Exploring Therapeutic Potential of Pleurotus ostreatus and Agaricus bisporus Mushrooms against Hyperlipidemia and Oxidative Stress Using Animal Model.

The mushrooms oyster (Pleurotus ostreatus) and white button (Agaricus bisporus) contain bioactive compounds that have potential beneficial effects on hypercholesterolemia and cardiovascular diseases. In this study, hypolipidemic and antioxidative potential of these mushrooms' extract were explored using hypercholesterolemic (HC) rats as animal model. For the study, 56 adult rats were divided into seven groups, i.e., G1 (negative control), G2 (positive control group), G3 (HC rats with statin drug orally), G4 and G5 (HC rats @ 100 and 200 mg/kg body weight (BW) dose of oyster mushroom extracts), and G6 and G7 (HC rats @ 100 and 200 mg/kg BW dose of white button mushroom extracts). The hypercholesterolemia was induced experimentally in fasted rats through a high-fat diet along with injection of triton WR-1339. After 48 h, the treatment groups were given extract for 28 days along with standard diet. At the trial termination, we analyzed the blood sugar levels, antioxidant parameters, lipid profile, and renal function, as well as conducting liver function tests of the rats. The results indicated that positive control group rats exhibited increased levels of total cholesterol (TC), triglycerides (TG), low-density level (LDL), and very-low-density level (vLDL) by 19%, 37%, 52%, and 32%, respectively, and 53% decrease in HDL, whereas treatment groups that received 200 mg oyster and white button mushroom extracts reported 15%, 34%, 22% reduction in TC, TG, vLDL, respectively, and 22% improvement in HDL level. The enzyme profiles of different groups showed non-significant differences, although both mushroom extracts provision reduced glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) levels. Overall, the results indicated that mushroom extracts were helpful in maintaining oxidative stress and have the potential to improve dyslipidemia in the tested rat animal model.

Relationship of stunting with water, sanitation, and hygiene (WASH) practices among children under the age of five: a cross-sectional study in Southern Punjab, Pakistan.

BACKGROUND: Reasons for undernutrition are food insufficiency, impaired child care, limited access to healthcare, and maternal lack of health literacy. In addition, there are several environmental factors, such as drinking water quality, poor sanitation, and hygienic practices that can lead to poor nutritional status in children. The present study aimed to compare household-associated risk factors, including water, sanitation, and hygiene (WASH) practices of mothers, with children's stunting under the age of five. METHODS: A face-to-face cross-sectional survey was conducted with mothers of children under the age of five in the Dera Ghazi Khan district in southern Punjab, Pakistan. World Health Organization criteria for stunting were used to identify the participants. A sociodemographic questionnaire was used to collect information from consented parents/guardians on children's age, feeding, and WASH practices. Pearson's chi-square, simple regression, and hierarchical regression models were applied for data analysis. RESULTS: A total of 204 mothers of children of both genders participated in this study. The children's mean (SD) age was 15.67 (± 10.2) months, their weight was 5.44 (± 1.45) kg, their height was 67.69 (± 10.05), and their mid-upper arm circumference was 9.75 (± 1.30) cm. Children's stunting was mild (z-score > -2) at 17.6%, moderate (z-score - 2 to -3) at 16.7%, and severe (z-score < -3) at 65.7% of participants. A simple regression model showed a strong association, r2 = 0.062, p = 0.013, with age as the most significant sociodemographic factor. The hierarchical regression model showed a combined value of r2 = 0.0128, p = 0.027, with hand pump and tank water as the significant source of drinking water that was related to stunting among children. CONCLUSION: Stunting can be associated with several risk factors, including WASH parameters. This study concluded that children aged under five years are susceptible to stunting in southern Punjab, Pakistan. The contamination of hand pumps and tank water resources was found to be the major contributing factor to stunting.

Double-Blind Parallel Treatment Randomized Controlled Trial of Prebiotics' Efficacy for Children Experiencing Severe Acute Malnutrition in Southern Punjab, Pakistan.

The prevalence of malnutrition among children under five is alarmingly high in Pakistan. However, there are ready-to-use therapeutic foods (RUTF) available which may be used to treat children with severe acute malnutrition (SAM). This study aims to assess the efficacy of prebiotics as a synergistic additive to RUTF to enhance blood parameters and anthropometric measurements in children with uncomplicated SAM living in Southern Punjab, Pakistan. A double-blind parallel treatment randomized controlled trial was conducted on 204 children aged 6-59 months. Participants were randomized and allocated to the placebo (n = 102) or experimental arms (n = 102) in a 1:1 ratio. One group of children was provided with RUTF and 4 g prebiotics, while the other group was given RUTF and starch as a placebo. Participants recruited for both arms were given treatment for eight weeks, and then their biochemical and anthropometric outcomes were evaluated. A substantial difference between the mean weight, mid-upper-arm circumference, haemoglobin, haematocrit, platelet count, mean corpuscular volume, mean corpuscular haemoglobin, and albumin was revealed. During the two-month follow-up phase of the trial, children who were enrolled in the treatment group gained about 20% of their initial weight (pre-study mean weight = 5.44 ± 1.35 kg; post-study mean weight = 6.53 ± 1.45 kg). The analysis showed a significant difference (p < 0.005) between the control and treatment groups for MUAC and complete blood counts. Conclusively, supplementation with RUTF and prebiotics has proven to be an efficient, effective, and safe therapy for children suffering from SAM to improve their growth and development indicators and reduce the dangers of malnutrition in comparison to RUTF alone.

Monitoring of frying process in canola oil blend using fourier transform infrared and chemometrics techniques.

The production of trans-fats and chemical changes during the process of frying are serious public health concerns and must be monitored efficiently. For this purpose, the canola oil was formulated with different ratio of extra virgin olive oil and palm olein using D-optimal mixture design, and the best formulation (67:22:11) based on free fatty acid (FFA) content, peroxide value (PV), and iodine value (IV) as responses was selected for multiple frying process. The data on FFA, PV, and IV along with Fourier transform-infrared (FT-IR) spectra were taken after each frying up to ten frying. The spectral data were preprocessed with standard normal variate followed by principal component analysis which is clearly showing the differentiation for various frying. Similarly, partial least square regression was applied to predict the FFA (0.37%-1.63%), PV (4.47-13.85 meqO2/kg), and IV (111.51-51.39 I2/100 g) which demonstrated high coefficient of determination (R2) 0.84, 0.83, and 0.81, respectively. It can be summarized that FT-IR can be used as a novel tool for fast and noninvasive quality determination of frying oils.

Evaluation of COVID-19 Disease Awareness and Its Relation to Mental Health, Dietary Habits, and Physical Activity: A Cross-Sectional Study from Pakistan.

Coronavirus infection (COVID-19) has significantly increased the mortality and morbidity rates worldwide. The present study was conducted to assess the general public's awareness of COVID-19 and its association with mental health, dietary habits, and physical activity. A web-based survey was conducted to gather information about demographics, knowledge about COVID-19, dietary habits, mental health, and anthropometry among the general public of Pakistan. Descriptive statistics, chi-square test, and multiple logistic regression were used for data analysis. The majority of the participants were suffering from anxiety (71.0%) and depression (52.0%) during the COVID-19 pandemic; 32.4% of participants had poor COVID-19-related knowledge. COVID-19 lockdown reduced the physical activity of 66.9% of participants and increased weight of 38.8% of the survey participants. Demographic variables, including age, gender, ethnicity, education, employment, family type, and geographical location, were significantly associated with knowledge about COVID-19 (P < 0.05). Depression was inversely associated with COVID-19 knowledge (P < 0.05). Healthy changes in dietary habits including decreased consumption of fast foods, soft and cola drinks, fruit drinks, cooked meat (outside the home), sugar, and fats, and were associated with increased knowledge of COVID-19. Vitamin C and immunity-boosting supplement consumption were significantly associated with increased knowledge regarding COVID-19 (< 0.05). Inadequate knowledge about COVID-19 and the presence of anxiety and depression were found among most of the study participants. There is a need to conduct educational seminars to limit the health consequences resulting from COVID-19 lockdown.

Assessment of aflatoxin in milk and feed samples and impact of seasonal variations in the Punjab, Pakistan.

The present study was designed to assess the incidence of aflatoxin contamination in animal feed and raw milk samples (total 240 each) collected from dairy farms during the complete year of 2015. These samples were collected through a cluster random sampling technique by dividing the province of Punjab, Pakistan into five clusters (north, south, east, west and central). Factors (environmental & physical) affecting aflatoxin contamination in milk and animal feed at farms were also studied. The AFM1 levels in raw milk & AFB1 levels in feed samples were analyzed by using the ELISA technique. Results demonstrated that overall about 53% raw milk samples from dairy farms were contaminated beyond the US MRL (0.50 µg/L) for AFM1 with than average level of 0.59 µg/L, while the 95% farm feed samples were exceeding the FDA MRL (20 µg/kg) of AFB1 with average level of 43 µg/kg. During winter season, the concentration of AFM1 was higher in all clusters with avg 0.68 µg/L, while the AFB1 contamination was highest in the spring season with avg 54 µg/kg. Market feed prices had negative correlation with AFB1 contamilevels, which were further supported by the positive correlation between quantity of feed at farms with AFM1 and AFB1 contamination. Results exhibited significantly positive impact of environmental factors on milk and feed aflatoxin contamination levels, whereas temperature showed an inverse relationship with AFM1 and AFB1 levels. The study recommends need of synergistic extension work to support dairy farms and highlight the contamination levels for regulatory bodies to introduce strategic policies for control measures.

Iron and prebiotic fortified flour improves the immune function of iron deficient women of childbearing age.

Micronutrient deficiencies (MNDs) are common worldwide, in both developing as well as developed countries. MNDs such as Iron Deficiency not only compromise the nutritional status of individuals but can also put them at an increased risk of developing various other diseases by negatively affecting their immunity. The objective of the current research was to determine the effects of prebiotics and iron fortificants on various immunoglobulins among iron deficient women belonging to childbearing age. To serve the purpose, a total of seventy five iron deficient women were selected and randomly divided into one control and four treatment groups. Accordingly, different types of fortified wheat flour were prepared, based on varying dosage of prebiotics and iron fortificants, to be fed to anemic women on daily basis for three months. Two iron salts (FeSO4 and NaFeEDTA) and two prebiotics (Galacto oligosaccharides and Inulin) were used to fortify wheat flour during the trials. Overnight fasted women were asked to give blood samples on monthly basis, up to three months. Four types of Immunoglobulins including IgA, IgE, IgG and IgM were determined at baseline, 30th, 60th and 90th day of trials using their respective protocols. The results of the study indicated that a statistically significant declining trend for IgA, IgE, IgG and IgM was present among the treatment groups (P-value < 0.05), compared to the control group. The study concluded that provision of iron and prebiotic fortified flour improved the immune function of iron deficient women.

Occurrence and Seasonal Variations of Aflatoxin M1 in Milk from Punjab, Pakistan.

The manifestation of aflatoxins in feed and food is a major issue in the world as its presence leads to some health problems. This study investigates the incidence of aflatoxin M1 (AFM1) contamination in raw milk samples which were collected from Punjab, Pakistan. The Cluster Random Sampling technique was used to collect 960 milk samples from five different regions, and samples were collected every month. The AFM1 level in raw milk was analyzed by the ELISA technique. The findings demonstrate that 70% of samples exceeded the United States permissible maximum residue limits (MRL 0.50 µg/L), with an overall AFM1 level that ranged from 0.3 to 1.0 µg/L. AFM1 contamination varied with the season: The highest average contamination was detected in winter (0.875 µg/L), followed by autumn (0.751 µg/L), spring (0.654 µg/L), and summer (0.455 µg/L). The Eastern region exhibited the highest average AFM1 contamination (0.705 µg/L). Milk samples from the Northern region were found to be widely contaminated, as 86.9% samples exceeded the US MRL, followed by the Eastern region, with 72.3% samples being contaminated with >0.5 µg/L AFM1. The study indicated that the raw milk supply chain was heavily contaminated. Recommendations and remedial measures need to be developed by regulatory authorities to improve the raw milk quality.

Immune modulatory and anti-oxidative effect of selenium against pulmonary tuberculosis.

Prevalence of pulmonary tuberculosis (TB) in Pakistan is due to poor living conditions, malnutrition and low immunity. The present project was conducted to show the role of selenium complement to enhance the immune status against TB. Total of 80 human TB patients were divided into treatment (selenium and anti-tuberculosis drug) and control groups (anti-tuberculosis drug). Levels of selenium, immunoglobulin and leukocyte count were determined before and after treatment. Selenium showed significant increase in levels of immunoglobulin and leukocyte count in patients as compared to control group. The level of SOD, catalase, glutathione and total antioxidants were remarkably lowered among control type group as compared to treatment type group (P<0.01). However, the values of lipid peroxidation products malondialdehyde (MDA) were notably higher in control group than treatment group.

Synergistic effect of galacto oligosaccharides and iron fortificants on serum iron, ferritin, transferrin and total iron binding capacity levels in anemic rats.

Iron deficiency anemia is one of the leading public health issues being faced by the global population currently. The present research was an attempt to determine the synergistic effect of Galacto Oligosaccharides and iron fortificants on serum iron, serum ferritin, serum transferrin and total iron binding capacity in anemic rats. To serve the purpose, eight different types of fortified feed were prepared with varying concentrations of Iron Fortificants (NaFeEDTA and FeSO4) while the varying dosage of galacto oligosaccharides was dissolved separately in water to be fed to anemic rats. Afterwards, animal trials were conducted for twelve weeks to determine the efficacy of Galacto Oligosaccharides & iron fortificants based feed against the aforementioned parameters. The results of the study suggested that both serum iron and serum ferritin levels were significantly improved when anemic rats were fed with iron and Galacto Oligosaccharides fortified feed. It was also observed that the levels of serum transferrin and total iron binding capacity steadily decreased over the study duration. It can be concluded that Galacto Oligosaccharides helped enhance the absorption of iron in anemic rats, reflected by increase in serum iron and serum ferritin levels and decrease in serum transferrin and total iron binding capacity.

White Sesame Seed Oil Mitigates Blood Glucose Level, Reduces Oxidative Stress, and Improves Biomarkers of Hepatic and Renal Function in Participants with Type 2 Diabetes Mellitus.

OBJECTIVES: The study was designed to investigate the impact of white sesame seed oil (WSSO) consumption on fasting blood glucose (GLU), insulin (INS), glycosylated hemoglobin (HbA1c), and hepatic antioxidant enzymes. A secondary aim was to check the influence on serum biochemistry, hepatic, cardiac, and renal functions. METHODS: Forty-six participants with type 2 diabetes were recruited and randomly divided into two equal groups: diabetic control (DCON) and diabetic sesame oil (DSO). At baseline and 30, 60, and 90 days, blood samples were drawn and analyzed. Two-way repeated-measures analysis of variance was used to evaluate the difference between groups and across time. RESULTS: In both groups, GLU, INS, and HbA1c were not significantly different at baseline (mean 187.07 ± 5.63 mg/dl, mean 12.12 ± 1.03 µU/ml, and mean 7.55 ± 0.37%, respectively). At 90 days, GLU was significantly (p < 0.05) decreased in DSO (137.83 ± 3.16 mg/dl) when compared with DCON (218.13 ± 5.92 mg/dl), while INS was significantly increased in DSO (23.13 ± 1.15 µU/ml) as compared to DCON (7.93 ± 0.38 µU/ml). At 90 days, HbA1c was significantly lower (p < 0.05) in DSO as compared to DCON. Thiobarbituric acid reactive substances were significantly lower (p < 0.05) in DSO (1.08 ± 0.05 [MDA] nmol/ml) as compared to DCON (2.26 ± 0.07 [MDA] nmol/ml). In DSO, activities of hepatic antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) increased while in DCON these activities decreased significantly (p < 0.05) across the time period. Biomarkers of liver, cardiac, and renal functions improved significantly in DSO as compared to DCON. CONCLUSION: WSSO as a functional food may play an important role in GLU regulation and against deleterious effects of diabetes in humans with type 2 diabetes.

Prevalence of anemia and associated risk factors among pregnant women in Lahore, Pakistan.

Maternal anemia is a frequent problem during pregnancy, especially in developing countries, such as Pakistan. In this context, the present study was conducted to determine factors associated with maternal anemia prevalent among women of different age groups visiting different hospitals in Lahore from February to April 2017. The current study used a cross-sectional design involving 390 pregnant women. Data related to demographic area, maternal age, gestational age/period, educational and income level, as well as socioeconomic status of all the participants were collected. Also, hemoglobin levels of the participants were obtained for assessment of maternal anemia. A total of 57.7% of participants were anemic (34.4 % mild and 23.3% moderate anemia). Bivariate analysis showed that less education (p = 0.01), occupation as housewife (p = 0.03), lower income (p = 0.001), rural residence (p = 0.028), and greater number of house members (p = 0.04) were significantly associated with maternal anemia. Results of multivariable logistic regression showed that low income was the only significant factor independently associated with maternal anemia. These results suggest that interventions, such as the Nutrition Support Program, to improve nutritional status of pregnant women by providing free food supplements with focus on the poor and marginalized communities are recommended to prevent anemia.

Nutraceutical, Anti-Inflammatory, and Immune Modulatory Effects of ß-Glucan Isolated from Yeast.

ß-Glucan is a dietary fibre, found in many natural sources, and controls chronic metabolic diseases effectively. However, ß-glucan from the yeast has rarely been investigated. Objectively, conditions were optimized to isolate ß-glucan from the yeast (max. 66% yield); those optimized conditions included 1.0 M NaOH, pH 7.0, and 90°C. The purity and identity of the isolated ß-glucan were characterized through FT-IR, SEM, DSC, and physicofunctional properties. The obtained results from DSC revealed highly stable ß-glucan (m.p., 125°C) with antioxidant activity (TAC value 0.240 ± 0.0021 µg/mg, H2O2 scavenging 38%), which has promising bile acid binding 40.463% and glucose control (in vitro). In line with these results, we evaluated the in vivo anti-inflammatory potential, that is, myeloperoxidase activity and reduction in MDA and NO; protective effect on proteins and keeping viscosity within normal range exhibited improvement. Also, the in vivo cholesterol binding and reduction in the skin thickness by ß-glucan were highly encouraging. Finally, our results confirmed that yeast ß-glucan is effective against some of the inflammatory and oxidative stress markers studied in this investigation. In general, the effect of 4% ß-glucan was more noticeable versus 2% ß-glucan. Therefore, our results support the utilization of ß-glucan as a novel, economically cheap, and functional food ingredient.

Impact of Novel Prebiotic Galacto-Oligosaccharides on Various Biomarkers of Colorectal Cancer in Wister Rats.

Colorectal cancer (CRC) is one of the leading causes of cancer deaths around the globe. Bioactive food ingredients such as prebiotics have protective potential in colon cancer. Data on galacto-oligosaccharides (GalOS) against CRC are very limited and GalOS used in this study have ß-1,6 and ß-1,3 as major glycosidic linkages and, to our best knowledge, were never used before against any cancer treatment. This study aims to investigate the protective role of novel GalOS against various biomarkers of CRC including aberrant crypt foci (ACF), bacterial enzymes and short chain fatty acids (SCFA) in a rodent model induced with 1,2-dimethylhydrazine dihydrochloride (DMH). Inulin group was taken as positive control in present study to compare novel GalOS protective effects. GalOS doses of 76-151 mg and inulin doses of 114 mg were given to different groups treated with DMH. Results showed that ACF formation was significantly (p ≤ 0.05) less in high dose GalOS group (27.3%). GalOS also had protective effects against DMH-induced body weight loss and showed higher level of cecal and fecal SCFA (acetate, propionate and butyrate). High doses of GalOS also resulted in significant (p ≤ 0.05) reduction of bacterial enzymatic activities. Increased populations of beneficial bacteria (bifidobacteria and lactobacilli) and decreased concentrations of harmful bacteria were observed in all prebiotics treatment groups. It can be concluded that novel GalOS exhibit robust protective activity against ACF formation in vivo.

Evaluation of White Sesame Seed Oil on Glucose Control and Biomarkers of Hepatic, Cardiac, and Renal Functions in Male Sprague-Dawley Rats with Chemically Induced Diabetes.

White sesame seed oil (WSSO) has been used in cooking and food preparations for centuries. It has many purported health benefits and may be a promising nutraceutical. The primary purpose of this study was to examine the effects of WSSO on fasting blood glucose (GLU) and insulin (INS) in male Sprague-Dawley rats with chemically induced diabetes. A secondary aim was to explore other hematological biomarkers of hepatic, cardiac, and renal function. Sixty-three male Sprague-Dawley rats were randomized into standard diet groups, normal control (NCON) (n = 21) and diabetic control (DCON) (n = 21), and a diabetic sesame oil (DSO) (n = 21) group, which were fed a diet containing 12% WSSO. Blood samples were analyzed at 0, 30, and 60 days. Differences between groups and across days were assessed with two-way repeated measures analysis of variance. At baseline, GLU and INS were similar in both diabetic groups, mean 248.4 ± 2.8 mg/dL and mean 23.4 ± 0.4 µU/mL, respectively. At 60 days, GLU was significantly (P < .05) higher in DCON (298.0 ± 2.3 mg/dL) compared with DSO (202.1 ± 1.0 mg/dL). INS showed similar favorable trends after WSSO supplementation. Consumption of WSSO significantly improved glucose control and other biomarkers of hepatic stress, as well as cardiac and renal health. WSSO may be a viable functional food to help reduce the detrimental effects of diabetes.

Novel Combination of Prebiotics Galacto-Oligosaccharides and Inulin-Inhibited Aberrant Crypt Foci Formation and Biomarkers of Colon Cancer in Wistar Rats.

The selectivity and beneficial effects of prebiotics are mainly dependent on composition and glycosidic linkage among monosaccharide units. This is the first study to use prebiotic galacto-oligosaccharides (GOS) that contains ß-1,6 and ß-1,3 glycosidic linkages and the novel combination of GOS and inulin in cancer prevention. The objective of the present study is to explore the role of novel GOS and inulin against various biomarkers of colorectal cancer (CRC) and the incidence of aberrant crypt foci (ACF) in a 1,2-dimethyl hydrazine dihydrochloride (DMH)-induced rodent model. Prebiotic treatments of combined GOS and inulin (57 mg each), as well as individual doses (GOS: 76-151 mg; inulin 114 mg), were given to DMH-treated animals for 16 weeks. Our data reveal the significant preventive effect of the GOS and inulin combination against the development of CRC. It was observed that inhibition of ACF formation (55.8%) was significantly (p ≤ 0.05) higher using the GOS and inulin combination than GOS (41.4%) and inulin (51.2%) treatments alone. This combination also rendered better results on short-chain fatty acids (SCFA) and bacterial enzymatic activities. Dose-dependent effects of prebiotic treatments were also observed on cecum and fecal bacterial enzymes and on SCFA. Thus, this study demonstrated that novel combination of GOS and inulin exhibited stronger preventive activity than their individual treatments alone, and can be a promising strategy for CRC chemoprevention.

Effect of Prebiotic Galacto-Oligosaccharides on Serum Lipid Profile of Hypercholesterolemics.

The present study focused on the role of galacto-oligosaccharides (GOS) against the development of hypercholesterolemia. In the current research, GOS synthesized from lactose solution were fed to hypercholesterolemic female Sprague-Dawley rats. Negative control group (G1) was fed on standard basal diet alone. Positive control group (G5) was fed on inulin (154 mg/250 g body weight), while treatment groups G2, G3 and G4 received 110 mg, 154 mg and 198.4 mg/250 g body weight, respectively, of GOS along with high-fat diet for a period of 60 days. Findings from this study revealed that animals belonging to prebiotic GOS (G2, G3 and G4)-fed group showed significantly decreased serum triglycerides, total cholesterol, LDL cholesterol and VLDL cholesterol as compared to control group (G0). The groups which were fed on different doses of GOS revealed a significant reduction in TC, TG, LDL, and VLDL levels and an increase in HDL level corresponding to the reference group that was fed on inulin, while G1 negative control group revealed increased levels of TC, TG, LDL and VLDL. In contrast to positive control group G5 (154 mg inulin), all doses of GOS lowered serum TC, TG and LDL-C and raised HDL-C; however, G4 (198.4 mg) proved to be more effective. Hence, GOS proved to be supportive in preventing hypercholesterolemia leading cause of cardiovascular disease and atherosclerosis. This study reported a significant reduction of serum TC, TG and LDL-C in female rats for 60 days as compared to control. Conclusively, GOS were found to be worthless against hypercholesterolemia.

Characterization of a heterodimeric GH2 ß-galactosidase from Lactobacillus sakei Lb790 and formation of prebiotic galacto-oligosaccharides.

The lacLM genes from Lactobacillus sakei Lb790, encoding a heterodimeric ß-galactosidase that belongs to glycoside hydrolase family GH2, were cloned and heterologously expressed in Escherichia coli . Subsequently, the recombinant ß-galactosidase LacLM was purified to apparent homogeneity and characterized. The enzyme is a ß-galactosidase with narrow substrate specificity because o-nitrophenyl-ß-D-galactopyranoside (oNPG) was efficiently hydrolyzed, whereas various structurally related oNP analogues were not. The K(m) and k(cat) values for oNPG and lactose were 0.6 mM and 180 s(-1) and 20 mM and 43 s(-1), respectively. The enzyme is inhibited competitively by its two end-products D-galactose and D-glucose (K(i) values of 180 and 475 mM, respectively). As judged by the ratio of the inhibition constant to the Michaelis constant, K(i)/K(m), this inhibition is only very moderate and much less pronounced than for other microbial ß-galactosidases. ß-Galactosidase from L. sakei possesses high transgalactosylation activity and was used for the synthesis of galacto-oligosaccharides (GalOS), employing lactose at a concentration of 215 g/L. The maximum GalOS yield was 41% (w/w) of total sugars at 77% lactose conversion and contained mainly non-lactose disaccharides, trisaccharides, and tetrasaccharides with approximately 38, 57, and 5% of total GalOS formed, respectively. The enzyme showed a strong preference for the formation of ß-(1→6)-linked transgalactosylation products, whereas ß-(1→3)-linked compounds were formed to a lesser extent and ß-(1→4)-linked reaction products could not be detected.

Cloning, purification, and characterization of ß-galactosidase from Bacillus licheniformis DSM 13.

The gene encoding homodimeric ß-galactosidase (lacA) from Bacillus licheniformis DSM 13 was cloned and overexpressed in Escherichia coli, and the resulting recombinant enzyme was characterized in detail. The optimum temperature and pH of the enzyme, for both o-nitrophenyl-ß-D: -galactoside (oNPG) and lactose hydrolysis, were 50°C and 6.5, respectively. The recombinant enzyme is stable in the range of pH 5 to 9 at 37°C and over a wide range of temperatures (4-42°C) at pH 6.5 for up to 1 month. The K(m) values of LacA for lactose and oNPG are 169 and 13.7 mM, respectively, and it is strongly inhibited by the hydrolysis products, i.e., glucose and galactose. The monovalent ions Na(+) and K(+) in the concentration range of 1-100 mM as well as the divalent metal cations Mg²(+), Mn²(+), and Ca²(+) at a concentration of 1 mM slightly activate enzyme activity. This enzyme can be beneficial for application in lactose hydrolysis especially at elevated temperatures due to its pronounced temperature stability; however, the transgalactosylation potential of this enzyme for the production of galacto-oligosaccharides (GOS) from lactose was low, with only 12% GOS (w/w) of total sugars obtained when the initial lactose concentration was 200 g/L.

beta-Galactosidase from Lactobacillus plantarum WCFS1: biochemical characterization and formation of prebiotic galacto-oligosaccharides.

Recombinant beta-galactosidase from Lactobacillus plantarum WCFS1, homologously over-expressed in L. plantarum, was purified to apparent homogeneity using p-aminobenzyl 1-thio-beta-d-galactopyranoside affinity chromatography and subsequently characterized. The enzyme is a heterodimer of the LacLM-family type, consisting of a small subunit of 35kDa and a large subunit of 72kDa. The optimum pH for hydrolysis of its preferred substrates o-nitrophenyl-beta-d-galactopyranoside (oNPG) and lactose is 7.5 and 7.0, and optimum temperature for these reactions is 55 and 60 degrees C, respectively. The enzyme is most stable in the pH range of 6.5-8.0. The K(m), k(cat) and k(cat)/K(m) values for oNPG and lactose are 0.9mM, 92s(-1), 130mM(-1)s(-1) and 29mM, 98s(-1), 3.3mM(-1)s(-1), respectively. The L. plantarum beta-galactosidase possesses a high transgalactosylation activity and was used for the synthesis of prebiotic galacto-oligosaccharides (GOS). The resulting GOS mixture was analyzed in detail, and major components were identified by using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as well as capillary electrophoresis. The maximal GOS yield was 41% (w/w) of total sugars at 85% lactose conversion (600mM initial lactose concentration). The enzyme showed a strong preference for the formation of beta-(1-->6) linkages in its transgalactosylation mode, while beta-(1-->3)-linked products were formed to a lesser extent, comprising approximately 80% and 9%, respectively, of the newly formed glycosidic linkages in the oligosaccharide mixture at maximum GOS formation. The main individual products formed were beta-d-Galp-(1-->6)-d-Lac, accounting for 34% of total GOS, and beta-d-Galp-(1-->6)-d-Glc, making up 29% of total GOS.