Saccharomyces cerevisiae T22, L. plantarum E5, and L. mesenteroides A27.     

Directed enzyme evolution via small and effective neutral drift libraries

Small libraries for directed evolution can be obtained by neutral drifts that maintain the protein's original function, yielding highly polymorphic, stable and evolvable variants. We describe methods for preparing such libraries, using serum paraoxonase (PON1). An optimized GFP variant fused to PON1 reported levels of soluble, functional enzyme, enabling selection by flow cytometry and identification of enzyme variants exhibiting improved specific and total activities toward several substrates, including toxic organophosphates.