A 21-day safety evaluation of biotechnologically produced 3-fucosyllactose (3-FL) in neonatal farm piglets to support use in infant formulas.

3-Fucosyllactose (3-FL) is one of the most abundant fucosylated oligosaccharides in human breast milk and is an approved infant formula ingredient world-wide. 3-FL functions as a prebiotic to promote early microbial colonization of the gut, increase pathogen resistance and modulate immune responses. To investigate safety and potential gut microbiota effects, 3-FL was fed for 21-days to farm piglets beginning on Postnatal Day (PND) 2. Fructooligosaccharide (FOS), an approved infant formula ingredient, was used as a reference control. Standard toxicological endpoints were evaluated, and the gut microbiota were assessed. Neither 3-FL (245.77 and 489.72 mg/kg/day for males and 246.57 and 494.18 mg/kg/day for females) nor FOS (489.44 and 496.33 mg/kg/day males and females, respectively) produced any adverse differences in growth, food intake or efficiency, clinical observations, or clinical or anatomic pathology changes. Differences in the gut microbiota after 3-FL consumption (versus control and FOS groups) included the absence of Bifidobacterium species from the piglets, enrichment of Prevotellamassilia timonensis, Blautia species, Mediterranea massiliensis, Lachnospiraceae incertae sedis, and Eubacterium coprostanoligens and lower relative abundance of Allisonella histaminiformans and Roseburia inulinivorans. This study further supports the safe use of 3-FL produced using biotechnology as a nutritional ingredient in foods.

The Effect of Human Milk Oligosaccharides and Bifidobacterium longum subspecies infantis Bi-26 on Simulated Infant Gut Microbiome and Metabolites.

Human milk oligosaccharides (HMOs) shape the developing infant gut microbiota. In this study, a semi-continuous colon simulator was used to evaluate the effect of 2 HMOs-2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL)-on the composition of infant faecal microbiota and microbial metabolites. The simulations were performed with and without a probiotic Bifidobacterium longum subspecies infantis Bi-26 (Bi-26) and compared with a control that lacked an additional carbon source. The treatments with HMOs decreased α-diversity and increased Bifidobacterium species versus the control, but the Bifidobacterium species differed between simulations. The levels of acetic acid and the sum of all short-chain fatty acids (SCFAs) trended toward an increase with 2'-FL, as did lactic acid with 2'-FL and 3-FL, compared with control. A clear correlation was seen between the consumption of HMOs and the increase in SCFAs (-0.72) and SCFAs + lactic acid (-0.77), whereas the correlation between HMO consumption and higher total bifidobacterial numbers was moderate (-0.46). Bi-26 decreased propionic acid levels with 2'-FL. In conclusion, whereas infant faecal microbiota varied between infant donors, the addition of 2'-FL and 3-FL, alone or in combination, increased the relative abundance and numbers Bifidobacterium species in the semi-continuous colon simulation model, correlating with the production of microbial metabolites. These findings may suggest that HMOs and probiotics benefit the developing infant gut microbiota.

Influence of 2'-Fucosyllactose and Bifidobacterium longum Subspecies infantis Supplementation on Cognitive and Structural Brain Development in Young Pigs.

Development of the gut-brain axis during early-life is an important contributor of brain structural and functional development. Human milk oligosaccharides and gut microbiota have potential beneficial effects on various aspects of development; however, the effects of 2'-fucosyllactose (2'-FL) and Bifidobacterium longum subsp. infantis Bi-26 (Bi-26) administration during infancy separately and combined are still not clear. Therefore, we investigated the effects of early administration of dietary 2'-FL and Bi-26 on brain structural and functional development in the young pig. From postnatal day (PND) 2-34 or 35, fifty-two intact male pigs were randomly assigned to treatment groups in a 2 × 2 factorial arrangement and provided ad libitum access to a nutritionally adequate milk replacer without or with 1.0 g of 2'-FL/L of reconstituted liquid. Pigs within each diet group were further stratified to receive a daily oral dose of glycerol stock without or with Bi-26 (109 CFU). Pigs were subjected to the novel object recognition (NOR) task from PND 27-31 to assess recognition memory and subsequently underwent magnetic resonance imaging procedures at PND 32 or 33 to assess brain macrostructure and microstructure. Pigs that received Bi-26 had smaller absolute brain volumes for 9 of 27 brain regions of interest, and smaller relative volumes for 2 regions associated with kinesthesia (P < 0.05). Synbiotic administration of 2'-FL and Bi-26 elicited interactive effects (P < 0.05) on several microstructural brain components, where dual supplementation negated the effects of each test article alone. Behavioral outcomes indicated that pigs did not express novelty preference, regardless of treatment group, demonstrating no effects of 2'-FL and Bi-26 on recognition memory when supplemented alone or in combination. Interactive effects (P < 0.05) were observed for the number of all object visits, latency to the first object visit, and number of familiar object visits. Pigs that did not receive Bi-26 supplementation exhibited less time interacting with the familiar object in total (P = 0.002) and on average (P = 0.005). In conclusion, supplementation of 2'-FL and/or Bi-26 elicited some alterations in object exploratory behaviors and macro/micro-structures of the brain, but changes in recognition memory were not observed. Specifically in brain microstructure, synbiotic administration of 2'-FL and Bi-26 appeared to negate effects observed when each dietary article was supplemented separately.

Regulation of hBD-2, hBD-3, hCAP18/LL37, and Proinflammatory Cytokine Secretion by Human Milk Oligosaccharides in an Organotypic Oral Mucosal Model.

Human milk oligosaccharides (HMOs), the third largest solid fraction in human milk, can modulate inflammation through Toll-like receptor signaling, but little is known about their immunomodulatory potential in the oral cavity. In this study, we determined whether the HMOs 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL) regulate human-beta defensin (hBD)-2 and -3, cathelicidin (hCAP18/LL-37), and cytokine responses in human gingival cells using a three-dimensional oral mucosal culture model. The model was incubated with 0.1% or 1% 2'-FL and 3-FL, alone and in combination, for 5 or 24 h, and hBD-2, hBD-3, and hCAP18/LL-37 were analyzed by immunohistochemistry. The expression profiles of interleukin (IL)-1, IL-1RA, IL-8, and monocyte chemoattractant protein (MCP)-1 were determined by LUMINEX immunoassay. The combination of 1% 2'-FL and 1% 3-FL, and 1% 3-FL alone, for 24 h upregulated hBD-2 protein expression significantly (p < 0.001 and p = 0.016, respectively). No changes in the other antimicrobial peptides or proinflammatory cytokines were observed. Thus, 3-FL, alone and in combination with 2'-FL, stimulates oral mucosal secretion of hBD-2, without effecting a proinflammatory response when studied in an oral mucosal culture model.

Selective Utilization of the Human Milk Oligosaccharides 2'-Fucosyllactose, 3-Fucosyllactose, and Difucosyllactose by Various Probiotic and Pathogenic Bacteria.

Prebiotic human milk oligosaccharides (HMOs) are found in human milk, which are not digested by infants but are metabolized by beneficial gut bacteria. We determined the ability of 57 bacterial strains within the Family Lactobacillaceae and genera Bifidobacterium and Bacteroides and potentially pathogenic bacteria to ferment the HMOs 2'-fucosyllactose, 3-fucosyllactose, and difucosyllactose. In addition, prebiotic galacto-oligosaccharides (GOS), lactose, fucose, and glucose were evaluated as carbon sources for these bacterial strains. Bacterial growth was monitored using the automatic Bioscreen C system. Only certain bifidobacteria, such as Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum, as well as Bacteroides fragilis, Bacteroides vulgatus, and Bacteroides thetaiotaomicron utilized the studied HMOs as their sole carbon source, whereas almost all studied bacterial strains were able to utilize GOS, lactose, and glucose. The selectivity in utilization of HMOs by only certain bacteria can be advantageous by promoting beneficial microbes but not supporting the harmful pathogens in contrast to other less selective prebiotics.

Evaluation of 2'-Fucosyllactose and Bifidobacterium longum Subspecies infantis on Growth, Organ Weights, and Intestinal Development of Piglets.

Human milk is rich in oligosaccharides that influence intestinal development and serve as prebiotics for the infant gut microbiota. Probiotics and 2'-fucosyllactose (2'-FL) added individually to infant formula have been shown to influence infant development, but less is known about the effects of their synbiotic administration. Herein, the impact of formula supplementation with 2'-fucosyllactose (2'-FL) and Bifidobacterium longum subsp. infantis Bi-26 (Bi-26), or 2'-FL + Bi-26 on weight gain, organ weights, and intestinal development in piglets was investigated. Two-day-old piglets (n = 53) were randomized in a 2 × 2 design to be fed a commercial milk replacer ad libitum without (CON) or with 1.0 g/L 2'-FL. Piglets in each diet were further randomized to receive either glycerol stock alone or Bi-26 (109 CFU) orally once daily. Body weights and food intake were monitored from postnatal day (PND) 2 to 33/34. On PND 34/35, animals were euthanized and intestine, liver and brain weights were assessed. Intestinal samples were collected for morphological analyses and measurement of disaccharidase activity. Dry matter of cecum and colon contents and Bifidobacterium longum subsp. infantis abundance by RT-PCR were also measured. All diets were well tolerated, and formula intake did not differ among the treatment groups. Daily body weights were affected by 2'-FL, Bi-26, and day, but no interaction was observed. There was a trend (p = 0.075) for greater total body weight gain in CON versus all other groups. Jejunal and ascending colon histomorphology were unaffected by treatment; however, there were main effects of 2'-FL to increase (p = 0.040) and Bi-26 to decrease (p = 0.001) ileal crypt depth. The addition of 2'-FL and/or Bi-26 to milk replacer supported piglet growth with no detrimental effects on body and organ weights, or intestinal structure and function.

Effect of Phytase on in Vitro Hydrolysis of Phytate and the Formation of myo-Inositol Phosphate Esters in Various Feed Materials.

Phytase is commonly used as a feed enzyme in monogastric animals to increase the bioavailability of phytate phosphorus and other nutrients. The accumulation of myo-inositol phosphate intermediates during phytate degradation in various segments of the gastrointestinal tract (GIT) is poorly understood. The aim of this study was to determine the efficacy of Buttiauxella spp. phytase in degrading the phytate in corn, soybean meal, and complete corn-soybean meal diet to myo-inositol phosphate esters (IP1-IP5) and completely dephosphorylated myo-inositol rings using an in vitro model of the poultry upper GIT. Our results show that the phytase hydrolyzes phytate efficiently to small IP esters, whereas the myo-inositol level remains constant between control and phytase treatments. Although the in vitro digestion model does not incorporate all factors that govern phytate hydrolysis, it is a valuable tool for evaluating phytase efficacy at various enzyme doses and with different feed ingredients.

The effect of 2'-fucosyllactose on simulated infant gut microbiome and metabolites; a pilot study in comparison to GOS and lactose.

Human milk oligosaccharides (HMOs) shape gut microbiota during infancy by acting as fermentable energy source. Using a semi-continuous colon simulator, effect of an HMO, 2'-fucosyllactose (2'-FL), on composition of the infant microbiota and microbial metabolites was evaluated in comparison to galacto-oligosaccharide (GOS) and lactose and control without additional carbon source. Data was analysed according to faecal sample donor feeding type: breast-fed (BF) or formula-fed (FF), and to rate of 2'-FL fermentation: fast or slow. Variation was found between the simulations in the ability to utilise 2'-FL. The predominant phyla regulated by 2'-FL, GOS and lactose were significant increase in Firmicutes, numerical in Actinobacteria, and numerical decrease in Proteobacteria compared to control. Verrucomicrobia increased in FF accounted for Akkermansia, whereas in fast-fermenting simulations Actinobacteria increased with trend for higher Bifidobacterium, and Proteobacteria decrease accounted for Enterobacteriaceae. Short-chain fatty acids and lactic acid with 2'-FL were produced in intermediate levels being between ones generated by the control and GOS or lactose. In 2'-FL fast-fermenting group, acetic acid specifically increased with 2'-FL, whereas lactose and GOS also increased lactic acid. The results highlight specificity of 2'-FL as energy source for only certain microbes over GOS and lactose in the simulated gut model.

Novel Genes and Metabolite Trends in Bifidobacterium longum subsp. infantis Bi-26 Metabolism of Human Milk Oligosaccharide 2'-fucosyllactose.

Human milk oligosaccharides (HMOs) function as prebiotics for beneficial bacteria in the developing gut, often dominated by Bifidobacterium spp. To understand the relationship between bifidobacteria utilizing HMOs and how the metabolites that are produced could affect the host, we analyzed the metabolism of HMO 2'-fucosyllactose (2'-FL) in Bifidobacterium longum subsp. infantis Bi-26. RNA-seq and metabolite analysis (NMR/GCMS) was performed on samples at early (A600 = 0.25), mid-log (0.5-0.7) and late-log phases (1.0-2.0) of growth. Transcriptomic analysis revealed many gene clusters including three novel ABC-type sugar transport clusters to be upregulated in Bi-26 involved in processing of 2'-FL along with metabolism of its monomers glucose, fucose and galactose. Metabolite data confirmed the production of formate, acetate, 1,2-propanediol, lactate and cleaving of fucose from 2'-FL. The formation of acetate, formate, and lactate showed how the cell uses metabolites during fermentation to produce higher levels of ATP (mid-log compared to other stages) or generate cofactors to balance redox. We concluded that 2'-FL metabolism is a complex process involving multiple gene clusters, that produce a more diverse metabolite profile compared to lactose. These results provide valuable insight on the mode-of-action of 2'-FL utilization by Bifidobacterium longum subsp. infantis Bi-26.

SUMO-1 regulates body weight and adipogenesis via PPARγ in male and female mice.

Properly functioning adipose tissue is essential for normal insulin sensitivity of the body. When mice are kept on high-fat diet (HFD), adipose tissue expands, adipocytes increase in size and number, and the mice become obese. Many of these changes are mediated by the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ), the activity of which is regulated by multiple posttranslational modifications, including SUMOylation. To address the role of small ubiquitin-like modifier-1 (SUMO-1) in PPARγ function in vivo, particularly in fat cell biology, we subjected Sumo1-knockout mice to HFD. Sumo1-null mice gained less weight and had smaller and fewer adipocytes in their gonadal fat tissue on HFD, but their glucose tolerance was similar to that of wild-type littermates. Adipogenesis was impaired in Sumo1-null cells, and expression of PPARγ target genes was attenuated. In addition, both Sumo1-null cells and Sumo1-null mice responded less efficiently to rosiglitazone, a PPARγ agonist. These findings indicate that SUMO-1 is important also for transcriptional activation by the PPARγ signaling pathway and not only for trans-repressive functions of PPARγ as previously reported.

Transcriptional activity of estrogen-related receptor γ (ERRγ) is stimulated by the phytoestrogen equol.

Estrogen-related receptor γ (ERRγ) is an orphan nuclear receptor lacking identified natural ligands. The synthetic estrogen receptor ligands 4-hydroxytamoxifen and diethylstilbestrol have, however, been shown to bind to and abolish the constitutive transcriptional activity of ERRγ. Certain phytoestrogens were recently reported to act as agonists of the related ERRα. We investigated whether phytoestrogens also modulated the transcriptional activity of ERRγ. We analyzed a selection of phytoestrogens for their potential agonistic or antagonistic activity on ERRγ. In transiently transfected PC-3 and U2-OS cells equol stimulated the transcriptional activity of ERRγ and enhanced its interaction with the coactivator GRIP1. The agonistic effect of equol was abolished by 4-hydroxytamoxifen. Equol induced a conformational change in the ERRγ ligand-binding domain. Based on structural models of the ERRγ ligand-binding domain, we were able to introduce mutations that modulated the agonistic potential of equol. Finally, equol enhanced the growth inhibitory effect of ERRγ on the prostate cancer PC-3 cells. In conclusion, we have demonstrated that the phytoestrogen equol acts as an ERRγ agonist.

Is longer waiting time for total knee replacement associated with health outcomes and medication costs? Randomized clinical trial.

BACKGROUND: The aim of this prospective randomized study was to evaluate the effect of waiting time (WT) on health-related quality of life (HRQoL), knee pain and physical function, and the use and costs of medication of patients awaiting total knee replacement. METHODS: When placed on the waiting list, 438 patients were randomized into a short waiting time (SWT ≤ 3 months) or a nonfixed waiting time (NFWT > 3 months) group. HRQoL was measured by the 15D, and pain and physical function by modified Knee Society Clinical Rating System at baseline, admission, and 3 and 12 months postoperatively. The costs of medication due to osteoarthritis were calculated at the same measurement points. All analyses were performed using the intention-to-treat principle. RESULTS: The mean WT was 94 and 239 days in the SWT and NFWT groups, respectively. Apart from higher weekly cost of medication in the SWT group at admission and better HRQoL in the NFWT group 1 year postoperatively, there were no statistically significant differences between the groups in other outcomes during the follow-up. CONCLUSION: Those in the SWT group had higher weekly costs of medication at admission, and reached better HRQoL 3 months earlier than those in the NFWT group, but the latter had better HRQoL after operation. Otherwise, the length of WT was not associated with different health and HRQoL outcomes in the groups.

The effect of waiting time on health-related quality of life, pain, and physical function in patients awaiting primary total hip replacement: a randomized controlled trial.

OBJECTIVE: This prospective randomized study assessed the effect of waiting time (WT) on health outcomes in Finnish patients admitted to hospital for primary total hip replacement (THR). METHODS: A total of 395 consecutive patients with a need for a primary THR because of osteoarthritis and who were placed on the waiting list between August 2002 and November 2003. After placement on the waiting list, the patients were randomly assigned to a short WT (

Is longer waiting time associated with health and social services utilization before treatment? A randomized study.

OBJECTIVE: To determine whether longer waiting time for major joint replacement is associated with health and social services utilization before treatment. METHODS: When placed on the waiting list, patients were randomized to short (

Evaluating waiting time effect on health outcomes at admission: a prospective randomized study on patients with osteoarthritis of the knee joint.

AIMS: To evaluate the effect of waiting on health-related quality of life (HRQoL), pain and physical function in patients awaiting primary total knee replacement (TKR) due to osteoarthritis. METHODS: Some 438 patients awaiting TKR were randomized to a short waiting time (WT) group (< or =3 months) or a non-fixed WT group. In the final assessment, 310 patients (213 women) with a mean age of 68 years were included. HRQoL was measured on being placed on the waiting list and again at hospital admission using the generic 15D. Patients' self-report pain and physical function were evaluated using a scale modified from the Knee Society Clinical Rating System. RESULTS: The median WTs for patients with short and non-fixed WT were 73 days (range 8-600 days) and 266 days (range 28-818 days), respectively. At admission, as assessed by the intention-to-treat analysis, there were no statistically significant differences between the groups in the 15D total score and disease-specific pain and function. CONCLUSIONS: Our study showed that longer WT did not result in worse pre-operative HRQoL.

The effect of co-morbidities on health-related quality of life in patients placed on the waiting list for total joint replacement.

BACKGROUND: Co-morbidity is a powerful predictor of health care outcomes and costs, as well as an important cofounder in epidemiologic studies. The effect of co-morbidities is generally related to mortality or complications. This study evaluated the association between co-morbidity and health-related quality of life (HRQoL) in patients awaiting total joint replacement. METHODS: A total of 893 patients were recruited to the study between August 2002 and November 2003 in four Finnish hospitals. The effect of co-morbidity on HRQoL was measured by the generic 15D instrument and by a Visual Analog Scale (VAS). Comparative variance analysis of socio-demographic and clinical characteristics was described by using either an independent samples t-test or the Chi-square test. The differences in each of the 15D dimensions and the overall 15D single index score for patients were calculated. Two-sided p-values were calculated with the Levene Test for Equality of Variances. RESULTS: Patients with co-morbidity totaled 649; the incidence of co-morbidity was 73%. The mean number of co-morbidities among the patients was two. At baseline the 15D score in patients with and without co-morbidity was 0.778 vs 0.816, respectively. The difference of the score (0.038) was clinically and statistically significant (P < 0.001). The patients' scores with and without co-morbidity on the different 15D dimensions related to osteoarthritis-moving, sleeping, usual activities, discomfort and symptoms, vitality and sexual activity-were low in both groups. Patients with co-morbidity scored lower on the dimensions of moving, vitality and sexual activity compared to the patients without co-morbidity. Co-morbidity was significantly associated with a reduced HRQoL. Patients without co-morbidity had poorer VAS, arthritis had strong effect to their quality of life compared to the patients with co-morbidity. CONCLUSION: Assessing co-morbidity in patients placed on the waiting list for joint replacement may be useful method to prioritization in medical decision-making for healthcare delivery. The assessment of co-morbidities during waiting time is important as well as evaluating how the co-morbidity may affect the final outcomes of the total joint replacement.

Health-related quality of life in patients waiting for major joint replacement. A comparison between patients and population controls.

BACKGROUND: Several quality-of-life studies in patients awaiting major joint replacement have focused on the outcomes of surgery. Interest in examining patients on the elective waiting list has increased since the beginning of 2000. We assessed health-related quality of life (HRQoL) in patients waiting for total hip (THR) or knee (TKR) replacement in three Finnish hospitals, and compared patients' HRQoL with that of population controls. METHODS: A total of 133 patients awaiting major joint replacement due to osteoarthritis (OA) of the hip or knee joint were prospectively followed from the time the patient was placed on the waiting list to hospital admission. A sample of controls matched by age, gender, housing and home municipality was drawn from the computerised population register. HRQoL was measured by the generic 15D instrument. Differences between patients and the population controls were tested by the independent samples t-test and between the measurement points by the paired samples t-test. A linear regression model was used to explain the variance in the 15D score at admission. RESULTS: At baseline, 15D scores were significantly different between patients and the population controls. Compared with the population controls, patients were worse off on the dimensions of moving (P < 0.001), sleeping (P < 0.001), sexual activity (P < 0.001), vitality (P < 0.001), usual activities (P < 0.001) and discomfort and symptoms (P < 0.001). Further, psychological factors--depression (P < 0.001) and distress (P = 0.004)--were worse among patients than population controls. The patients showed statistically significantly improved average scores at admission on the dimensions of moving (P = 0.026), sleeping (P = 0.004) and discomfort and symptoms (P = 0.041), but not in the overall 15D score compared with the baseline. In patients, 15D score at baseline (P < 0.001) and body mass index (BMI) (P = 0.020) had an independent effect on patients' 15D score at hospital admission. CONCLUSION: Although patients' HRQoL did not deteriorate while waiting, a consistently worse HRQoL was observed in patients waiting for major joint replacement compared with population controls.