Fundamental studies of sourdoughs fermented with Weissella cibaria and Lactobacillus plantarum: influence on baking characteristics, sensory profiles and in vitro starch digestibility of gluten-free breads

The application of sourdough can improve texture, structure, nutritional value, staling rate and shelf life of wheat and gluten-free breads. These quality improvements are associated with the formation of organic acids, exopolysaccharides (EPS), aroma or antifungal compounds. Initially, the suitability of two lactic acid bacteria strains to serve as sourdough starters for buckwheat, oat, quinoa, sorghum and flours was investigated. Wheat flour was chosen as a reference. The obligate heterofermentative lactic acid bacterium (LAB) Weissella cibaria MG1 (Wc) formed the EPS dextran (a α-1,6-glucan) from sucrose in situ with a molecular size of 106 to 107 kDa. EPS formation in all breads was analysed using size exclusion chromatography and highest amounts were formed in buckwheat (4 g/ kg) and quinoa sourdough (3 g/ kg). The facultative heterofermentative Lactobacillus plantarum FST1.7 (Lp) was identified as strong acidifier and was chosen due to its ubiquitous presence in gluten-free as well as wheat sourdoughs (Vogelmann et al. 2009). Both Wc and Lp, showed highest total titratable acids in buckwheat (16.8 ml; 26.0 ml), teff (16.2 ml; 24.5 ml) and quinoa sourdoughs (26.4 ml; 35.3 ml) correlating with higher amounts of fermentable sugars and higher buffering capacities. Sourdough incorporation reduced the crumb hardness after five days of storage in buckwheat (Wc -111%), teff (Wc -39%) and wheat (Wc -206%; Lp -118%) sourdough breads. The rate of staling (N/ day) was reduced in buckwheat (Ctrl 8 N; Wc 3 N; Lp 6 N), teff (Ctrl 13 N; Wc 9 N; Lp 10 N) and wheat (Ctrl 5 N; Wc 1 N; Lp 2 N) sourdough breads. Bread dough softening upon Wc and Lp sourdough incorporation accounted for increased crumb porosity in buckwheat (+10.4%; +4.7), teff (+8.1%; +8.3%) and wheat sourdough breads (+8.7%; +6.4%). Weissella cibaria MG1 sourdough improved the aroma quality of wheat bread but had no impact on aroma of gluten-free breads. Microbial shelf life however, was not prolonged in any of the breads regardless of the starter culture used. Due to the high prevalence of insulin-dependent diabetes mellitus particular amongst coeliac patients, glycaemic control is of great (Berti et al. 2004). The in vitro starch digestibility of gluten-free breads with and without sourdough addition was analysed to predict the GI (pGI). Sourdough can decrease starch hydrolysis in vitro, due to formation of resistant starch and organic acids. Predicted GI of gluten-free control breads were significantly lower than for the reference white wheat bread (GI=100). Starch granule size was investigated with scanning electron microscopy and was significantly smaller in quinoa flour (<2 μm). This resulted in higher enzymatic susceptibility and hence higher pGI for quinoa bread (95). Lowest hydrolysis indexes for sorghum and teff control breads (72 and 74, respectively) correlate with higher gelatinisation peak temperatures (69°C and 71°C, respectively). Levels of resistant starch were not increased by addition of Weissella cibaria MG1 (weak acidifier) or Lactobacillus plantarum FST1.7 (strong acidifier). The pGI was significantly decreased for both wheat sourdough breads (Wc 85; Lp 76). Lactic acid can promote starch interactions with gluten hence decreasing starch susceptibility (Östman et al. 2002). For most gluten-free breads, the pGI was increased upon sourdough addition. Only sorghum and teff Lp sourdough breads (69 and 68, respectively) had significantly decreased pGI. Results suggest that the increase of starch hydrolysis in gluten-free breads was related to mechanism other than presence of organic acids and formation of resistant starch.

Characterisation and application of fruit by-products as novel ingredients in gluten-free products

The physicochemical and nutritional properties of two fruit by-products were initially studied. Apple pomace (AP) contained a high level of fibre and pectin. The isolated AP pectin had a high level of methylation which developed viscous pastes. Orange pomace also had high levels of fibre and pectin, and it was an abundant source of minerals such as potassium and magnesium. Due to the fibrous properties of orange pomace flour, proofing and water addition were studied in a bread formulation. When added at levels greater than 6%, the loaf volume decreased. An optimised formulation and proofing time was derived using the optimisation tool; these consisted of 5.5% orange pomace, 94.6% water inclusion and with 49 minutes proofing. These optimised parameters doubled the total dietary fibre content of the bread compared to the original control. Pasting results showed how orange pomace inclusions reduced the final viscosity of the batter, reducing the occurrence of starch gelatinisation. Rheological properties i.e. the storage modulus (G') and complex modulus (G*) increased in the orange pomace batter compared to the control batter. This demonstrates how the orange pomace as an ingredient improved the robustness of the formulation. Sensory panellists scored the orange pomace bread comparably to the control bread. Milled apple pomace was studied as a potential novel ingredient in an extruded snack. Parameters studied included apple pomace addition, die head temperature and screw speed. As screw speed increased the favourable extrudate characteristics such as radical expansion ratio, porosity and specific volume decreased. The inclusion of apple pomace had a negative effect on extrudate characteristics at levels greater than 8% addition. Including apple pomace reduced the hardness and increased the crispiness of the snack. The optimised and validated formulation and extrusion process contained the following parameters: 7.7% apple pomace, 150°C die head temperature and a screw speed of 69 rpm.

Isolation and characterisation of antifungal compounds from lactic acid bacteria and their application in wheat and gluten-free bread

As part of the “free-from” trend, biopreservation for bread products has increasingly become important to prevent spoilage since artificial preservatives are more and more rejected by consumers. A literature review conducted as part of this thesis revealed that the evaluation of more suitable antifungal strains of lactic acid bacteria (LAB) is important. Moreover, increasing the knowledge about the origin of the antifungal effect is fundamental for further enhancement of biopreservation. This thesis addresses the investigation of Lactobacillus amylovorus DSM19280, Lb. brevis R2: and Lb. reuteri R29 for biopreservation using in vitro trials and in situ sourdough fermentations of quinoa, rice and wheat flours as biopreservatives in breads. Their contribution to quality and shelf life extension on bread was compared and related to their metabolic activity and substrate features. Moreover, the quantity of antifungal carboxylic acids produced during sourdough fermentation was analysed. Overall a specific profile of antifungal compounds was found in the sourdough samples which were strain and substrate dependently different. The best preservative effect in quinoa sourdough and wheat sourdough bread was achieved when Lb. amylovorus DSM19280 fermented sourdough was used. However, the concentration of the antifungal compounds found in these biopreservatives were much lower when compared with Lb. reuteri R29 as the highest producer. Nevertheless, the artificial application of the highest concentration of these antifungal compounds in chemically acidified wheat sourdough bread succeeded in a longer shelf life than achieved only by acidifying the dough. This evidences their partial contribution to the antifungal activity and their synergy. Additionally, a HRGC/MS method for the identification and quantification of the antifungal active compounds cyclo(Leu-Pro), cyclo(Pro-Pro), cyclo(Met-Pro) and cyclo(Phe-Pro) was successfully developed by using stable isotope dilutions assays with the deuterated counterparts. It was observed that the concentrations of cyclo(Leu-Pro), cyclo(Pro-Pro), and cyclo(Phe-Pro) increased only moderately in MRS-broth and wort fermentation by the activity of the selected microorganism, whereas the concentration of cyclo(Met-Pro) stayed unchanged.

Reliability of antitransglutaminase antibodies as predictors of gluten-free diet compliance in adult celiac disease.

OBJECTIVE: Strict lifelong compliance to a gluten-free diet (GFD) minimizes the long-term risk of mortality, especially from lymphoma, in adult celiac disease (CD). Although serum IgA antitransglutaminase (IgA-tTG-ab), like antiendomysium (IgA-EMA) antibodies, are sensitive and specific screening tests for untreated CD, their reliability as predictors of strict compliance to and dietary transgressions from a GFD is not precisely known. We aimed to address this question in consecutively treated adult celiacs. METHODS: In a cross-sectional study, 95 non-IgA deficient adult (median age: 41 yr) celiacs on a GFD for at least 1 yr (median: 6 yr) were subjected to 1) a dietician-administered inquiry to pinpoint and quantify the number and levels of transgressions (classified as moderate or large, using as a cutoff value the median gluten amount ingested in the overall noncompliant patients of the series) over the previous 2 months, 2) a search for IgA-tTG-ab and -EMA, and 3) perendoscopic duodenal biopsies. The ability of both antibodies to discriminate celiacs with and without detected transgressions was described using receiver operating characteristic curves and quantified as to sensitivity and specificity, according to the level of transgressions. RESULTS: Forty (42%) patients strictly adhered to a GFD, 55 (58%) had committed transgressions, classified as moderate (< or = 18 g of gluten/2 months; median number 6) in 27 and large (>18 g; median number 69) in 28. IgA-tTG-ab and -EMA specificity (proportion of correct recognition of strictly compliant celiacs) was 0.97 and 0.98, respectively, and sensitivity (proportion of correct recognition of overall, moderate, and large levels of transgressions) was 0.52, 0.31, and 0.77, and 0.62, 0.37, and 0.86, respectively. IgA-tTG-ab and -EMA titers were correlated (p < 0.001) to transgression levels (r = 0.560 and R = 0.631, respectively) and one to another (p < 0.001) in the whole patient population (r = 0.834, N = 84) as in the noncompliant (r = 0.915, N = 48) group. Specificity and sensitivity of IgA-tTG-ab and IgA-EMA for recognition of total villous atrophy in patients under a GFD were 0.90 and 0.91, and 0.60 and 0.73, respectively. CONCLUSIONS: In adult CD patients on a GFD, IgA-tTG-ab are poor predictors of dietary transgressions. Their negativity is a falsely secure marker of strict diet compliance.

Evaluation of tumor response, disease control, progression-free survival, and time to progression as potential surrogate end points in metastatic breast cancer.

PURPOSE: Overall survival (OS) can be observed only after prolonged follow-up, and any potential effect of first-line therapies on OS may be confounded by the effects of subsequent therapy. We investigated whether tumor response, disease control, progression-free survival (PFS), or time to progression (TTP) could be considered a valid surrogate for OS to assess the benefits of first-line therapies for patients with metastatic breast cancer. PATIENTS AND METHODS: Individual patient data were collected on 3,953 patients in 11 randomized trials that compared an anthracycline (alone or in combination) with a taxane (alone or in combination with an anthracycline). Surrogacy was assessed through the correlation between the end points as well as through the correlation between the treatment effects on the end points. RESULTS: Tumor response (survival odds ratio [OR], 6.2; 95% CI, 5.3 to 7.0) and disease control (survival OR, 5.5; 95% CI, 4.8 to 6.3) were strongly associated with OS. PFS (rank correlation coefficient, 0.688; 95% CI, 0.686 to 0.690) and TTP (rank correlation coefficient, 0.682; 95% CI, 0.680 to 0.684) were moderately associated with OS. Response log ORs were strongly correlated with PFS log hazard ratios (linear coefficient [rho], 0.96; 95% CI, 0.73 to 1.19). Response and disease control log ORs and PFS and TTP log hazard ratios were poorly correlated with log hazard ratios for OS, but the confidence limits of rho were too wide to be informative. CONCLUSION: No end point could be demonstrated as a good surrogate for OS in these trials. Tumor response may be an acceptable surrogate for PFS.

Investigation of the shape of the R(0) absorption line in ν3, N2O recorded from an axisymmetric supersonic free jet expansion

The hydrodynamic structure of an axisymmetric supersonic expansion can be regarded as a series of concentric divergent cones, with decreasing particle densities as the cone angle increases. Different groups of molecules therefore contribute to high-resolution absorption line shapes when optically probing the expansion in a direction perpendicular to the jet axis. These groups are distinguished by the cone angle, inducing a specific Doppler shift, and by the particle density, contributing a specific weight to the absorption intensity. As a result different broader line profiles are observed compared to room temperature spectra. This effect is investigated here selecting as the working example the R(0), ν3 absorption line in N2O recorded using a Fourier transform interferometer. Independent impact pressure and quadrupole mass spectrometric measurements are performed leading to two complementary maps of the expansion, allowing the recorded absorption line shape to be quantitatively modeled. © 2006 Elsevier Ltd. All rights reserved.

Potent and broad neutralizing activity of a single chain antibody fragment against cell-free and cell-associated HIV-1.

Several human monoclonal antibodies (hmAbs) exhibit relatively potent and broad neutralizing activity against HIV-1, but there has not been much success in using them as potential therapeutics. We have previously hypothesized and demonstrated that small engineered antibodies can target highly conserved epitopes that are not accessible by full-size antibodies. However, their potency has not been comparatively evaluated with known HIV-1-neutralizing hmAbs against large panels of primary isolates. We report here the inhibitory activity of an engineered single chain antibody fragment (scFv), m9, against several panels of primary HIV-1 isolates from group M (clades A-G) using cell-free and cell-associated virus in cell line-based assays. M9 was much more potent than scFv 17b, and more potent than or comparable to the best-characterized broadly neutralizing hmAbs IgG(1) b12, 2G12, 2F5 and 4E10. It also inhibited cell-to-cell transmission of HIV-1 with higher potency than enfuvirtide (T-20, Fuzeon). M9 competed with a sulfated CCR5 N-terminal peptide for binding to gp120-CD4 complex, suggesting an overlapping epitope with the coreceptor binding site. M9 did not react with phosphatidylserine (PS) and cardiolipin (CL), nor did it react with a panel of autoantigens in an antinuclear autoantibody (ANA) assay. We further found that escape mutants resistant to m9 did not emerge in an immune selection assay. These results suggest that m9 is a novel anti-HIV-1 candidate with potential therapeutic or prophylactic properties, and its epitope is a new target for drug or vaccine development.

Reproduction and population growth in free-ranging mantled howling monkeys.

Free-ranging mantled howling monkey (Alouatta palliata Gray) females experienced a regular estrus cycle averaging 16.3 days, demonstrated sexual skin changes, and participated in multiple matings before becoming pregnant. Gestation averaged 186 days. The average interval between births was 22.5 months. Sexual maturity occurred at approximately 36 and 42 months for females and males, respectively. Female age at first birth was about 3 1/2 years. Births were scattered during some years and clustered during others. The age, rank, and parity of the females affected infant survival. More female than male infants survived to one year of age. Increased population size was the result of immigration rather than births.

Plasma lipoproteins of free-ranging howling monkeys (Alouatta palliata).

1. Plasma lipids and lipoproteins of free-ranging howling monkeys from Costa Rica (Alouatta palliata), aged 5 months to 23 years, were characterized. 2. High density lipoproteins were lipid-rich, similar to HDL2 of human plasma. 3. Fatty acid compositions of major lipid classes of very low, low and high density lipoproteins differed among social groups, possibly due to both dietary and genetic factors. 4. Low and high density lipoprotein phospholipids were enriched in phosphatidylethanolamine. 5. Howler plasma cross reacted with antihuman apoA-I antibodies but not with antihuman LDL antibodies. 6. No dimeric form of apoA-II was present, unlike human apoA-II.

Infant-nonmother interactions of free-ranging mantled howlers (Alouatta palliata) in Costa Rica

Proximate and ultimate explanations of interactions between infants and nonmothers vary depending upon the relatedness of the interactors. We investigated interactions of infants and nonmothers from a 22-month continuous study and from the long-term monitoring of the mantled howler population of La Pacifica, Guanacaste Province, Costa Rica. Relatedness is low or absent in these mantled howler groups. Juvenile females appeared to practice care skills with older infants, but as most first infants died, they failed to benefit. Infant positive interactions with adults occurred with the mother and probable father. Other adult females behaved aggressively toward the youngest infants. Mothers were retentive of infants and responded negatively to these interactions, suggesting that they perceived them as threatening. Interactions with infants appear to reflect competition in groups of unrelated adults. A review of other populations of Alouatta palliata and other species of howlers indicate variability in social group size and suggest variability in intragroup relatedness. We suggest that further study will confirm that social behavior (including interactions with infants) will vary by resource availability (group size) and associated demographic patterns (male and female migration) that affect relatedness in howler social groups.