Effect of Selected Oligosaccharides on the Viability and Fermentation Kinetics of Lactobacillus acidophilus and Lactobacillus casei in Cultured Milk

The study aimed to investigate the effect of fructo-oligosaccharides (FOS) on the growth and fermentation kinetics of Lactobacillus casei LC-01 (LC) and Lactobacillus acidophilus LA5 (LA) in cultured milk. Two commercially available FOS with different degree of polymerization (DP), namely Fibrulose F97 (DP, 2-20) and Fibruline Instant (DP, 3-60) were used at 4% (w/v) and 8% (w/v) respectively during fermentation and storage of cultured milk. Physicochemical properties and acidification kinetic of milk were measured throughout the fermentation. The concentration and DP values of the FOS do not seem to affect the growth of both probiotics during fermentation. Nevertheless, the pH and total soluble solid of milk fermented by both probiotics supplemented with FOS decreased tremendously during fermentation. It is noted that the percentage of lactic acid produced in L. acidophilus is higher than L. casei owing to the metabolic characteristic of the strain. The kinetic of maximum acidification rate Vmax of cultured milk was significantly higher with the addition of FOSs at 4%. However, FOS with lower DP seemed to enhance (p<0.05) the stability of LA in cultured milk during cold storage, but no significant effect on LC. The results of this work indicate that FOS could significantly improve the survival of probiotics in cultured milk especially during refrigerated storage

Microbial Metabolic Networks at the Mucus Layer Lead to Diet-Independent Butyrate and Vitamin B12 Production by Intestinal Symbionts

Peer reviewe

Deciphering the trophic interaction between Akkermansia muciniphila and the butyrogenic gut commensal Anaerostipes caccae using a metatranscriptomic approach

Host glycans are paramount in regulating the symbiotic relationship between humans and their gut bacteria. The constant flux of host-secreted mucin at the mucosal layer creates a steady niche for bacterial colonization. Mucin degradation by keystone species subsequently shapes the microbial community. This study investigated the transcriptional response during mucin-driven trophic interaction between the specialised mucin-degrader Akkermansia muciniphila and a butyrogenic gut commensal Anaerostipes caccae. A. muciniphila monocultures and co-cultures with non-mucolytic A. caccae from the Lachnospiraceae family were grown anaerobically in minimal media supplemented with mucin. We analysed for growth, metabolites (HPLC analysis), microbial composition (quantitative reverse transcription PCR), and transcriptional response (RNA-seq). Mucin degradation by A. muciniphila supported the growth of A. caccae and concomitant butyrate production predominantly via the acetyl-CoA pathway. Differential expression analysis (DESeq 2) showed the presence of A. caccae induced changes in the A. muciniphila transcriptional response with increased expression of mucin degradation genes and reduced expression of ribosomal genes. Two putative operons that encode for uncharacterised proteins and an efflux system, and several two-component systems were also differentially regulated. This indicated A. muciniphila changed its transcriptional regulation in response to A. caccae. This study provides insight to understand the mucin-driven microbial ecology using metatranscriptomics. Our findings show that the expression of mucolytic enzymes by A. muciniphila increases upon the presence of a community member. This could indicate its role as a keystone species that supports the microbial community in the mucosal environment by increasing the availability of mucin sugars.Peer reviewe

Malaysian Females With Congenital Adrenal Hyperplasia: Surgical Outcomes and Attitudes

Background: Girls born with congenital adrenal hyperplasia have virilized external genitalia. There is considerable debate regarding both the outcomes of feminizing genitoplasty and timing of the surgery in this population.Objective: To investigate outcomes of females 46,XX individuals with CAH in Malaysia, the surgical outcomes of feminizing genitoplasty (FG) and their attitudes toward surgery.Study Design: This is a cross-sectional study involving the two main tertiary centers in Malaysia. All 46,XX patients with CAH and raised female, who had undergone FG were identified and invited to participate. Data on socio-demographic, medical profiles, and attitudes toward surgery were collected. A standardized evaluation of the external genitalia was undertaken including the anatomic and cosmetic evaluation by independent gynecologists.Results: Of 61 individuals identified, 59 participated—consisting of children (n = 12), adolescents (n = 29) and adults (n = 18). All but one had classical CAH (98.3%) and had undergone FG (n = 55, 93.2%) with surgery mostly undertaken by pediatric surgeons trained in DSD work (n = 44, 74.6%). Complications overall were low (20.3%), with repeat surgery rate of 9.1%. External genital examination was performed in 38 participants. Overall 36.8% had absent clitoral glands and 39.5% had a persistent urogenital sinus and in 10.5%, no vaginal orifices were seen. Poor cosmetic outcomes were present in 42.1% with 55.3% recommended for further assessment under general anesthetic. Almost half participants did not venture an opinion on FG, those who did varied from having a positive attitude toward it (18 participants) to 3 opining that it should not be done, or avoided or delayed. From the participants, 35.5% preferred FG to be done early in life compared to 44.0% of the parents.Conclusions: The reoperation rates of the feminizing genitoplasty surgeries were low however due to the anatomic and cosmetic outcomes, reassessment of the external genitalia of these CAH patients may be required once they consider becoming sexually active as they may require further treatment. Many factors such as cultural sensitivities and access to medical treatment and late diagnoses have an impact on attitudes toward FG

Cross-feeding interactions of gut symbionts driven by human milk oligosaccharidesand mucins

The co-evolution of the human host and the gut microbiota has led to bacterial adaption to forage on host-produced glycans such as human milk oligosaccharides (HMOS) and mucins. In early life, the HMOS that are present in mother milk contribute to the establishment of a healthy gut microbiota. Furthermore, mucins that cover the intestinal lining create a stable niche for bacterial colonization throughout a person’s life. Due to the highly complex nature of these host-secreted glycans, bacteria equipped with specific glycan-degrading enzymes can exploit them as substrate for growth. Subsequently, the glycan-degrading bacteria can drive microbial networks via cross-feeding. The glycan-foraging microbial population exerts a large influence on the host physiology, by influencing the immune, metabolic, and neurological development early in life, and by conferring colonisation resistance throughout life. The work described in this thesis aims to improve our understanding of the metabolic dependencies between the milk- and mucin-degrading microbes (Akkermansia muciniphila, Bifidobacterium spp. and Bacteroides spp.) and butyrate-producing bacteria (Anaerostipes caccae, Eubacterium hallii, and Faecalibacterium prausnitzii). Bacteria-derived butyrate is the preferred energy source for host epithelial cells and is associated with a range of beneficial effects including enhancement of colonic barrier function, increased satiety, and protection against inflammation, and cancer. In the introductory chapter, the role of human milk and mucin glycans in fostering the gut microbial network is discussed. As such, the molecular mechanisms of glycan-foraging by key microbial species and the potential butyrate-inducing interaction among gut symbionts is presented. In Chapter 2 and 3, the role of HMOS as selective substrates for microbial growth that drive the establishment of the infant gut microbiota was investigated. At this developmental stage, HMOS promote the dominance of bifidobacteria from the Actinobacteria phylum. Upon weaning, the gut microbiota shifts towards an adult gut microbiota composition that is predominantly comprised of bacteria from the Firmicutes and Bacteroidetes phyla. In chapter 2, the interaction between a HMOS-degrader, Bifidobacterium infantis (Actinobacteria phylum) and a butyrogenic non-HMOS-degrader, Anaerostipes caccae (a member of the Lachnospiraceae from the Firmicutes phylum) was studied. Anaerostipes caccae in monoculture was not able to metabolise lactose or HMOS but its growth and concomitant butyrate production were detected in co-cultures with Bifidobacterium infantis. Anaerostipes caccae was sustained by cross-feeding on the monosaccharides, lactate and acetate derived from Bifidobacterium infantis. Bifidobacterium infantis fully degraded lactose and the complete range of low molecular weight HMOS, pointing towards the key ecological role of bifidobacteria in providing substrates for other important emerging species in the infant gut. The gradual shift of the microbiota composition in the ecosystem contributing to the slow induction of butyrate could also be important for gut maturation. In chapter 3, the microbial network formation in the infant gut driven by another HMOS-degrading species, namely Bacteroides thetaiotaomicron was studied. We showed that Bacteroides thetaiotaomicron could drive the butyrogenic trophic chain with Anaerostipes caccae. Bacteroides thetaiotaomicron could metabolise lactose and HMOS. The bacterium showed different preference for specific HMOS structures when grown in co-culture. Subsequently, Anaerostipes caccae cross-fed on Bacteroides thetaiotaomicron-derived monosaccharides, lactate and acetate for growth and butyrate production. Bacteroides thetaiotaomicron might drive the establishment of the microbial network in the infant gut, leading to the sequential establishment of adult-like functional groups such as lactate-utilising and butyrate-producing bacteria. Furthermore, we observed stereospecific lactate isomer production in which Bacteroides spp. and Bifidobacterium spp. produced predominantly D- and L-lactate, respectively. The distinct lactate isomer production by these major glycans-degrading genera might affect the gut microbiota compositions by differential cross-feeding interaction with the lactate-utilisers. Chapter 4 and 5 studied the role of mucins in creating a micro-environment that leads to the formation of a microbial network at the intestinal mucosal layer. In chapter 4, we demonstrated that Akkermansia muciniphila, a gut symbiont specialised in mucin degradation, could support the growth of the butyrate-producing cross-feeders Anaerostipes caccae, Eubacterium hallii, and Faecalibacterium prausnitzii. Akkermansia muciniphila metabolised the complex mucin glycans into short chain fatty acids including acetate, propionate and 1,2-propanediol as well as the mucin-derived sugars. Subsequently, acetate and the liberated sugars could be used by the butyrate-producers for growth and concomitant butyrate production. Interestingly, a bidirectional cross-feeding was observed between Akkermansia muciniphila and Eubacterium hallii. Pseudo-vitamin B12 produced by Eubacterium hallii facilitated propionate production by Akkermansia muciniphila via the methylmalonyl-CoA pathway. Propionate could be beneficial to the human host by regulating satiety and lipid biosynthesis in the liver, indicative of a mutualistic host-microbial interaction driven by mucin glycans. In chapter 5, we studied the molecular mechanism of cross-feeding between Akkermansia muciniphila and Anaerostipes caccae by metatranscriptomics. We observed a differential transcriptional response of Akkermansia muciniphila grown in monoculture as compared to a co-culture together with Anaerostipes caccae. In particular, the expression of the extracellular mucin-degrading enzymes by Akkermansia muciniphila was heightened in co-cultures. As a result, the monosaccharides liberated from the breakdown of mucin oligosaccharides chain could support the central metabolism of both Akkermansia muciniphila and the butyrate-producer. This suggested that Akkermansia muciniphila plays a key role in supporting the microbial community at the mucosal environment of the intestine by increasing the availability of substrates. In summary, this thesis demonstrated the key functional role of milk- and mucin-degrading symbionts in fostering a butyrogenic microbial network via cross-feeding. Ecologically, HMOS-degraders are critical to drive the establishment of a healthy infant gut microbiota, whilst mucin-degraders are vital to maintain a beneficial mucosal community. A better understanding of the complex nature of both the microbial network and the host-secreted glycans could aid in the design of nutritional intervention for health improvement. To this end, innovative avenues using novel probiotic strains (key species including Akkermansia muciniphila, Bacteroides spp. and butyrate-producing Clostridium), prebiotics (HMOS in early nutrition) and microbiota-targeted nutrients (iron and vitamin B12) deem promising

Milk protein coagulation under gastric conditions : A review

Milk protein coagulation is not only important during the processing of milk into various dairy products, but also during digestion of milk. This review focusses on the gastric coagulation of milk proteins. During this process, coagulation of casein micelles and milk fat globules can occur due to pepsin-induced hydrolysis of the proteins that provide steric stabilisation. The gastric coagulation leads to delayed gastric emptying of casein and fat. Native whey proteins are not susceptible to gastric coagulation or delayed gastric emptying. Both heat treatment and homogenisation of milk lead to weaker gastric curds being formed, which are broken down more easily due to proteolysis and deformation. Incorporation of denatured whey proteins in gastric curds of heated milk delays their emptying. Understanding gastric coagulation and digestion behaviour allows tailoring of gastric transit via compositional differences or processing

Deciphering the trophic interaction between Akkermansia muciniphila and the butyrogenic gut commensal Anaerostipes caccae using a metatranscriptomic approach

Host glycans are paramount in regulatingthe symbiotic relationship between humans and their gutbacteria. The constant flux of host-secreted mucin at themucosal layer creates a steady niche for bacterialcolonization. Mucin degradation by keystone speciessubsequently shapes the microbial community. Thisstudy investigated the transcriptional response duringmucin-driven trophic interaction between the specialisedmucin-degrader Akkermansia muciniphila and a butyrogenicgut commensal Anaerostipes caccae. A.muciniphila monocultures and co-cultures with nonmucolyticA. caccae from the Lachnospiraceae familywere grown anaerobically in minimal media supplementedwith mucin. We analysed for growth, metabolites(HPLC analysis), microbial composition(quantitative reverse transcription PCR), and transcriptionalresponse (RNA-seq). Mucin degradation by A.muciniphila supported the growth of A. caccae andconcomitant butyrate production predominantly via theacetyl-CoA pathway. Differential expression analysis(DESeq 2) showed the presence of A. caccae inducedchanges in the A. muciniphila transcriptional responsewith increased expression of mucin degradation genesand reduced expression of ribosomal genes. Twoputative operons that encode for uncharacterised proteinsand an efflux system, and several two-componentsystemswere also differentially regulated.This indicatedA. muciniphila changed its transcriptional regulation inresponse to A. caccae. This study provides insight tounderstand the mucin-driven microbial ecology usingmetatranscriptomics. Our findings show that the expressionof mucolytic enzymes by A. muciniphila increasesupon the presence of a community member. This couldindicate its role as a keystone species that supports themicrobial community in the mucosal environment byincreasing the availability of mucin sugars.Keywords Butyrate Cross feeding Keystonespecies Microbiome Mucin Transcriptionalregulation Verrucomicrobi

Deciphering the trophic interaction between Akkermansia muciniphila and the butyrogenic gut commensal Anaerostipes caccae using a metatranscriptomic approach

Host glycans are paramount in regulatingthe symbiotic relationship between humans and their gutbacteria. The constant flux of host-secreted mucin at themucosal layer creates a steady niche for bacterialcolonization. Mucin degradation by keystone speciessubsequently shapes the microbial community. Thisstudy investigated the transcriptional response duringmucin-driven trophic interaction between the specialisedmucin-degrader Akkermansia muciniphila and a butyrogenicgut commensal Anaerostipes caccae. A.muciniphila monocultures and co-cultures with nonmucolyticA. caccae from the Lachnospiraceae familywere grown anaerobically in minimal media supplementedwith mucin. We analysed for growth, metabolites(HPLC analysis), microbial composition(quantitative reverse transcription PCR), and transcriptionalresponse (RNA-seq). Mucin degradation by A.muciniphila supported the growth of A. caccae andconcomitant butyrate production predominantly via theacetyl-CoA pathway. Differential expression analysis(DESeq 2) showed the presence of A. caccae inducedchanges in the A. muciniphila transcriptional responsewith increased expression of mucin degradation genesand reduced expression of ribosomal genes. Twoputative operons that encode for uncharacterised proteinsand an efflux system, and several two-componentsystemswere also differentially regulated.This indicatedA. muciniphila changed its transcriptional regulation inresponse to A. caccae. This study provides insight tounderstand the mucin-driven microbial ecology usingmetatranscriptomics. Our findings show that the expressionof mucolytic enzymes by A. muciniphila increasesupon the presence of a community member. This couldindicate its role as a keystone species that supports themicrobial community in the mucosal environment byincreasing the availability of mucin sugars.Keywords Butyrate Cross feeding Keystonespecies Microbiome Mucin Transcriptionalregulation Verrucomicrobi

Bacteroides thetaiotaomicron fosters the growth of butyrate-producing anaerostipes caccae in the presence of lactose and total human milk carbohydrates

The development of infant gut microbiota is strongly influenced by nutrition. Human milk oligosaccharides (HMOSs) in breast milk selectively promote the growth of glycan-degrading microbes, which lays the basis of the microbial network. In this study, we investigated the trophic interaction between Bacteroides thetaiotaomicron and the butyrate-producing Anaerostipes caccae in the presence of early-life carbohydrates. Anaerobic bioreactors were set up to study the monocultures of B. thetaiotaomicron and the co-cultures of B. thetaiotaomicron with A. caccae in minimal media supplemented with lactose or a total human milk carbohydrate fraction. Bacterial growth (qPCR), metabolites (HPLC), and HMOS utilization (LC-ESI-MS2) were monitored. B. thetaiotaomicron displayed potent glycan catabolic capability with differential preference in degrading specific low molecular weight HMOSs, including the neutral trioses (2′-FL and 3-FL), neutral tetraoses (DFL, LNT, LNnT), neutral pentaoses (LNFP I, II, III, V), and acidic trioses (3′-SL and 6′-SL). In contrast, A. caccae was not able to utilize lactose and HMOSs. However, the signature metabolite of A. caccae, butyrate, was detected in co-culture with B. thetaiotaomicron. As such, A. caccae cross-fed on B. thetaiotaomicron-derived monosaccharides, acetate, and d-lactate for growth and concomitant butyrate production. This study provides a proof of concept that B. thetaiotaomicron could drive the butyrogenic metabolic network in the infant gut.</p

Discordance of epidermal growth factor receptor mutation between primary lung tumor and paired distant metastases in non-small cell lung cancer: A systematic review and meta-analysis.

PurposeTo evaluate the rate of discordance of epidermal growth factor receptor (EGFR) mutation between primary lung tumor and paired distant metastases in non-small-cell lung cancer (NSCLC).MethodsWe performed a meta-analysis of 17 studies (518 cases) assessing discordance rates of EGFR mutation in primary tumors and paired distant metastases. We performed subgroup analyses based on EGFR mutation status in primary tumor (mutant or wildtype), site of distant metastasis (bone, central nervous system (CNS) or lung/ pleural), methods of testing (direct sequencing or allele-specific testing) and timing of metastasis (synchronous or metachronous).ResultsThe overall discordance rate in EGFR mutation was low at 10.36% (95% CI = 4.23% to 18.79%) and varied widely between studies (I2 = 83.18%). The EGFR discordance rate was statistically significantly higher in bone metastases (45.49%, 95% CI = 14.13 to 79.02) than CNS (17.26%, 95% CI = 7.64 to 29.74; P = 0.002) and lung/ pleural metastases (8.17%, 95% CI = 3.35 to 14.85; P ConclusionThe overall discordance rate in EGFR mutation between primary lung tumor and paired distant metastases in NSCLC is low, although higher discordance rates were observed in bone metastases compared with CNS and lung/pleural metastases. Future studies assessing the impact of EGFR mutation discordance on treatment outcomes are required