L-arabinose isomerase (L-AI) is the key enzyme that isomerizes D-galactose to D-tagatose. In this study, to improve the activity of L-arabinose isomerase on D-galactose and its conversion rate in biotransformation, an L-arabinose isomerase from Lactobacillus fermentum CGMCC2921 was recombinantly expressed and applied in biotransformation. Moreover, its substrate binding pocket was rationally designed to improve the affinity and catalytic activity on D-galactose. We show that the conversion of D-galactose by variant F279I was increased 1.4 times that of the wild-type enzyme. The K_m and k_cat values of the double mutant M185A/F279I obtained by superimposed mutation were 530.8 mmol/L and 19.9 s^-1, respectively, and the catalytic efficiency was increased 8.2 times that of the wild type. When 400 g/L lactose was used as the substrate, the conversion rate of M185A/F279I reached a high level of 22.8%, which shows great application potential for the enzymatic production of tagatose from lactose.
Galactose/metabolism* ; Limosilactobacillus fermentum/genetics* ; Lactose ; Hexoses/metabolism* ; Aldose-Ketose Isomerases/genetics* ; Hydrogen-Ion Concentration

OBJECTIVETo investigate relations between milk consumption and lactose intolerance (LI) in adults and to explore the effect of milk consumption on lactase activity.

METHODSTotal of 182 subjects aged 20-70 years were recruited and interviewed by questionnaires, and their accumulative cow's milk intake (AMI) was calculated. LI was evaluated by hydrogen breath test (HBT).

RESULTSA negative correlation was found between AMI and severity of observed LI symptom (r=-0.2884; P<0.05). Binary logistic regression analysis showed a negative correlation between LI and duration and frequency of milk consumption (OR, 0.317 and 0.465, respectively; both P<0.05) and a positive correlation between LI and amount of milk consumed per sitting (OR, 6.337; P<0.05).

CONCLUSIONLI is related to various milk consumption behaviors. Most Chinese adults with LI may tolerate moderate milk consumption <160 mL.

Adult ; Animals ; Breath Tests ; China ; epidemiology ; Drinking Behavior ; Humans ; Hydrogen ; Lactase ; metabolism ; Lactose ; Lactose Intolerance ; diagnosis ; epidemiology ; prevention & control ; Milk ; Prevalence

Infant formula is classified into standard cow's milk-based and special formulas. This review aimed at summarizing the types of special milk formulas currently sold in Korea, and the appropriate indications for the use of these formulas; lactose free formula, soy-based formula, protein hydrolysate formula, amino acid-based formula, preterm formula, medium chain triglyceride formula, low-phosphorus formula, protein-energy-enriched formula, and formulas for inborn errors of metabolism.
Humans ; Infant Formula* ; Infant* ; Korea* ; Lactose ; Metabolism, Inborn Errors ; Milk ; Protein Hydrolysates ; Soybeans ; Triglycerides

BACKGROUND/AIMS: Lactose-free milk (LFM) is available for nutrient supply for those with lactose intolerance (LI). However, there are no consistent results of the efficacy of LFM in LI subjects. We aimed to examine the changes of gastrointestinal (GI) symptoms and hydrogen breath test (HBT) values after ingestion of lactose contained milk (LCM) vs. LFM. METHODS: From May 2015 to September 2015, thirty-five healthy adults with history of LCM-induced GI symptoms were recruited at a tertiary hospital. For the diagnosis of LI, HBT with LCM 550 mL (lactose 25 g) was performed every 20 minutes for 3 hours. The test was defined as "positive" when H2 peak exceeded 20 ppm above baseline values (DeltaH2>20 ppm). When the subjects are diagnosed as LI, the second HBT using LFM 550 mL (lactose 0 g) was performed 7 days later. Subjects were asked to complete a questionnaire about the occurrence and severity of GI symptoms. RESULTS: Among a total of 35 subjects, 31 were diagnosed with LI at first visit, and their LCM-related symptoms were abdominal pain (98.6%), borborygmus (96.8%), diarrhea (90.3%), and flatus (87.1%). The DeltaH2 value in subjects taking LCM (103.7+/-66.3 ppm) significantly decreased to 6.3+/-4.9 ppm after ingesting LFM (p<0.0001). There were also significant reduction in total symptom scores and the severity of each symptom when LCM was changed to LFM (p<0.0001). CONCLUSIONS: This is the first report that LFM reduce LCM-related GI symptoms and H2 production in Korean adults. LFM can be an effective alternative for LCM in adults with LI.
Abdominal Pain/pathology ; Adult ; Aged ; Animals ; Asian Continental Ancestry Group ; Breath Tests ; Diarrhea/pathology ; Female ; Humans ; Hydrogen/metabolism ; Lactose/chemistry/metabolism ; Lactose Intolerance/*diagnosis ; Male ; Middle Aged ; Milk/chemistry ; Republic of Korea ; Severity of Illness Index ; Surveys and Questionnaires ; Tertiary Care Centers

Calcium is an essential nutrient that is necessary for many functions in human health. Calcium is the most abundant mineral in the body with 99% found in teeth and bone. Only 1% is found in serum. The serum calcium level is tightly monitored to remain within normal range by a complex metabolic process. Calcium metabolism involves other nutrients including protein, vitamin D, and phosphorus. Bone formation and maintenance is a lifelong process. Early attention to strong bones in childhood and adulthood will provide more stable bone mass during the aging years. Research has shown that adequate calcium intake can reduce the risk of fractures, osteoporosis, and diabetes in some populations. The dietary requirements of calcium and other collaborative nutrients vary slightly around the world. Lactose intolerance due to lactase deficiency is a common cause of low calcium intake. Strategies will be discussed for addressing this potential barrier to adequate intake. The purpose of this narrative review is a) to examine the role of calcium in human health, b) to compare nutrient requirements for calcium across lifecycle groups and global populations, c) to review relationships between calcium intake, chronic disease risk, and fractures, and d) to discuss strategies to address diet deficiencies and lactose intolerance.
Aging* ; Calcium* ; Chronic Disease ; Diet ; Humans ; Lactase ; Lactose Intolerance ; Metabolism ; Nutritional Requirements ; Osteogenesis ; Osteoporosis ; Phosphorus ; Reference Values ; Tooth ; Vitamin D

Escherichia coli strain DC1515, deficient in glucose phosphotransferase (ptsG), lactate dehydrogenase (ldhA) and pyruvate:formate lyase (pflA), is a promising candidate for the fermentative production of succinate. To further improve the succinate producing capability of DC1515, we constructed plasmid pTrchisA-pyc with heterogenous pyruvate carboxylase (pyc) from Bacillus subtilis 168 under the Trc promoter and introduced it into DC1515. We used lactose as a substitute of IPTG to induce pyc. We optimized the culture conditions such as the lactose addition time, the lactose concentration and the culture temperature after induction for succinate production. We also explored the effect of lactose supplement during the fermentation. The results showed that pyc can be expressed under lactose induction in the fermentative medium with 15 g/L glucose due to the deficient of ptsG in DC1515. Under optimized conditions, the final succinate concentration reached to 15.17 g/L, which was 1.78-fold higher than that of control strain. If complementing lactose twice to the concentration of 1 g/L during the fermentation, the final succinate concentration could further reach to 17.54 g/L. This work might provide valuable information for gene expression in E. coli strains using lactose as inducer for succinate production in a glucose-medium. Due to the reduced cost, E. coli is becoming a more promising strain for succinate production through fermentation.
Bacillus subtilis ; enzymology ; Culture Media ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Lactose ; pharmacology ; Promoter Regions, Genetic ; Pyruvate Carboxylase ; biosynthesis ; genetics ; Succinic Acid ; metabolism

To produce recombinant phospholipase A(1) (PLA(1)) by Escherichian coli, the pla gene encoding PLA(1) was amplified from Serratia liquefaciens by PCR and cloned into two vectors pET20-b(+) and pET28-a(+). The two recombinant plasmids were then transformed into E. coli BL21 (DE3) individually to express PLA(1). E. coli BL21(DE3)/pET28a-pla yielded extracellular PLA(1) with an activity of 40.8 U/mL in batch cultivations of shaken flasks by auto-induction, which was accounted for 91% of total enzyme activity. On the basis of primal auto-induction medium, the optimized fermentation medium of PLA(1) contained tryptone 10 g/L, yeast extract 5 g/L, glucose 0.8 g/L, lactose 5 g/L, Na2HPO4 25 mmol/L, KH2PO4 25 mmol/L and 1 mmol/L MgSO4 (final concentration). Glycine (7.5 g/L) was added 6 h after inoculated. After incubated at 37 degrees C for 24 h, extracellular enzyme activity reached 128.7 U/mL.
Cloning, Molecular ; Culture Media ; Escherichia coli ; genetics ; growth & development ; metabolism ; Fermentation ; Lactose ; pharmacology ; Phospholipases A1 ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Serratia liquefaciens ; enzymology

To improve the yield of sucrose isomerase from Pantoea dispersa UQ68J, we studied the effect of different signal peptides and fermentation conditions on sucrose isomerase expression in Escherichia coli. The gene of sucrose isomerase was optimized and expressed in E. coli BL21 (DE3) with native signal peptide which was named as ORI strain. The total and extracellular enzyme activity was 85 and 65 U/mL in the flask, respectively. The mature protein, which started from the 22th amino acid, was connected with the PelB and OmpA signal peptide to construct P22 and O22 strain, respectively. The total activity of P22 reached 138 U/mL, which was 1.6 times of ORI strain. The total activity of O22 strain was similar to that of ORI strain. Induced by 3.0 g/L lactose, the total activity of P22 strain increased to 168 U/mL. In 3 L fermentor, the effects of glycine concentration and induction time were studied. Induction when the DCW reached 18 g/L (OD₆₀₀=30), with 0.5% glycine, the extracellular enzyme activity reached 1 981 U/mL, and the total enzyme activity reached 2 640 U/mL, which is the highest activity of sucrose isomerase that was expressed in recombinant E. coli.
Bacterial Proteins ; biosynthesis ; Bioreactors ; Escherichia coli ; metabolism ; Fermentation ; Gene Expression ; Glucosyltransferases ; biosynthesis ; Lactose ; Pantoea ; enzymology ; Protein Sorting Signals ; Recombinant Proteins ; biosynthesis

OBJECTIVETo construct four recombinant Lactococcus lactis strains exhibiting high beta-galactosidase activity in fusion or non-fusion ways, and to study the influence factors for their protein expression and secretion.

METHODSThe gene fragments encoding beta-galactosidase from two strains of Lactobacillus bulgaricus, wch9901 isolated from yogurt and 1.1480 purchased from the Chinese Academy of Sciences, were amplified and inserted into lactococcal expression vector pMG36e. For fusion expression, the open reading frame of the beta-galactosidase gene was amplified, while for non-fusion expression, the open reading frame of the beta-galactosidase gene was amplified with its native Shine-Dalgarno sequence upstream. The start codon of the beta-galactosidase gene partially overlapped with the stop codon of vector origin open reading frame. Then, the recombinant plasmids were transformed into Escherichia coli DH5 alpha and Lactococcus lactis subsp. lactis MG1363 and confirmed by determining beta-galactosidase activities.

RESULTSThe non-fusion expression plasmids showed a significantly higher beta-galactosidase activity in transformed strains than the fusion expression plasmids. The highest enzyme activity was observed in Lactococcus lactis transformed with the non-fusion expression plasmids which were inserted into the beta-galactosidase gene from Lactobacillus bulgaricus wch9901. The beta-galactosidase activity was 2.75 times as high as that of the native counterpart. In addition, beta-galactosidase expressed by recombinant plasmids in Lactococcus lactis could be secreted into the culture medium. The highest secretion rate (27.1%) was observed when the culture medium contained 20 g/L of lactose.

CONCLUSIONDifferent properties of the native bacteria may have some effects on the protein expression of recombinant plasmids. Non-fusion expression shows a higher enzyme activity in host bacteria. There may be a host-related weak secretion signal peptide gene within the structure gene of Lb. bulgaricus beta-galactosidase, and its translation product may introduce the enzyme secretion out of cells in special hosts.

Erythromycin ; pharmacology ; Gene Expression Regulation, Bacterial ; Lactobacillus ; drug effects ; enzymology ; genetics ; Lactococcus lactis ; drug effects ; enzymology ; genetics ; Lactose ; metabolism ; pharmacology ; Recombinant Proteins ; genetics ; metabolism ; Time Factors ; beta-Galactosidase ; genetics ; metabolism

The purpose of this study was to evaluate the capacity of a lactic acid bacteria (LAB) inoculum to protect calves with or without lactose supplements against Salmonella Dublin infection by evaluating histopathological lesions and pathogen translocation. Fifteen calves were divided into three groups [control group (C-G), a group inoculated with LAB (LAB-G), and a group inoculated with LAB and given lactose supplements (L-LAB-G)] with five, six, and four animals, respectively. The inoculum, composed of Lactobacillus (L.) casei DSPV 318T, L. salivarius DSPV 315T, and Pediococcus acidilactici DSPV 006T, was administered with milk replacer. The LAB-G and L-LAB-G received a daily dose of 109 CFU/kg body weight of each strain throughout the experiment. Lactose was provided to the L-LAB-G in doses of 100 g/day. Salmonella Dublin (2 x 1010 CFU) was orally administered to all animals on day 11 of the experiment. The microscopic lesion index values in target organs were 83%, 70%, and 64.3% (p < 0.05) for the C-G, LAB-G, and L-LAB-G, respectively. Administration of the probiotic inoculum was not fully effective against infection caused by Salmonella. Although probiotic treatment was unable to delay the arrival of pathogen to target organs, it was evident that the inoculum altered the response of animals against pathogen infection.
Administration, Oral ; Animals ; Animals, Newborn ; Cattle ; Cattle Diseases/*drug therapy/microbiology/pathology ; Dietary Supplements/*analysis ; Feces/microbiology ; Lactobacillus/metabolism ; Lactose/*metabolism ; Male ; Pediococcus/metabolism ; Probiotics/*therapeutic use ; Salmonella Infections, Animal/*drug therapy/microbiology/pathology ; Salmonella enterica/*drug effects/growth & development ; Tissue Distribution