Isolation of obligate anaerobic rumen bacteria capable of degrading the neurotoxin β‐ODAP (β‐N‐oxalyl‐L‐α,β‐diaminopropionic acid) as evaluated by a liquid chromatography/biosensor analysis system

Six pure strains of obligate anaerobes capable of degrading the toxin β‐N‐oxalyl‐L ‐α, β‐diaminopropionic acid (β‐ODAP) contained in grass pea (Lathyrus sativus) have been isolated from cow rumen. The new isolates were identified as Megasphaera elsdenii (five different genotypes) and Clostridium bifermentans using 16S rDNA analysis. The β‐ODAP degrading efficiency of the isolates was evaluated by measuring the amount of β‐ODAP in the growth medium, which contained β‐ODAP as the only carbon source, before and after incubation with the microbes. The method of analysis was liquid chromatography employing bioelectrochemical detection. The biosensor is based on co‐immobilising two enzymes, glutamate oxidase (GlOx) and horseradish peroxidase (HRP), on the end of a spectrographic graphite electrode. β‐ODAP is oxidised by GlOx to form H₂O₂, which in turn is bioelectrocatalytically reduced by HRP through a mediated reaction using a polymeric mediator incorporating Os^{2 + /3+} functionalities rapidly shuttling electrons with the electrode_giving rise to the analytical signal. On the basis of this analysis system, the new isolates are capable of utilising β‐ODAP as sole carbon source to a maximum of 90–95% within 5 days with concomitant increase in cell protein. Copyright © 2005 Society of Chemical Industry     

The influence of tempeh fermentation and conventional cooking on anti‐nutrient level and protein bioavailability (in vitro test) of grass‐pea seeds

BACKGROUND: The solid state fermentation tempeh type is known to result in the decomposition of anti‐nutrients and the improvement of nutritional value of legume seeds. The aim of the research was to study the influence of tempeh fermentation and conventional cooking on (1) levels of 3‐N‐oxalyl‐L ‐2,3‐diaminopropionic acid (ODAP), soluble phenols, trypsin inhibitors and inositol phosphates; and (2) the in vitro bioavailability of proteins in grass‐pea seeds. RESULTS: Tempeh fermentation reduced the level of ODAP, trypsin inhibitors and phytates by 93, 99 and 22%, respectively, and increased protein bioavailability by about 25%. Protein bioavailability from conventionally cooked seeds was higher than from fermented seeds. However, during the in vitro test more soluble protein (approx. 10%) was released from 100 g tempeh product than from cooked seeds. CONCLUSIONS: Solid state fermentation and cooking resulted in seeds of comparable quality but the tempeh contained much less ODAP, which is very promising in the context of popularising grass pea. Copyright © 2008 Society of Chemical Industry     

Non-volatile components of several novel species of edible fungi in China

A nonprotein neurotoxic amino acid, β-N-oxalyl-l-2,3-diaminopropionic acid (ODAP), found in Lathyrus sativus (grass pea or chickling vetch) seeds is known to be relatively heat stable. The present study aims at development of a kinetic model for degradation of ODAP in Lathyrus sativus subjected to a defined set of processing conditions. This study was carried out at pH 4.0 and 9.2. Isothermal condition experiments were carried out over a temperature range of 60–120 °C. For nonisothermal conditions, three different cooking methods viz., – open pan, pressure cooking and cooking in recently developed and patented fuel efficient ‘EcoCooker’ were used. The degradation of ODAP was adequately modeled by Arrhenius type of equation. A mathematical model based on the time temperature data of the nonisothermal heat process and isothermal kinetic rate parameters has been developed to predict the degradation of ODAP in any nonisothermal heating process of known time temperature profiles.     

Solid-state fermentation of Lathyrus sativus seeds using Aspergillus oryzae and Rhizopus oligosporus sp T-3 to eliminate the neurotoxin β-ODAP without loss of nutritional value

The presence of a neurotoxic amino acid, 3-N-oxalyl-L-2,3-diaminopropanoic acid (β-ODAP), in the seeds of grass pea (Lathyrus sativus) is the cause of an irreversible spastic paraparesis, neurolathyrism, after over-consumption. This is a negative factor in an otherwise very tasty, nutritious, easily cultivated and hardy food crop in Asia and Africa. The reduction of β-ODAP to less than 10% of the original content has been reached by fermenting L sativas Jamalpur seeds with Aspergillus oryzae NRRL 1988 for 48 h, followed by fermentation with Rhizopus oligosporus sp T-3 for 48 h. Other nutritional qualities were also improved in the fermented seed meal: increased content of protein, higher amino acid scores for sulphur-containing and aromatic amino acids, better resistance to high temperature and to oxidation, and drastic reduction of the flatulence factors.     

Effects of Processing on the Reduction of β-ODAP (β-N-Oxalyl-L-2,3-diaminopropionic acid) and Anti-Nutrients of Khesari Dhal,Lathyrus sativus

Effects of different processing techniques on the neurotoxin, β-ODAP (β- N -oxalyl-L-2,3-diaminopropionic acid), and the anti-nutritional compounds (phytate, polyphenols, trypsin and amylase inhibitors, and lectins) within four lines of Lathyrus sativus (high-, medium- and low-ODAP, and so-called ODAP-free) were investigated. Soaking of seeds in various media reduced the contents of these compounds to a varying and significant extent; losses were higher in freshly boiled water, alkaline and tamarind solutions than after soaking in drinking water. The highest losses in boiled water (65–70%) were observed for β-ODAP, followed by trypsin inhibitors (42–48%) and polyphenols (30–37%). Ordinary cooking and pressure cooking of pre-soaked seeds were found to be most effective in reducing the levels of all the natural toxicants examined, whilst fermentation and germination were more effective in destroying both of the enzyme inhibitors (amylase inhibitors by 69–71%; trypsin inhibitors by 65–66%) than either phytates or polyphenols. Lectins were not affected by most of these processes except by pressure cooking and fermentation. Dehusking of pre-soaked seeds significantly reduced β-ODAP levels, but this reduction was lower for the anti-nutrients. These findings and the high water solubility suggest that a simple and effective means of detoxifying Lathyrus by removing this neurotoxic amino acid may be practicable.     

Detection of Lathyrus sativus in processed chickpea‐ and red gram‐based products by thin layer chromatography

Excessive consumption of Lathyrus sativus leads to crippling, irreversible paralysis of both lower limbs, mainly in males. This is attributed to the presence of a non‐protein amino acid, β‐N‐oxalyl‐L ‐2,3‐diaminopropionic acid or β‐N‐oxalylamino‐L ‐alanine. Using a thin layer chromatography method developed in our laboratory, adulteration of chickpea and red gram with L sativus in pressure‐cooked batters could be detected at levels of 100 and 200 g kg^?1 respectively. When processed as a curried liquid dal, L sativus could be detected at 200 g kg^?1 in chickpea and 100 g kg^?1 in red gram. Processing into fried bhajiyas resulted in a detection limit of 200 g kg^?1 in both red gram and chickpea. © 2003 Society of Chemical Industry     

The contents of the neuro-excitatory amino acid β-ODAP (β-N-oxalyl-l-α,β-diaminopropionic acid), and other free and protein amino acids in the seeds of different genotypes of grass pea (Lathyrus sativus L.)

The free and protein amino acids of nine different genotypes of grass pea (Lathyrus sativus L.) seeds were analysed by HPLC with pre-column PITC (phenyl isothiocyanate) derivatisation. Among the free amino acids, homoarginine was quantitatively the most important (up to 0.8% seed weight) and stable while the neuro-excitatory amino acid β-ODAP (β-N-oxalyl-l-α,β-diaminopropionic acid) showed highest variation (0.02–0.54%) in the nine genotypes examined. Among protein amino acids, glutamic acid was quantitatively most significant, followed by aspartic acid, arginine, leucine, lysine and proline. The sulphur amino acid, methionine, showed the lowest concentration in all the L. sativus genotypes, and also in lentil (Lens culinaris) and in soybean (Glycine max) seeds analysed at the same time.     

Effect of processing on the content of β-N-oxalyl-α, β-diaminopropionic acid (gb-ODAP) in grass pea (Lathyrus sativus) seeds and flour as determined by flow injection analysis

The effect of cooking, roasting, autoclaving and fermentation on the content of β-ODAP in the whole seeds and flour of grass pea (Lathyrus sativus) were studied at different levels of temperature, time, pH, degree of soaking and moisture content. The method of determination used was flow injection analysis, with immobilised glutamate oxidase and horseradish peroxidase. The whole seeds flour was found to contain about 922 mg 100g⁻¹ β-ODAP in dry weight basis. The reduction of β-ODAP content, in samples which were cooked for 60min and roasted (150 °C for 60min) was 57% and 82%, respectively. The content of β-ODAP in dry seeds autoclaved for 30 min also showed a significant (p = 0.05) reduction by 39%, as compared to that of raw whole seeds. Similarly, by cooking of presoaked seeds the content of β-ODAP was reduced by up to 67%. Neither the back-slopped fermentation process nor the spontaneous fermentation were effective in reducing the content of β-ODAP. Whereas roasting and autoclaving of the milled samples caused significant (p = 0.05) reduction in the content of β-ODAP up to 30% and 50%, respectively, compared to that of raw whole seeds.     

Fatty acids and all‐trans‐β‐carotene are correlated in differently colored rice landraces

A set of 54 rice landrace samples was compiled from various Asian countries, including six red/brownish and eight black/purple varieties. Brown rice samples were analyzed for lipid content and fatty acid profile, as well as all‐trans‐β‐carotene content. Black/purple varieties were found to be higher in crude lipid content than the red/brownish and colorless varieties. They also had a higher β‐carotene content than the other two color classes. The highest β‐carotene content determined was 0.22 mg kg⁻¹. Black/purple varieties tended to have a higher proportion of saturated fatty acids in their lipid fraction and a lower proportion of unsaturated fatty acids. The differences were statistically significant (P < 0.05) for oleic acid, which accounted for 42.1% of the lipid fraction in black/purple varieties and for 45.3% and 46.3% in red/brownish and colorless varieties, respectively. β‐Carotene content showed a significantly positive correlation with the crude lipid content (P < 0.001) and the content of saturated fatty acids (P < 0.001) on a dry matter basis. However, it was not correlated with the unsaturated fatty acids content on a dry matter basis. Within the total lipid extract, β‐carotene showed a significantly positive correlation with the proportion of saturated fatty acids (P < 0.01), especially palmitic acid (P < 0.01), and a significantly negative correlation with unsaturated fatty acids (P < 0.001), especially oleic acid (P < 0.01). Copyright © 2005 Society of Chemical Industry     

Effect of applying formic acid,heterolactic bacteria or homolactic and heterolactic bacteria on the fermentation of bi‐crops of peas and wheat

This work aimed to compare the effectiveness of bacterial inoculants or a chemical additive for preserving whole‐crop silages made from wheat, two pea varieties (cv Magnus or Setchey) or intercrops of wheat and both pea varieties. The forages were harvested when the wheat and peas were at the late milk and yellow wrinkled stages respectively, and conserved in five replicate mini silos without treatment (control) or after treatment with 2.5 g kg^?1 of formic acid (FA) or 1 × 10⁶ cfu g^?1 fresh forage of either of two bacterial inoculants (WholeCrop Gold (WCG) or WholeCrop Legume (WCL), Biotal Ltd, Cardiff, UK). WCG contained Lactobacillus buchneri, while WCL contained L buchneri, L plantarum and Pediococcus pentosaceus. Chemical composition, fermentation characteristics and in vitro digestibility were measured after 65 days of ensiling. Additive‐treated bi‐crops had lower (P < 0.05) concentrations of soluble N, ammonia N and lactic acid than the controls. Inoculant‐treated bi‐crops had higher (P < 0.001) acetic acid and lower (P < 0.001) residual water‐soluble carbohydrate (WSC) concentrations than FA‐treated bi‐crops. WCL‐treated bi‐crops had similar residual WSC concentrations to and higher (P < 0.05) starch concentrations than WCG‐treated bi‐crops. Unlike Magnus pea bi‐crops, Setchey pea bi‐crops treated with WCL had lower concentrations of ammonia N (P < 0.01) and acetic acid (P < 0.001) and higher concentrations of starch (P < 0.001) and lactic acid (P < 0.05) than those treated with WCG. For both bi‐crops, FA‐treated bi‐crops were more stable (P < 0.05) than inoculant‐treated or untreated silages, and the stability of inoculant‐treated and untreated silages was similar. Formic acid treatment was also the most effective at reducing WSC losses in the bi‐crop and pea silages. Inoculant treatment reduced proteolysis in these forages but did not prevent spoilage in the bi‐crops. Additive treatment reduced yeast counts but did not improve the fermentation in wheat silages. Copyright © 2004 Society of Chemical Industry     

Interaction of β‐casein with (−)‐epigallocatechin‐3‐gallate assayed by fluorescence quenching: effect of thermal processing temperature

The interactions between the flavan‐3‐ol (?)‐epigallocatechin‐3‐gallate (EGCG) and bovine β‐casein in phosphate‐buffered saline (PBS) of pH 6.5 subjected to thermal processing at various temperatures (25–100 °C) were investigated using fluorescence quenching. The results indicated that different temperatures had different effects on the structural changes and EGCG‐binding ability of β‐casein. At temperatures below 60 °C, the β‐casein–EGCG interaction changed little (P > 0.05) with increasing temperature. At temperatures above 80 °C, native assemblies of β‐casein in solution dissociated into individual β‐casein molecules and unfolded, as demonstrated by a red shift of the maximum fluorescence emission wavelength (λ_max) of up to 8.8 nm. The highest quenching constant (K_q) and the number of binding sites (n) were 0.92 (±0.01) × 10¹³ m ^?1 s^?1 and 0.73 (±0.02) (100 °C), respectively. These results provide insight into the potential of interactions between β‐casein–EGCG that may modulate bioactivity or bioavailability to be altered during thermal process.     

The Formation of β‐Damascenone in Sweet Potato Shochu

The formation of β‐damascenone during shochu manufacture was investigated by quantifying β‐damascenone at each stage of manufacturing. Steamed sweet potato has a low level of free β‐damascenone (0.02–0.1 μg/g). During fermentation, β‐damascenone was produced in small quantities that were degraded by yeast. Thus, the second mash accumulates little free β‐damascenone (approximately 17 μg/L). The concentration profile in the fractionated distillate showed that β‐damascenone was produced during heating. Most β‐damascenone in shochu was formed during distillation, not during steam heating and fermentation. It is suggested that the level of β‐damascenone in shochu could be increased by reducing the pH of the second mash and prolonging the distillation period. Sweet potato cultivars differed in total free and hydrolyzed β‐damascenone content and there was a strong association between each cultivar and its shochu β‐damascenone content. The selection of the sweet potato cultivar is important for determining the quantity of β‐damascenone in a shochu brew.     

Physicochemical Characterization and Sensory Analysis of Yeast‐leavened and Sourdough Soy Breads

Sourdough fermentation has been shown to have numerous beneficial effects on bread quality, and nutritionally enhance soy‐supplemented bread by altering isoflavone chemical forms. Given this, the objective of this study was to compare the loaf quality and shelf life of sourdough and yeast‐leavened soy breads by various physical, thermal, and sensorial methods, and to assess the effects of fermentation by various microorganisms on isoflavone profile in dough and breads using high‐performance liquid chromatography analysis. Sourdough fermentation yielded a less extensible dough compared to yeast‐leavened soy dough (P < 0.001), and resulted in a harder bread crumb (P < 0.05) and lighter crust color (P < 0.001), compared to yeast‐leavened soy bread (Y‐B). Sensory analysis revealed a significantly higher overall liking of Y‐B compared to sourdough soy bread (SD‐B) (P < 0.001). Segmentation analysis of the cohort suggests that overall liking and bread consumption frequency may be determinants of Y‐B or SD‐B preference. SD‐B and Y‐B exhibited similar shelf‐life properties. Despite significantly different enthalpies associated with the melting of amylose‐lipid complexes, thermal analysis of the 2 soy breads stored for 10 d (ambient conditions) demonstrated no significant difference in water distribution and starch retrogradation (P < 0.05). Lastly, SD‐B was determined to have 32% of total isoflavones occurring in the aglycone form compared to 17% in Y‐B. These findings warrant further investigation of sourdough fermentation as a processing technique for quality and nutritional enhancement of soy‐based baked goods.     

Vitamin E levels,thiobarbituric acid test and sensory evaluation of breast muscles from broilers fed α‐tocopheryl acetate‐ and β‐carotene‐supplemented diets

The objective of this study was to investigate the effect of dietary α‐tocopheryl acetate and β‐carotene supplementation on lipid oxidation of breast meat from broilers fed lard as the fat source. Supplementation of broilers with 100 mg kg^?1 α‐tocopheryl acetate increased the vitamin E levels in raw breast samples significantly (p < 0.05), whereas the presence of 1.5 mg kg^?1 dietary β‐carotene tended to decrease vitamin E deposition. The presence of vitamin E delayed lipid oxidation significantly, but thiobarbituric acid values of samples from broilers fed the β‐carotene‐supplemented diet did not differ from those of control samples. Vitamin E reduced sensory meat rancidity, whilst vitamin E, β‐carotene and their combination modified meat texture. The results show the effectiveness of dietary α‐tocopheryl acetate supplementation in protecting broiler meat against lipid oxidation. Copyright © 2004 Society of Chemical Industry     

Terpene profiles in Cantal and Saint‐Nectaire‐type cheese made from raw or pasteurised milk

Terpene profiles in cheese can be considered a ‘terroir’ fingerprint as the information contained in it should enable the pastures on which the animals were fed to be recognised. Yet a certain elasticity of the signature must be taken into account when determining authentication strategies, since products acknowledged as containing a common signature may have undergone certain procedures, such as cheese making and milk pasteurisation, that could have potentially altered their terpene profiles. In this study, Cantal and Saint‐Nectaire‐type cheeses were made from both raw and pasteurised milk from the same herd of dairy cows that had been grazed on natural grassland. Cheeses from raw and pasteurised milk were made from the same milking on the same days. Cantal and Saint‐Nectaire‐type cheeses were made on 4 different days, alternatively over four weeks. The terpenes in the cheese fat were analysed by dynamic headspace/gas chromatography/mass spectrometry. A great diversity of monoterpenes, sesquiterpenes and oxygen‐containing derivatives were identified. The major terpenes identified in most cheeses were β‐caryophyllene, α‐ and β‐pinene and limonene. Milk pasteurisation did not induce changes in the terpene profile of the cheese. Significant differences (p < 0.001) were observed between Cantal and Saint‐Nectaire cheeses: α‐pinene, β‐myrcene and β‐phellandrene were, respectively, three, five and five times more abundant in Cantal cheese, while tricyclene, α‐phellandrene and geraniol were found exclusively in Cantal cheese. In contrast, unidentified sesquiterpenes with retention indices (KI) = 1342 and 1511, α‐cubebene, longifolene and γ‐elemene were more abundant or exclusively found in Saint‐Nectaire cheese. A significant relationship with the date of milking (p < 0.01) was observed for α‐pinene and tricyclene in Cantal, for β‐myrcene, δ‐3‐carene, p‐cymene and α‐terpinene in Saint‐Nectaire cheese. Copyright © 2005 Society of Chemical Industry     

Optimisation of culture conditions and development of a novel fed‐batch strategy for high production of β‐galactosidase by Kluyveromyces lactis

The aim of this study was to enhance β‐galactosidase production by Kluyveromyces lactis CICC1773. Firstly, the optimum culture conditions were obtained by response surface methodology, and the maximum β‐galactosidase activity reached 20.6 U mL^?1, about two‐fold increase than that of the initial conditions (initial fermentation medium and conditions). To further improve β‐galactosidase production, a new fed‐batch strategy based on pH feedback control was developed successfully in a 7‐L fermenter, using 400 g L^?1 lactose as feeding medium. As a result, the β‐galactosidase activity and productivity reached up to 111.61 U mL^?1 and 5.31 U/(mL·h), enhanced by 15.3‐fold and 17.6‐fold superior than the results of initial conditions, respectively. To our knowledge, β‐galactosidase activity obtained was the highest value among the results reported by nonrecombinant strains. These results demonstrated that the new fed‐batch strategy based on optimum culture conditions could be automatic control easily and be conductive to further scale up for industrial fermentation.     

Emulsifying Properties of Legume Proteins Compared to β‐Lactoglobulin and Tween 20 and the Volatile Release from Oil‐in‐Water Emulsions

The emulsifying properties of plant legume protein isolates (soy, pea, and lupin) were compared to a milk whey protein, β‐lactoglobulin (β‐lg), and a nonionic surfactant (Tween 20). The protein fractional composition was characterized using sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. The following emulsion properties were measured: particle diameter, shear surface ζ‐potential, interfacial tension (IT), and creaming velocity. The effect of protein preheat treatment (90 °C for 10 min) on the emulsifying behavior and the release of selected volatile organic compounds (VOCs) from emulsions under oral conditions was also investigated in real time using proton transfer reaction‐mass spectrometry. The legume proteins showed comparable results to β‐lg and Tween 20, forming stable, negatively charged emulsions with particle diameter d_3,2 < 0.4 μm, and maintained stability over 50 d. The relatively lower stability of lupin emulsions was significantly correlated with the low protein surface hydrophobicity and IT of the emulsion. After heating the proteins, the droplet size of pea and lupin emulsions decreased. The VOC release profile was similar between the protein‐stabilized emulsions, and greater retention was observed for Tween 20‐stabilized emulsions. This study demonstrates the potential application of legume proteins as alternative emulsifiers to milk proteins in emulsion products.     

Integrated community‐based growth monitoring and vegetable gardens focusing on crops rich in β‐carotene: Project evaluation in a rural community in the Eastern Cape,South Africa

BACKGROUND: Cultivation in β‐carotene vegetables was promoted in a crop‐based intervention in Lusikisiki, Eastern Cape, South Africa. The project also included nutrition education and community‐based growth monitoring, and focused on technology transfer and mobilisation of local organisations. Three years after initiation of the project, participating (n = 219) and non‐participating (n = 223) households were compared in terms of child morbidity, nutritional knowledge, dietary intake and gardening practices. RESULTS: Compared to control households, more caregivers from project households thought that yellow fruit and vegetables were good for their children (73% vs. 45%; P < 0.001), were familiar with the term ‘vitamin A’ (89% vs. 63%; P < 0.001), knew that vitamin A is a nutrient in food (83% vs. 53%; P < 0.001), and could name three food sources rich in vitamin A (56% vs. 27%; P < 0.001). Fewer 1–5‐year‐old children in the project households reportedly experienced vomiting (6% vs. 13%; P = 0.012), fever (30% vs. 42%; P = 0.008), sores on the skin (6% vs. 19%; P < 0.001), continuous runny nose (20% vs. 33%; P = 0.002), diarrhoea (2% vs. 7%; P = 0.026) and poor appetite (7% vs. 14%; P = 0.016). Children from project households consumed carrot (P = 0.008), butternut (P = 0.002), spinach (P = 0.004) and orange‐fleshed sweetpotato (P = 0.028) more frequently than children from control households. CONCLUSION: The agricultural intervention, combined with nutrition education and community‐based growth monitoring, showed a favourable effect on child morbidity, nutritional knowledge and dietary intake of β‐carotene‐rich vegetables. Agricultural interventions can therefore contribute significantly towards nutritional outcomes. Copyright © 2008 Society of Chemical Industry