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1.
Production of HMOs using microbial hosts - from cell engineering to large scale production.
Bych, K, Mikš, MH, Johanson, T, Hederos, MJ, Vigsnæs, LK, Becker, P
Current opinion in biotechnology. 2019;:130-137
Abstract
Human Milk Oligosaccharides (HMOs) constitute an important, highly abundant part of mothers' milk delivering many health benefits to the neonate. Until recently, limited availability of HMOs has prevented their use in infant nutrition and impeded research into their biological effects. The shift from chemical synthesis to biotechnological manufacturing has made them accessible in quantities and at prices that are within reach for commercial applications, including infant formula. It accelerated the studies in the field of pre-clinical and clinical HMO biology. This review gives a short overview of HMO manufacturing from the design and optimization of the microbial cell factory and the production of HMOs in the industrial fermentation process to the purification in the downstream process necessary to obtain a final product. Moreover, the transition from chemistry to biotechnology and the current regulatory landscape and commercialization progress are briefly reviewed.
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2.
Synthesis of Human Milk Oligosaccharides: Protein Engineering Strategies for Improved Enzymatic Transglycosylation.
Zeuner, B, Teze, D, Muschiol, J, Meyer, AS
Molecules (Basel, Switzerland). 2019;(11)
Abstract
Human milk oligosaccharides (HMOs) signify a unique group of oligosaccharides in breast milk, which is of major importance for infant health and development. The functional benefits of HMOs create an enormous impetus for biosynthetic production of HMOs for use as additives in infant formula and other products. HMO molecules can be synthesized chemically, via fermentation, and by enzymatic synthesis. This treatise discusses these different techniques, with particular focus on harnessing enzymes for controlled enzymatic synthesis of HMO molecules. In order to foster precise and high-yield enzymatic synthesis, several novel protein engineering approaches have been reported, mainly concerning changing glycoside hydrolases to catalyze relevant transglycosylations. The protein engineering strategies for these enzymes range from rationally modifying specific catalytic residues, over targeted subsite -1 mutations, to unique and novel transplantations of designed peptide sequences near the active site, so-called loop engineering. These strategies have proven useful to foster enhanced transglycosylation to promote different types of HMO synthesis reactions. The rationale of subsite -1 modification, acceptor binding site matching, and loop engineering, including changes that may alter the spatial arrangement of water in the enzyme active site region, may prove useful for novel enzyme-catalyzed carbohydrate design in general.
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3.
Association of Maternal Secretor Status and Human Milk Oligosaccharides With Milk Microbiota: An Observational Pilot Study.
Cabrera-Rubio, R, Kunz, C, Rudloff, S, García-Mantrana, I, Crehuá-Gaudiza, E, Martínez-Costa, C, Collado, MC
Journal of pediatric gastroenterology and nutrition. 2019;(2):256-263
Abstract
BACKGROUND AND OBJECTIVES Breast milk contains several bioactive factors including human milk oligosaccharides (HMOs) and microbes that shape the infant gut microbiota. HMO profile is determined by secretor status; however, their influence on milk microbiota is still uncovered. This study is aimed to determine the impact of the FUT2 genotype on the milk microbiota during the first month of lactation and the association with HMO. METHODS Milk microbiota from 25 healthy lactating women was determined by quantitative polymerase chain reaction and 16S gene pyrosequencing. Secretor genotype was obtained by polymerase chain reaction-random fragment length polymorphisms and by HMO identification and quantification. RESULTS The most abundant bacteria were Staphylococcus and Streptococcus, followed by Enterobacteriaceae-related bacteria. The predominant HMO in secretor milk samples were 2'FL and lacto-N-fucopentaose I, whereas non-secretor milk was characterized by lacto-N-fucopentaose II and lacto-N-difucohexaose II. Differences in microbiota composition and quantity were found depending on secretor/non-secretor status. Lactobacillus spp, Enterococcus spp, and Streptococcus spp were lower in non-secretor than in secretor samples. Bifidobacterium genus and species were less prevalent in non-secretor samples. Despite no differences on diversity and richness, non-secretor samples had lower Actinobacteria and higher relative abundance of Enterobacteriaceae, Lactobacillaceae, and Staphylococcaceae. CONCLUSIONS Maternal secretor status is associated with the human milk microbiota composition and is maintained during the first 4 weeks. Specific associations between milk microbiota, HMO, and secretor status were observed, although the potential biological impact on the neonate remains elusive. Future studies are needed to reveal the early nutrition influence on the reduction of risk of disease.
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4.
Exit Gluten-Free and Enter Low FODMAPs: A Novel Dietary Strategy to Reduce Gastrointestinal Symptoms in Athletes.
Lis, DM
Sports medicine (Auckland, N.Z.). 2019;(Suppl 1):87-97
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Abstract
Exercise-associated physiological disturbances alter gastrointestinal function and integrity. These alterations may increase susceptibility to dietary triggers, namely gluten and a family of short-chain carbohydrates known as FODMAPs (fermentable oligo-, di-, monosaccharides and polyols). A recent surge in the popularity of gluten-free diets (GFDs) among athletes without celiac disease has been exacerbated by unsubstantiated commercial health claims and high-profile athletes citing this diet to be the secret to their success. Up to 41% of athletes at least partially adhere to a GFD diet, with the belief that gluten avoidance improves exercise performance and parameters influencing performance, particularly gastrointestinal symptoms (GIS). In contrast to these beliefs, seminal work investigating the effects of a GFD in athletes without celiac disease has demonstrated no beneficial effect of a GFD versus a gluten-containing diet on performance, gastrointestinal health, inflammation, or perceptual wellbeing. Interestingly, the subsequent reduction in FODMAPs concurrent with the elimination of gluten-containing grains may actually be the factors affecting GIS improvement, not gluten. Pre-existent in the gastrointestinal tract or ingested during exercise, the osmotic and gas-producing effects of variably absorbed FODMAPs may trigger or increase the magnitude of exercise-associated GIS. Research using FODMAP reduction to address gastrointestinal issues in clinically healthy athletes is emerging as a promising strategy to reduce exercise-associated GIS. Applied research and practitioners merging clinical and sports nutrition methods will be essential for the effective use of a low FODMAP approach to tackle the multifactorial nature of gastrointestinal disturbances in athletes.
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Non-digestible carbohydrates in infant formula as substitution for human milk oligosaccharide functions: Effects on microbiota and gut maturation.
Akkerman, R, Faas, MM, de Vos, P
Critical reviews in food science and nutrition. 2019;(9):1486-1497
Abstract
Human milk (HM) is the golden standard for nutrition of newborn infants. Human milk oligosaccharides (HMOs) are abundantly present in HM and exert multiple beneficial functions, such as support of colonization of the gut microbiota, reduction of pathogenic infections and support of immune development. HMO-composition is during lactation continuously adapted by the mother to accommodate the needs of the neonate. Unfortunately, for many valid reasons not all neonates can be fed with HM and are either totally or partly fed with cow-milk derived infant formulas, which do not contain HMOs. These cow-milk formulas are supplemented with non-digestible carbohydrates (NDCs) that have functional effects similar to that of some HMOs, since production of synthetic HMOs is challenging and still very expensive. However, NDCs cannot substitute all HMO functions. More efficacious NDCs may be developed and customized for specific groups of neonates such as pre-matures and allergy prone infants. Here current knowledge of HMO functions in the neonate in view of possible replacement of HMOs by NDCs in infant formulas is reviewed. Furthermore, methods to expedite identification of suitable NDCs and structure/function relationships are reviewed as in vivo studies in babies are impossible.
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Effects of oligosaccharide-sialic acid (OS) compound on maternal-newborn gut microbiome, glucose metabolism and systematic immunity in pregnancy: protocol for a randomised controlled study.
Wang, S, Peng, R, Qin, S, Liu, Y, Yang, H, Ma, J
BMJ open. 2019;(9):e026583
Abstract
INTRODUCTION The gut microbiota participates in multiple human biological processes, including metabolism and immune responses. During pregnancy, the dynamics of gut microbiota is involved in physiological adaptation. The disturbed profile of microbiome is associated with maternal complications, such as gestational diabetes mellitus (GDM), which further transfers to the offspring and influence their metabolic and immunological functions in the long term. Prebiotics targeting the gut microbiota and modulating metabolic and immune functions have been shown to be effective in non-pregnant populations with metabolic syndrome. Hence, we propose the use of a prebiotic supplement, oligosaccharide-sialic acid (OS) from the first trimester until delivery in pregnant women, can benefit maternal/new-born gut microbiome, glucose metabolism and innate immunity. METHODS AND ANALYSIS In this prospective double-blinded randomised clinical trial, recruited singleton pregnancies will be stratified by body mass index (BMI) and randomly assigned to consume the OS preparation or placebo daily from the first trimester. At seven later time points (before and after recruitment in the first trimester, in the middle and third trimesters, before delivery, at birth and 42 days postpartum), compliance will be evaluated and/or biological samples will be collected. Along with maternal clinical information, questionnaires on lifestyle and infant development will be recorded. The primary outcomes are the effect of OS on the maternal-offspring gut microbiome and GDM incidence. The secondary outcomes are maternal glycolipid biochemical parameters, cytokine profiles, weight gain during pregnancy and infant morbidities, growth and development. The study aims to validate the effects of OS on reducing maternal morbidity within different BMI groups. The multiple dimensional dataset generated from the study includes clinical and lifestyle-related information, various biological markers and associated protective or risk factors for morbidity and prognosis. An extended follow-up through 42 days after birth could further explore the intrauterine influence on the long-term health of offspring. ETHICS AND DISSEMINATION This protocol has been approved by Peking University First Hospital, National Unit of Clinical Trial Ethics Committee (reference number: 164). The results are expected to be published in scientific manuscripts by 2021. TRIAL REGISTRATION NUMBER ChiCTR1800017192.
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Varied Pathways of Infant Gut-Associated Bifidobacterium to Assimilate Human Milk Oligosaccharides: Prevalence of the Gene Set and Its Correlation with Bifidobacteria-Rich Microbiota Formation.
Sakanaka, M, Gotoh, A, Yoshida, K, Odamaki, T, Koguchi, H, Xiao, JZ, Kitaoka, M, Katayama, T
Nutrients. 2019;(1)
Abstract
The infant's gut microbiome is generally rich in the Bifidobacterium genus. The mother's milk contains natural prebiotics, called human milk oligosaccharides (HMOs), as the third most abundant solid component after lactose and lipids, and of the different gut microbes, infant gut-associated bifidobacteria are the most efficient in assimilating HMOs. Indeed, the fecal concentration of HMOs was found to be negatively correlated with the fecal abundance of Bifidobacterium in infants. Given these results, two HMO molecules, 2'-fucosyllactose and lacto-N-neotetraose, have recently been industrialized to fortify formula milk. As of now, however, our knowledge about the HMO consumption pathways in infant gut-associated bifidobacteria is still incomplete. The recent studies indicate that HMO assimilation abilities significantly vary among different Bifidobacterium species and strains. Therefore, to truly maximize the effects of prebiotic and probiotic supplementation in commercialized formula, we need to understand HMO consumption behaviors of bifidobacteria in more detail. In this review, we summarized how different Bifidobacterium species/strains are equipped with varied gene sets required for HMO assimilation. We then examined the correlation between the abundance of the HMO-related genes and bifidobacteria-rich microbiota formation in the infant gut through data mining analysis of a deposited fecal microbiome shotgun sequencing dataset. Finally, we shortly described future perspectives on HMO-related studies.
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Histological improvement of non-alcoholic steatohepatitis with a prebiotic: a pilot clinical trial.
Bomhof, MR, Parnell, JA, Ramay, HR, Crotty, P, Rioux, KP, Probert, CS, Jayakumar, S, Raman, M, Reimer, RA
European journal of nutrition. 2019;(4):1735-1745
Abstract
PURPOSE In obesity and diabetes the liver is highly susceptible to abnormal uptake and storage of fat. In certain individuals hepatic steatosis predisposes to the development of non-alcoholic steatohepatitis (NASH), a disease marked by hepatic inflammation and fibrosis. Although the precise pathophysiology of NASH is unknown, it is believed that the gut microbiota-liver axis influences the development of this disease. With few treatment strategies available for NASH, exploration of gut microbiota-targeted interventions is warranted. We investigated the therapeutic potential of a prebiotic supplement to improve histological parameters of NASH. METHODS In a placebo-controlled, randomized pilot trial, 14 individuals with liver-biopsy-confirmed NASH [non-alcoholic fatty liver activity score (NAS) ≥ 5] were randomized to receive oligofructose (8 g/day for 12 weeks followed by 16 g/day for 24 weeks) or isocaloric placebo for 9 months. The primary outcome measure was the change in liver biopsy NAS score and the secondary outcomes included changes in body weight, body composition, glucose tolerance, inflammatory markers, and gut microbiota. RESULTS Independent of weight loss, oligofructose improved liver steatosis relative to placebo and improved overall NAS score (P = 0.016). Bifidobacterium was enhanced by oligofructose, whereas bacteria within Clostridium cluster XI and I were reduced with oligofructose (P < 0.05). There were no adverse side effects that deterred individuals from consuming oligofructose for treatment of this disease. CONCLUSIONS Independent of other lifestyle changes, prebiotic supplementation reduced histologically-confirmed steatosis in patients with NASH. Larger follow-up studies are warranted. CLINICAL TRIAL This trial was registered at Clinicaltrials.com as NCT03184376.
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Downscale fermentation for xylooligosaccharides production by recombinant Bacillus subtilis 3610.
Amorim, C, Silvério, SC, Gonçalves, RFS, Pinheiro, AC, Silva, S, Coelho, E, Coimbra, MA, Prather, KLJ, Rodrigues, LR
Carbohydrate polymers. 2019;:176-183
Abstract
The global demand of prebiotics such as xylooligosaccharides (XOS) has been growing over the years, motivating the search for different production processes with increased efficiency. In this study, a cloned Bacillus subtilis 3610, containing the xylanase gene xyn2 of Trichoderma reesei coupled with an endogenous secretion tag, was selected for XOS production through direct fermentation of beechwood xylan. A mixture of XOS with a degree of polymerization ranging from 4 to 6 was obtained, presenting high stability after a static in vitro digestion (98.5 ± 0.2%). The maximum production yield expressed as total XOS per amount of xylan (306 ± 4 mg/g) was achieved after 8 h of fermentation operating under one-time impulse fed-batch. The optimal conditions found were pH 6.0 and 42.5 °C, using 2.5 g/L of initial concentration of xylan increased up to 5.0 g/L at 3 h. Xylopentaose was the major oligosaccharide produced, representing 47% of the total production yield.
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Lignocellulose derived functional oligosaccharides: production, properties, and health benefits.
Bhatia, L, Sharma, A, Bachheti, RK, Chandel, AK
Preparative biochemistry & biotechnology. 2019;(8):744-758
Abstract
Lignocellulosic biomass (LB) is the renewable feedstock for the production of fuel/energy, feed/food, chemicals, and materials. LB could also be the versatile source of the functional oligosaccharides, which are non-digestible food ingredients having numerous applications in food, cosmetics, pharmaceutical industries, and others. The burgeoning functional food demand is expected to be more than US$440 billion in 2022. Because of higher stability at low pH and high temperature, oligosaccharides stimulate the growth of prebiotic bifidobacteria and lactic acid bacteria. Xylooligosaccharides (XOS) are major constituents of oligosaccharides consisting of 2-7 xylose monomeric units linked via β-(1,4)-linkages. XOS can be obtained from various agro-residues by thermochemical pretreatment, enzymatic or chemoenzymatic methods. While thermochemical methods are fast, reproducible, enzymatic methods are substrate specific, costly, and produce minimum side products. Enzymatic methods are preferred for the production of food grade and pharmaceutically important oligosaccharides. XOS are potent prebiotics having antioxidant properties and enhance the bio-adsorption of calcium and improving bowel functions, etc. LB can cater to the increasing demand of oligosaccharides because of their foreseeable amount and the advancements in technology to recover oligosaccharides. This paper summarizes the methods for oligosaccharides production from LB, classification, and benefits of oligosaccharides on human health.