Secretion of non-cell-bound phytase by the yeast Pichia kudriavzevii TY13.

AIMS: Mineral deficiencies cause several health problems in the world, especially for populations consuming cereal-based diets rich in the anti-nutrient phytate. Our aim was to characterize the phytate-degrading capacity of the yeast Pichia kudriavzevii TY13 and its secretion of phytase. METHODS AND RESULTS: The phytase activity in cell-free supernatants from cultures with 100% intact cells was 35-190 mU ml(-1) depending on the media. The Km was 0.28 mmol l(-1) and the specific phytase activity 0.32 U mg(-1) total protein. The phytase activity and secretion of extracellular non-cell-bound phytase was affected by the medium phosphate concentrations. Further, addition of yeast extract had a clearly inducing effect, resulting in over 60% of the cultures total phytase activity as non-cell-bound. CONCLUSIONS: Our study reveals that it is possible to achieve high extracellular phytase activity from the yeast P. kudriavzevii TY13 by proper composition of the growth medium. SIGNIFICANCE AND IMPACT OF THE STUDY: TY13 could be a promising future starter culture for fermented foods with improved mineral bioavailability. Using strains that secrete phytase to the food matrix may significantly improve the phytate degradation by facilitating the enzyme-to-substrate interaction. The secreted non-cell-bound phytase activities by TY13 could further be advantageous in industrial production of phytase.

Risk factors for anaemia in schoolchildren in Tanga Region, Tanzania.

Anaemia is one of the major public health problems affecting more than half of school children along the coast in Tanzania. Due to the multiplicity of its causes it sometimes becomes difficult to find appropriate intervention measures. In order to assist schools in implementing appropriate public health measures for anaemia in Tanga Region of Tanzania risk factors were investigated in school children. A total of 845 schoolchildren age 7-14 years were randomly selected in a cross-sectional survey conducted in 20 randomly selected schools for inclusion in the investigations. Socio-economic, environmental and biological data were collected, as well as academic information, health care and feeding practices. Diagnosis of anaemia was based on haemoglobin concentration below 115 g/L determined by HemoCue meter. Serum Retinol was determined by High performance liquid chromatography and serum ferritin by an Enzyme linked immunosorbent assay. Urine from each child was tested for blood using a haematest reagent strip and those testing positive were examined microscopically by filtration method for Schistosoma haematobium ova. A faecal sample collected from them was also examined microscopically for ova and larvae of intestinal worms. To analyse variables associated with anaemia a stepwise multiple regression model was used. The prevalence of anaemia was 79.6%. Micronutrient deficiencies were highly prevalent. Iron deficiency (SF <20 microg/dl) was affecting 33%, vitamin A deficiency (SR < 20 microg/dL) 31.9% and 25% of the children had mild iodine deficiency (UIE < 20 microg/L). Intestinal helminths were also highly prevalent; 68% of children had hookworm and 54% had urinary schistosomiasis. Inadequate diet was a feature in >50% of children. About 10% of households had no latrines and multiple infection rank score was high especially in older age children. The risk of having anaemia was two times higher in children with iron deficiency (RR=2.1) and 49% higher in those with vitamin A deficiency. These deficiencies correlated significantly with the anaemia (P<0.05). Vitamin A deficiency and infections with hookworm and schistosomiasis were the most significant factors predicting for anaemia (r=0.318 and r2=0.101). We therefore conclude that high prevalence of infections and nutritional deficiencies are important risk factors for anaemia in this community. The high attributable fractions for hookworm, schistosomiasis, iron deficiency and vitamin A confirms that these are significant risk factors to be considered when designing public health measures for anaemia prevention in this community.

In vitro accessibility and intake of beta-carotene from cooked green leafy vegetables and their estimated contribution to vitamin A requirements.

Provitamin A carotenoids from green leafy vegetables constitute a major dietary source of vitamin A in Tanzania. The efficiency of different vegetable preparations in contributing to the daily safe intake level of vitamin A for children was evaluated in the present study. The amount of bioaccessible (available for absorption) all-trans-beta-carotene from green leafy vegetable dishes prepared either traditionally in households or with an improved method was estimated by an in vitro digestion method that simulates human digestion. The median vegetable portion size consumed by 34 children (2-12 years old) in the surveyed households was determined by weighing. The amount of all-trans-beta-carotene available for absorption from the same type of vegetables cooked without oil by traditional or a modified method ranged from 5 to 12% and 4 to 15%, respectively. Vegetables cooked with modified method with oil had 2-5 times higher amount of in vitro accessible all-trans-beta-carotene than vegetables cooked without oil. A median portion (84 g) of such a relish then provided 88-477% of the daily safe intake level of vitamin A, while portions cooked without oil only provided between 23 and 46%. In this study, a wide variation in all-trans-beta-carotene content and bioaccessibility among studied green leaves was obtained. Still, to supply daily safe intake level of vitamin A for children from a median vegetable relish portion it is necessary to add oil in the preparation.

Estimation of carotenoid accessibility from carrots determined by an in vitro digestion method.

OBJECTIVE: To develop an in vitro digestion method to assess the impact of heat treatment, particle size and presence of oil on the accessibility (available for absorption) of alpha- and beta-carotene in carrots. DESIGN: Raw and cooked carrots were either homogenized or cut into pieces similar to chewed items in size. The carrot samples, with or without added cooking oil, were exposed to an in vitro digestion procedure. Adding a pepsin-HCl solution at pH 2.0 simulated the gastric phase. In the subsequent intestinal phase, pH was adjusted to 7.5 and a pancreatin-bile extract mixture was added. Carotenoids released from the carrot matrix during the digestion were extracted and quantified on high-performance liquid chromatography (HPLC). RESULTS: Three percent of the total beta-carotene content was released from raw carrots in pieces. When homogenized (pulped) 21% was released. Cooking the pulp increased the accessibility to 27%. Addition of cooking oil to the cooked pulp further increased the released amount to 39%. The trends for alpha-carotene were similar to those for beta-carotene. CONCLUSION: The described in vitro digestion method allows a rapid estimation of carotene accessibility in processed carrots, which may reliably predict in vivo behavior. SPONSORSHIP: This study was supported by the Swedish International Development Cooperation Agency (SIDA) and the International Program in the Chemical Sciences (IPICS), Uppsala University, Sweden.

Oxidation of polyphenols in phytate-reduced high-tannin cereals: effect on different phenolic groups and on in vitro accessible iron.

After reduction of phytate with phytase, water slurries of two high-tannin cereal flours were incubated with polyphenol oxidase (mushroom tyrosinase), and the effects on different phenolic groups and on in vitro accessible iron were studied. Enzyme incubation was also performed after cooking, soaking, and germination of the cereals. Phytase incubation significantly decreased the phytate content, and incubation with polyphenol oxidase had a reducing effect on the total phenol content, as well as on the amount of catechol and resorcinol groups. The in vitro accessible iron increased when the cereals were incubated with phytase and polyphenol oxidase, and the highest accessibility of iron was obtained when the germinated samples were incubated. The results from this study imply that oxidation of polyphenols in high-tannin cereals, after reduction of phytate, may be used to increase the bioavailability of iron in foods prepared from these cereals.

Enteropathogenic bacteria in faecal swabs of young children fed on lactic acid-fermented cereal gruels.

The influence of consumption of a lactic acid-fermented cereal gruel togwa with pH < or = 4 on the presence of faecal enteric bacteria such as campylobacter, enterohaemorrhagic Escherichia coli (EHEC:O157), enterotoxigenic Escherichia coli (ETEC), salmonella and shigella was evaluated. Under 5 years old healthy children listed in an ascending order of age were alternatively assigned and given either a lactic-acid fermented cereal gruel togwa (test diet) or an unfermented cereal gruel uji (control diet) once a day for 13 consecutive days. The presence of the enteropathogens was examined in rectal swabs collected from the children at baseline (before feeding session started), on days 7 and 13, and additionally 14 days (follow-up day) after the feeding session had stopped. The swabs were cultured on to different optimal media for respective enteropathogen and confirmed by standard microbiological and serological methods. Campylobacter spp. dominated among the enteropathogens (62% out of total) followed by Salmonella spp., ETEC and Shigella spp. Children with isolated enteropathogens in the togwa group was significantly reduced (P < 0.001) from 27.6% at baseline to 7.8, 8.2 and 12.7% on days 7, 13 and follow-up day, respectively. The effect was more pronounced in those children taking togwa > 6 times during the study period. In the control group, there was a slight decrease from 16.7% at baseline to 11.4% on day 7 and 8.1% on day 13. On the follow-up day, enteropathogens were found in 22.6% of the children, which was significantly higher than in those children taking togwa > 6 times. We conclude, that regular consumption of togwa with pH < or = 4, once a day, three times a week may help to control intestinal colonization with potential diarrhoea-causing pathogens in young children.

PIP: Campylobacter jejuni/coli, enteropathogenic E. coli, enterotoxigenic E. coli, Salmonella spp., Shigella spp., and rotavirus are consistently implicated with diarrhea in children under age 5 years in developing countries. The influence of consumption of a lactic acid-fermented cereal gruel (togwa) with pH of less than or equal to 4 upon the presence of such fecal enteric bacteria was evaluated. 151 healthy children aged 6-60 months in Majohe village listed in an ascending order of age were alternatively assigned and given either togwa or an unfermented cereal gruel (uji) once a day for 13 consecutive days. The presence of enteropathogens was then examined in rectal swabs collected from the children at baseline before the feeding session started, on days 7 and 13, and 14 days later after the feeding session had stopped. 62% of the enteropathogens identified were of campylobacter spp., followed by Salmonella and Shigella spp. The proportion of children with isolated enteropathogens in the togwa group was significantly reduced from 27.6% at baseline to 7.8%, 8.2%, and 12.7% on days 7, 13, and the follow-up day, respectively. The effect was more pronounced among children taking togwa more than 6 times/day during the study period. In the control group, there was a slight decrease from 16.7% at baseline to 11.4% on day 7 and 8.1% on day 13. On the follow-up day, enteropathogens were found in 22.6% of the children, which was significantly higher than in those children taking togwa more than 6 times. The regular consumption of togwa with pH of less than or equal to 4, once per day, 3 times per week may help to control intestinal colonization with potential diarrhea-causing pathogens in young children.

Processing of quinoa (Chenopodium quinoa, Willd): effects on in vitro iron availability and phytate hydrolysis.

The effect of different processing techniques was studied on in vitro iron availability and phytate hydrolysis in high and low saponin content quinoa (Chenopodium quinoa, Willd) seeds. Water slurries of ungerminated and germinated quinoa flour were processed by cooking, soaking, and fermentation using Lactobacillus plantarum as starter. Iron solubility under physiological conditions (in vitro) was measured and used as an estimation of iron availability. Phytate (inositol hexaphosphate/IP6) and its degradation products were analysed by an HPLC method. The IP6 + IP5 content was reduced by cooking with 4 to 8%, germination with 35 to 39%, soaking with 61 to 76% and by fermentation with 82 to 98%. The highest reduction, about 98%, was obtained after fermentation of the germinated flour. Cooking had no effect on the amount of soluble iron. Iron solubility increased, however, two to four times after soaking and germination, three to five times after fermentation and five to eight times after fermentation of the germinated flour samples and was highly correlated to the reduction of IP6 + IP5 (P < 0.001). There was no difference between the quinoa varieties with regard to phytate reduction and iron solubility. The pH in fermented samples was reduced from 6.5 to about 3.5, due to lactic acid formation.

Low dietary iron availability is a major cause of anemia: a nutrition survey in the Lindi District of Tanzania.

A nutrition survey was conducted in the rural Lindi District of Tanzania to determine the magnitude of anemia and iron deficiency in different age and sex groups as related to nutritional status, parasitic infections, food iron intake, and socioeconomic factors. In a 30-cluster sampling design, 660 households were randomly selected and a total of 2320 subjects aged 6 mo to 65 y were examined. Iron status was assessed by measuring hemoglobin and erythrocyte protoporphyrin in a finger-prick sample: 55% of the subjects had anemia and 61% of the anemia was associated with iron deficiency (erythrocyte protoporphyrin > 125 micromol/mol heme). Preschool children (aged < 5 y) were the most affected; 84% were anemic (hemoglobin < 110 g/L). Fifty percent of the nonanemic preschool children and approximately 90% of all the severely anemic subjects were iron deficient. Hemoglobin was lower in schoolchildren (aged 5-14 y) and in adolescent and adult males (aged > or = 15 y) with a low body mass index. Parasitic infections were only associated with anemia and iron deficiency in schoolchildren and adolescent and adult males. Malaria was associated with anemia (P < 0.001), whereas schistosomiasis was associated with anemia and iron deficiency (P < 0.001 and P < 0.05, respectively). Hookworm infestation was associated with iron deficiency (P < 0.05) and with anemia (P < 0.01) only in adolescents and adults. A mainly cereal-based diet with additional legumes and green vegetables was found by in vitro tests to contain high amounts of total iron but of low bioavailability. Estimation of the amount of iron absorbed confirmed inadequate iron nutrition. Although anemia is a result of a synergism of a variety of causes, iron deficiency remains the major cause.

Inhibition of different strains of enteropathogens in a lactic-fermenting cereal gruel.

Twenty-eight strains of enteropathogens, including Campylobacter, Salmonella, Shigella, enterotoxigenic Escherichica coli (ETEC), Staphylococcus and Bacillus were added to cereal gruels prepared from low-tannin sorghum and inoculated with a lactic acid starter culture. Campylobacter strains were not detectable after 6 h, and Salmonella, Shigella and Staphylococcus strains not after 12 h. No viable cells of Bacillus strains were detected after 16 h of fermentation and the ETEC strains were completely inhibited after 24 h. No strain variability was observed within the different genera. In control gruels (no starter culture added), all the enteropathogens increased in number during incubation at 32°C except for the Campylobacter strains which decreased after 12 h of incubation.

pH and acidity in lactic-fermenting cereal gruels: effects on viability of enteropathogenic microorganisms.

Survival of Bacillus cereus, Campylobacter jejuni, enterotoxigenic Escherichia coli, Salmonella typhimurium and Shigella flexneri during lactic acid fermentation of cereal gruels prepared from low-tannin (white) and high-tannin (red) sorghum varieties was studied. A previously fermented gruel (starter culture, SC) recycled daily or stored for 7, 14 or 28 days, germinated cereal flour (power flour, PF), or a combination of PF and SC (PF+SC) were used as starters. At 24 h, the pH of all gruels with added starter was ≤4; the pH in control gruels without starter was ≥5.2. pH decrease was significantly faster in gruels made with PF+SC than with either PF or SC alone (P<0.05). A daily recycled SC resulted in a significantly faster decrease in pH (P<0.05) than SC stored for more than 7 days. Acid production was correlated with pH decrease (r=-0.94; P<0.01). In control gruels, the enteropathogens remained at the inoculation level or increased in number. Their growth was inhibited within 24 to 48 h in the fermented gruels, in the order: Bacillus > Campylobacter > Escherichia coli > Salmonella > Shigella. The inhibition rate was significantly faster in fermenting gruel with PF+SC (P<0.05) than in gruel with PF or SC alone and correlated with pH development (r=0.71; P<0.01). Both white and red sorghum gruels gave similar results. Using PF+SC as a starter resulted in a faster decrease in pH as well as a more rapid inhibition of enteropathogenic microorganisms. The effect is optimal if the SC is transferred daily.

Inhibited growth of common enteropathogenic bacteria in lactic-fermented cereal gruels.

A natural lactic fermentation of mixtures of water and whole flour of either maize or high-tannin sorghum was obtained either before or after cooking to a weaning gruel: The preparations had a final pH of about 3.8 (range 3.67 to 4.00) and a ratio of lactic acid to acetic acid of 9∶1 (w/w). The growth of added (about 10(7) c.f.u./g gruel) Gram-negative intestinal pathogenic bacteria, enterotoxigenicEscherichia coli, Campylobacter jejuni, Shigella flexneri andSalmonella typhimurium, was strongly inhibited in the sour gruels, and the effect could primarily be explained by the low pH caused by the formation of lactic and acetic acids during the fermentation process. Of the added Gram-positive bacteria,Bacillus cereus andStaphylococcus aureus showed similar inhibited growth up to 7h after inoculation in the sour gruels. The strain ofStaphylococcus, however, showed only a continued reduction in growth in the fermented gruel samples, which had a viable lactic bacteria culture indicating the presence of a bacteriocin. This implies that a low pH (< 4.0) alone is not sufficient to sustain the inhibition of the growth ofStaphylococcus aureus. The survival studies were carried out at optimal temperatures for each respective enteropathogen.

Noncollagenous proteins of rat compact bone.

In order to obtain a comprehensive overview of the noncollagenous proteins (NCPs) of bone matrix, the NCPs were extracted from rat compact bone and fractionated using methods aiming to prevent artifactual degradation and losses of protein. The NCP content of rat bone was found to be similar to that of rat dentin in several respects but different in others. The soluble NCPs of bone fell into four categories: acidic glycoproteins, gamma-carboxyglutamate-containing proteins, phosphoproteins, and proteoglycans. With the exception of the gamma-carboxyglutamate-containing proteins, the majority of NCPs had apparent molecular weights exceeding 50,000. As in rat dentin, several gamma-carboxyglutamate-containing proteins could be demonstrated in rat bone. Earlier studies have only taken one molecular species into consideration. No highly phosphorylated phosphoprotein could be demonstrated in bone. However, at least two phosphoproteins with a low degree of phosphorylation were found to be present. No plasma proteins could be demonstrated in any of the chromatographic fractions from the EDTA extracted NCPs by means of double diffusion. The NCPs, remaining firmly associated with the collagenous matrix after thorough demineralization and extraction, were analyzed after CNBr and collagenase degradation of the matrix. Much smaller amounts of phosphoprotein were recovered after CNBr digestion than reported earlier. Collagenase digestion released small amounts of acidic glycoprotein, phosphoprotein, and proteoglycan. The results give additional evidence that this small remainder might be explained, not by any covalent linkage to collagen, but by an inefficient extraction.

Plasma amino acid response to single test meals in humans. VI. Determination of available lysine.

The plasma amino acid response to single test meals in young adults was used to determine the amount of physiologically available lysine in heat-treated milk samples. The plasma amino acid responses were evaluated as PAA ratios according to a modified calculation procedure based on the method proposed by Longenecker and Hause. The amount of available lysine was determined by two separate series of single-meal tests. In the first, a relationship was established between the plasma amino acid response (PAA ratio) and the dietary level of lysine (given as synthetic amino acid mixtures). This relationship could be described by linear regression (r = 0.90). This regression line was then used to quantitatively evaluate the lysine PAA ratios obtained from single test meals with heat-treated milk samples. The heat-treatment of the milk samples caused a marked reduction of the amount of physiologically available lysine. The quantitative estimates obtained by the present plasma amino acid technique were in good agreement with determinations of available lysine in the same milk samples based on rat bioassay and chemical analysis.