CRISPR/Cas9-Based Genome Editing for Protein Expression and Secretion in Kluyveromyces lactis.

The budding yeast Kluyveromyces lactis has emerged as a promising microbial chassis in industrial biotechnology. However, a lack of efficient molecular genetic manipulation tools and strategies has hindered the development of K. lactis as a biomanufacturing platform. In this study, we developed and applied a CRISPR/Cas9-based genome editing method to K. lactis. Single-gene editing efficiency was increased to 80% by disrupting the nonhomologous end-joining-related gene KU80 and performing a series of process optimizations. Subsequently, the CRISPR/Cas9 system was explored based on different sgRNA delivery modes for simultaneous multigene editing. With the aid of the color indicator, the editing efficiencies of two and three genes reached 73.3 and 36%, respectively, in the KlΔKU80 strain. Furthermore, the CRISPR/Cas9 system was used for multisite integration to enhance lactase production and combinatorial knockout of TMED10 and HSP90 to characterize the extracellular secretion of lactase in K. lactis. Generally, genome editing is a powerful tool for constructing K. lactis cell factories for protein and chemical production.

A cost-effective enzyme kinetics and inhibition model for biochemistry education and research.

Enzyme kinetics and inhibition studies are crucial in biochemistry education and research. Conventional methods often require expensive equipment and reagents, potentially limiting their accessibility in limited resource settings. Our approach sought to develop a cost-effective experimental design for studying enzyme kinetics and inhibition. Lactase was chosen as a protein model and its activity was investigated by measuring glucose production from lactose hydrolysis. In the study, commercially available lactase pills were used as an enzyme source, while milk was used as a substrate. Instead of scientific equipment, glucometers were used to measure lactase activity. Enzyme kinetics were evaluated using Michaelis-Menten and Lineweaver-Burk plots. In the study, the effects of temperature, pH, and inhibitors were also investigated. The results of our study aligned with established enzyme kinetics theories and previous studies. Lactase showed temperature and pH-dependent activity, with decreased activity observed at both low and high extremes. Results also showed that galactose acts as a competitive inhibitor of lactase. The approach presented here offers a cost-effective procedure for studying enzyme kinetics and inhibition. It can act as a valuable tool for educational purposes and for preliminary research in settings with limited resources.

Prebiotic Strategies to Manage Lactose Intolerance Symptoms.

Lactose intolerance, which affects about 65-75% of the world's population, is caused by a genetic post-weaning deficiency of lactase, the enzyme required to digest the milk sugar lactose, called lactase non-persistence. Symptoms of lactose intolerance include abdominal pain, bloating and diarrhea. Genetic variations, namely lactase persistence, allow some individuals to metabolize lactose effectively post-weaning, a trait thought to be an evolutionary adaptation to dairy consumption. Although lactase non-persistence cannot be altered by diet, prebiotic strategies, including the consumption of galactooligosaccharides (GOSs) and possibly low levels of lactose itself, may shift the microbiome and mitigate symptoms of lactose consumption. This review discusses the etiology of lactose intolerance and the efficacy of prebiotic approaches like GOSs and low-dose lactose in symptom management.

Added Value of 13C Analysis in Breath Tests in H2-Negative Subjects to Diagnose Lactose Malabsorption: A Proof of Concept Study.

INTRODUCTION: Diagnosing lactose malabsorption is usually based on hydrogen excretion in breath after a lactose challenge. However, a proportion of subjects with lactose malabsorption will not present a rise in hydrogen. Measuring excretion of methane or stable isotope labeled 13CO2 after ingestion of 13C-lactose has been proposed to mitigate this problem. OBJECTIVE: The aim of the study was to assess the performance of measuring methane and 13CO2 in individuals with normal hydrogen excretion compared to a genetic lactase non-persistence test. METHODS: Individuals referred for lactose breath testing and healthy controls were included. Participants received 13C-enriched lactose, performed breath testing, and underwent genotyping for a marker of lactase non-persistence (13910C*T). Using genotype as gold standard, the performance of measuring methane and 13CO2 excretion was assessed. RESULTS: 151 subjects participated in the study, 50 of which presented a lactase non-persistent genotype. Of these, 72% were correctly diagnosed through hydrogen excretion of ≥ 20 ppm above baseline. In subjects with normal hydrogen excretion, cumulative 13C excretion had an area under the curve (AUC) of the receiver operating characteristics (ROC) curve of 0.852. Sensitivity was 93% and specificity was 51% for the current cutoff of 14.5%. The optimal cutoff was 12.65% (sensitivity 93%, specificity 70%). The ROC curve of peak methane had an AUC of 0.542 (sensitivity of 14%, specificity of 91% for cutoff ≥ 10 ppm). CONCLUSIONS: In individuals with genetically demonstrated lactase non-persistence and negative hydrogen breath test, the use of 13C-lactose with measurement of 13CO2 excretion and hydrogen is a well-performing test to detect the lactose malabsorption and performs better than methane in our cohort.

Genetically Determined Circulating Lactase/Phlorizin Hydrolase Concentrations and Risk of Colorectal Cancer: A Two-Sample Mendelian Randomization Study.

Previous research has found that milk is associated with a decreased risk of colorectal cancer (CRC). However, it is unclear whether the milk digestion by the enzyme lactase-phlorizin hydrolase (LPH) plays a role in CRC susceptibility. Our study aims to investigate the direct causal relationship of CRC risk with LPH levels by applying a two-sample Mendelian Randomization (MR) strategy. Genetic instruments for LPH were derived from the Fenland Study, and CRC-associated summary statistics for these instruments were extracted from the FinnGen Study, PLCO Atlas Project, and Pan-UK Biobank. Primary MR analyses focused on a cis-variant (rs4988235) for LPH levels, with results integrated via meta-analysis. MR analyses using all variants were also undertaken. This analytical approach was further extended to assess CRC subtypes (colon and rectal). Meta-analysis across the three datasets illustrated an inverse association between genetically predicted LPH levels and CRC risk (OR: 0.92 [95% CI, 0.89-0.95]). Subtype analyses revealed associations of elevated LPH levels with reduced risks for both colon (OR: 0.92 [95% CI, 0.89-0.96]) and rectal cancer (OR: 0.92 [95% CI, 0.87, 0.98]). Consistency was observed across varied analytical methods and datasets. Further exploration is warranted to unveil the underlying mechanisms and validate LPH's potential role in CRC prevention.

Lactose Intolerance versus Cow's Milk Allergy in Infants: A Clinical Dilemma.

Due to its very early introduction, cow's milk is one of the first foods that can cause adverse reactions in human beings. Lactose intolerance (LI) and cow's milk allergy (CMA) are the most common adverse reactions to cow's milk. While LI is due to insufficient small intestinal lactase activity and/or a large quantity of ingested lactose, CMA is an aberrant immune reaction to cow's milk proteins, particularly casein or ß-lactoglobulin. However, the clinical manifestations of LI and CMA, particularly their gastrointestinal signs and symptoms, are very similar, which might lead to misdiagnosis or delayed diagnosis as well as nutritional risks due to inappropriate dietary interventions or unnecessary dietary restriction. Formula-fed infants with LI should be treated with formula with reduced or no lactose, while those with CMA should be treated with formula containing extensive hydrolyzed cow's milk protein or amino acids. This review is therefore written to assist clinicians to better understand the pathophysiologies of LI and CMA as well as to recognize the similarities and differences between clinical manifestations of LI and CMA.

Safety evaluation of the food enzyme ß-galactosidase from the non-genetically modified Papiliotrema terrestris strain AE-BLC.

The food enzyme ß-galactosidase (ß-d-galactoside galactohydrolase; EC 3.2.1.23) is produced with the non-genetically modified Papiliotrema terrestris strain AE-BLC by Amano Enzyme Inc. The food enzyme was considered free from viable cells of the production organism. It is intended to be used in the production of galacto-oligosaccharides (GOS) from lactose. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.441 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1800 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 4082. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Lactase can target cellular differentiation of Acanthamoeba castellanii belonging to the T4 genotype.

The free living Acanthamoeba spp. are ubiquitous amoebae associated with potentially blinding disease known as Acanthamoeba keratitis (AK) and a fatal central nervous system infection granulomatous amoebic encephalitis (GAE). With the inherent ability of cellular differentiation, it can phenotypically transform to a dormant cyst form from an active trophozoite form. Acanthamoeba cysts are highly resistant to therapeutic agents as well as contact lens cleaning solutions. One way to tackle drug resistance against Acanthamoeba is by inhibiting the formation of cysts from trophozoites. The biochemical analysis showed that the major component of Acanthamoeba cyst wall is composed of carbohydrate moieties such as galactose and glucose. The disaccharide of galactose and glucose is lactose. In this study, we analyzed the potential of lactase enzyme to target carbohydrate moieties of cyst walls. Amoebicidal assessment showed that lactase was ineffective against trophozoite of A. castellanii but enhanced amoebicidal effects of chlorhexidine. The lactase enzyme did not show any toxicity against normal human keratinocyte cells (HaCaT) at the tested range. Hence, lactase can be used for further assessment for development of potential therapeutic agents in the management of Acanthamoeba infection as well as formulation of effective contact lens disinfectants.

Lactose Intolerance - Single Nucleotide Polymorphisms and Treatment.

The majority (about 70%) of the world's population suffers from lactose intolerance. Lactose intolerance leads to long-term discomfort when consuming milk and dairy products, and hence, to their avoidance. Consequently, the intake of important nutrients is reduced, which potentially has a negative impact on the overall health. Knowing the condition - lactose intolerance - will prevent people from unnecessarily restricting dairy products in their diets. In this study, lactose synthesis and catabolism in the human body are presented, also the types of lactose intolerance, as well as the methods of diagnosing this condition, are discussed. Special attention is paid to the genetic causes of this discomfort and to the tests that can be performed. Solutions for the treatment of lactose intolerance have also been proposed, both up-to-date and easily applicable, as well as future developments.

This review highlights the lactose pathway ­ from the mammary gland production to recipient gut hydrolysis.Lactose intolerance associated SNPs known so far are presented and discussed.Advice for people with lactose intolerance is presented in the form of possible treatments and healthy feeding behaviors.

Nonallergic Diseases Associated With Foods.

Clinicians are faced with evaluating real and alleged reactions to foods that may be allergic or nonallergic. Pathogenesis, diagnosis, and treatment of various non-IgE-mediated diseases are discussed in this review. These food-related conditions range from mild to severe. Referral for an allergy workup may be pursued despite the lack of IgE-mediated symptoms. Diagnostic testing is available for defined non-IgE-mediated food diseases that are either immunologic or nonimmunologic. These include celiac disease and related disorders, carbohydrate maldigestion, pancreatic insufficiency, and histamine intolerance. In contrast, there is a paucity of definitive studies to prove food intolerance diseases. There are no definitive diagnostic criteria or testing for nonceliac gluten sensitivity. Functional gastrointestinal disorders, such as irritable bowel syndrome, are better stratified diagnostically but still lack reliable testing. Both nonceliac gluten sensitivity and irritable bowel syndrome are linked to dietary triggers including fermentable oligosaccharides, disaccharides, monosaccharides, and polyols. Therefore, dietary alteration alone may be diagnostic and therapeutic when all other conditions are ruled out. These conditions are important considerations when evaluating a patient with history of a food reaction. There is little evidence that foods are causative in other ailments such as acne, migraines, and nasal congestion and hypersecretion.

Changes in gut microbiota and lactose intolerance symptoms before and after daily lactose supplementation in individuals with the lactase nonpersistent genotype.

BACKGROUND: Approximately 70%-100% of the Asian adult population is lactase nonpersistent (LNP). The literature shows that many individuals with the LNP-genotype can consume ≤12 g of lactose without experiencing gastrointestinal discomfort. Repetitive consumption of lactose may reduce intolerance symptoms via adaptation of the gut microbiota. OBJECTIVE: This study aimed to assess the effects of daily consumption of incremental lactose doses on microbiota composition and function, and intolerance symptoms. METHODS: Twenty-five healthy adults of Asian origin, carrying the LNP-genotype were included in this 12-wk before and after intervention trial. Participants consumed gradually increasing lactose doses from 3 to 6 g to 12 g twice daily, each daily dose of 6 g, 12 g, or 24 g being provided for 4 consecutive weeks. Participants handed-in repeated stool samples and underwent a 25 g lactose challenge hydrogen breath test (HBT) before and after the 12-wk intervention. Daily gastrointestinal symptoms and total symptom scores (TSSs) during the lactose challenge were recorded. RESULTS: A significant increase from 5.5% ± 7.6% to 10.4% ± 9.6% was observed in Bifidobacterium relative abundance after the intervention (P = 0.009), accompanied by a 2-fold increase (570 ± 269 U/g; P < 0.001) in fecal ß-galactosidase activity compared with baseline (272 ± 158 U/g). A 1.5-fold decrease (incremental area under the curve; P = 0.01) in expired hydrogen was observed during the second HBT (38 ± 35 ppm·min), compared with the baseline HBT (57 ± 38 ppm·min). There was a nonsignificant decrease in TSS (10.6 ± 8.3 before compared with 8.1 ± 7.2 after intervention; P = 0.09). Daily consumption of lactose was well tolerated, with mild to no gastrointestinal complaints reported during the intervention. CONCLUSIONS: Increased levels of Bifidobacterium indicate an adaptation of the gut microbiota upon repetitive consumption of incremental doses of lactose, which was well tolerated as demonstrated by reduced expired hydrogen concentrations during the second 25-g lactose HBT. Bifidobacteria metabolize lactose without gas production thereby potentially reducing intestinal gas formation in the gut of individuals with the LNP-genotype. This increased lactose tolerance possibly lifts the necessity to remove nutrient-rich dairy foods completely from the diet. The trial is registered at the International Clinical Trials Registry Platform: NL9516. The effect of dietary lactose in lactase nonpersistent individuals on gut microbiota.

Asian Variant Intravascular Lymphoma: A Look at the Laboratory Data of Lactase Dehydrogenase and Soluble Interleukin-2 Receptor.

Asian variant intravascular lymphoma (AIVL) is a rare type of intravascular lymphoma that occurs in Asian populations. Syndrome of inappropriate antidiuretic hormone selection (SIADH) frequently occurs in patients with AIVL. Because it remains difficult to diagnose and has a poor prognosis, markers for early diagnosis are required. Although lactase dehydrogenase (LD) and soluble interleukin-2 receptor (sIL-2R) are diagnostic candidates, these markers do not appear to have been used often in prior studies. We present the case of an 87-year-old Japanese man with AIVL complicated by unexplained SIADH with a complaint of anorexia. Computer tomography showed splenomegaly but no lymphadenopathy. Elevated LD and sIL-2R were detected in the blood. The patient was diagnosed with AIVL through a random skin biopsy and was successfully treated with chemotherapy. When a patient presents with SIADH, we should actively look at or measure blood LD and sIL-2R for early diagnosis of ALVL. Further cases are warranted to determine these observations.

Characterization of an isolated lactase enzyme produced by Bacillus licheniformis ALSZ2 as a potential pharmaceutical supplement for lactose intolerance.

Introduction: Lactose intolerance is a widespread problem that affects people of many different races all over the world. The following pharmacological supplements can improve the lives of those who suffer from this issue. Methods: This work focused on lactase producer isolation and statistical design (Plackett-Burman, and BOX-Behnken) to maximize the effectiveness of environmental factors. A lactase-producing bacterium was chosen from a discovery of 100 strains in soil that had previously been polluted with dairy products. Plackett-Burman investigated fifteen variables. Results: The most critical variables that lead to increased lactase synthesis are glucose, peptone, and magnesium sulfate (MgSO4). The ideal process conditions for the creation of lactase yield among the stated variables were then determined using a BOX-Benken design. To establish a polynomial quadratic relationship between the three variables and lactase activity, the Box-Behnken design level was used. The EXCEL-solver nonlinear optimization technique was used to predict the best form for lactase production. The ideal temperature and pH levels have been determined, both before and after the lactase purification process, to achieve the highest performance of isolated lactase. Conclusion: According to this study, Bacillus licheniformis is a perfect supply of the lactase enzyme (ß -Galactosidase), It can be used as a product to assist people who have health issues due to lactose intolerance.

Genetic and cultural adaptations underlie the establishment of dairy pastoralism in the Tibetan Plateau.

BACKGROUND: Domestication and introduction of dairy animals facilitated the permanent human occupation of the Tibetan Plateau. Yet the history of dairy pastoralism in the Tibetan Plateau remains poorly understood. Little is known how Tibetans adapted to milk and dairy products. RESULTS: We integrated archeological evidence and genetic analysis to show the picture that the dairy ruminants, together with dogs, were introduced from West Eurasia into the Tibetan Plateau since ~ 3600 years ago. The genetic admixture between the exotic and indigenous dogs enriched the candidate lactase persistence (LP) allele 10974A > G of West Eurasian origin in Tibetan dogs. In vitro experiments demonstrate that - 13838G > A functions as a LP allele in Tibetans. Unlike multiple LP alleles presenting selective signatures in West Eurasians and South Asians, the de novo origin of Tibetan-specific LP allele - 13838G > A with low frequency (~ 6-7%) and absence of selection corresponds - 13910C > T in pastoralists across eastern Eurasia steppe. CONCLUSIONS: Results depict a novel scenario of genetic and cultural adaptations to diet and expand current understanding of the establishment of dairy pastoralism in the Tibetan Plateau.

Improving Biohydrogen Production by Dark Fermentation of Milk Processing Wastewater by Physicochemical and Enzymatic Pretreatments.

Biohydrogen is considered an alternative energy reserve. Dark fermentation is one of the important green hydrogen production techniques that utilizes organic waste as raw material. It is a promising bioconversion, easy, not expensive, and cost-effective process. Milk processing wastewater (MPWW) is an organic effluent generated in large volumes on a daily basis and disposed directly into the environment. In this research, the study of biochemical hydrogen potential (BHP) test of MPWW was evaluated and used as substrate (S). A waste sludge was used as an inoculum (I) and source of bacteria. Both substrate and inoculum were analyzed and the study was based mainly on the ratio of volatile solids (VS) of inoculum and substrate subsequently, which was noted as I/S. Different substrate pretreatments were performed: ultrasonic, thermal, chemical, and enzymatic hydrolysis. The I/S ratio impact was investigated and evaluated the hydrogen production improvement. Modified Gompertz and modified Logistic kinetic models were employed for the kinetic modeling of cumulative hydrogen production values. Results show that I/S ratio of 1/4 gVS/gVS resulted from the best hydrogen production of 59.96 mL during 30 days of MPWW fermentation without pretreatment. It was also shown that all the adopted pretreatments enhanced hydrogen production, whereas ultrasonic pretreatment for 5 min increased the production by only 14.84%. Heat pretreatment was more efficient, where the hydrogen production increased from 60 to 162 mL (170% of improvement) using heat shock at 90 °C for 30 min. The impact of chemical pretreatment was different from a reagent to another. Pretreatment using calcium hydroxide resulted in the biggest hydrogen production of 165.3 mL (175.5%) compared to the other chemical pretreatments. However, the best hydrogen production was given by the biological pretreatment using enzymatic hydrolysis (Lactase) resulting in 254 mL of hydrogen production, which is equivalent to 323.62% of production improvement. Modified Gompertz and Logistic kinetic models fitted well with experimental data. Thus, the enzymatic hydrolysis of MPWW proved to be a promising technique for biohydrogen production enhancement.

Advances in Low-Lactose/Lactose-Free Dairy Products and Their Production.

With increasing health awareness worldwide, lactose intolerance has become a major concern of consumers, creating new market opportunities for low-lactose/lactose-free dairy foods. In recent years, through innovating processes and technologies, dairy manufacturers have significantly improved the variety, and functional and sensory qualities of low-lactose and lactose-free dairy products. Based on this, this paper first covers the pathology and epidemiology of lactose intolerance and market trends. Then, we focus on current advantages and disadvantages of different lactose hydrolysis technologies and improvements in these technologies to enhance nutritional value, and functional, sensory, and quality properties of lactose-free dairy products. We found that more and more cutting-edge technologies are being applied to the production of lactose-free dairy products, and that these technologies greatly improve the quality and production efficiency of lactose-free dairy products. Hopefully, our review can provide a theoretical basis for the marketing expansion and consumption guidance for low-lactose/lactose-free dairy products.

The cow's milk allergy that wasn't: allergy to supplemental oral lactase enzyme.

BACKGROUND: Allergy to supplemental lactase is sparsely reported in the literature with only one prior case of anaphylaxis documented [2]. Reactions to this agent can occur following cow's milk ingestion which could lead to an erroneous diagnosis of cow's milk allergy in the absence of another explanation. CASE PRESENTATION: Our patient, a 48-year-old male with eczema, exercise-induced asthma and rhinoconjunctivitis, presented with four episodes of systemic reactions characterized by mucosal swelling and asthma symptoms following ice-cream exposure. It was later recognized that he had been taking a lactase enzyme supplement just prior to all of his reactions. Epicutaneous testing was strongly positive to a saline slurry of the lactase supplement he had been using. The patient has been avoiding supplemental lactase since with no subsequent reactions. DISCUSSION: Our patient was diagnosed with an allergy to supplemental lactase enzyme on the basis of convincing Immunoglobulin E (IgE) mediated symptoms and positive skin testing. He continued to eat cow's milk products, ruling out an IgE-mediated allergy to cow's milk. In the literature, there is one prior case of anaphylaxis documented. Another case of localized oropharyngeal symptoms described in the literature was thought to be a form of oral allergy syndrome as the patient had positive epicutaneous testing to Aspergillus oryzae-derived lactase as well as Aspergillus species. Occupational sensitization, rhinitis/asthma, and protein contact dermatitis have also been detailed in the literature. Although rare, this case highlights the importance of a thorough history of over-the-counter supplements when assessing a patient with features of anaphylaxis.