An Accurate Estimate of the Amino Acid Content of Human Milk Collected from Chinese Women Adjusted for Differences in Amino Acid Digestibility.

BACKGROUND: The amino acid (AA) composition of human milk is used to define the AA requirements of the infant. Thus, it is important that estimates of composition be as complete and accurate as possible. When determining AA composition using standard hydrolysis methods, some AAs are progressively destroyed while others are incompletely released. For accuracy, AA composition needs to be determined using multiple hydrolysis times. The true ileal digestibility of AAs also needs to be taken into consideration. OBJECTIVE: The objective was to bring together AA compositional (determined using multiple hydrolysis intervals) and digestibility data determined using the piglet to give an estimate of the absorbed AA profile of human milk with reference in particular to Asian females. METHODS: Mature milk was collected from Chinese females. AA analysis using multiple hydrolysis intervals and a nonlinear regression model was used to accurately estimate AA composition. Human milk, as well as a protein-free diet, were fed to piglets (n = 6), and ileal digesta were collected (piglet age, 21 d) to determine the true ileal AA digestibility of AAs in human milk. RESULTS: True ileal AA digestibility coefficients ranged from (mean ± standard error of the mean) 0.61 ± 0.081 for tyrosine to 1.01 ± 0.030 for tryptophan, with a digestibility for total nitrogen of 0.90 ± 0.013. Convergence criteria were met for the modeling for each AA, and the model had a level of significance of P < 0.0001 for each AA. The amount of available AAs (total AA content as per the model prediction multiplied by the true ileal AA digestibility coefficient determined in the piglet) are reported. CONCLUSIONS: An estimate of the absorbed AA profile of mature milk collected from Chinese females is provided. For the first time, data is presented for cysteine.

Predominance of non-covalent interactions of polyphenols with milk proteins and their health promoting properties.

Polyphenols have widely accepted health benefits which are limited by their low uptake, low bioavailability, and rapid degradation in the gut. While milk proteins are excellent carriers for polyphenols, the specific interactions of the polyphenols with the milk proteins, need to be understood to facilitate the utilization of these delivery systems in food and pharmaceutical applications. We have evaluated the relevance of different factors affecting milk protein-polyphenol interactions and the subsequent impact on the bioavailability and health promoting aspects of polyphenols. Hydrophobic forces are the primary binding forces of polyphenols to milk proteins. The significant factors affecting the interactions and binding affinity are the molecular weight and the hydrophobicity of the polyphenols. The interaction of polyphenols with milk proteins improved the antioxidant activity in comparison to milk proteins, while conflicting results exists for comparisons with polyphenols. In-vitro and cell line studies demonstrated enhanced bioavailability of polyphenols in the presence of milk proteins as well as higher anti-cancer and anti-allergy benefits. Overall, this work will pave the way for better understanding of polyphenol interactions with milk proteins and enable the tailoring of complexes through sustainable green processes, enabling higher bioavailability and health promoting effects of the polyphenols in food and pharmaceutical applications.

Epigenetic Changes in Saccharomyces cerevisiae Alters the Aromatic Profile in Alcoholic Fermentation.

Epigenetic changes in genomics provide phenotypic modification without DNA sequence alteration. This study shows that benzoic acid, a common food additive and known histone deacetylase inhibitor (HDACi), has an epigenetic effect on Saccharomyces cerevisiae. Benzoic acid stimulated formation of epigenetic histone marks H3K4Me2, H3K27Me2, H3K18ac, and H3Ser10p in S. cerevisiae and altered their phenotypic behavior, resulting in increased production of phenylethyl alcohol and ester compounds during alcoholic fermentation using wine as a representative model system. Our study demonstrates the HDACi activity of certain dietary compounds such as sodium butyrate, curcumin and anacardic acid, suggests the potential use of these dietary compounds in altering S. cerevisiae phenotypes without altering host-cell DNA. This study highlights the potential to use common dietary compounds to exploit epigenetic modifications for various fermentation and biotechnology applications as an alternative to genetic modification. These findings indicate that benzoic acid and other food additives may have potential epigenetic effects on human gut microbiota, in which several yeast species are involved. IMPORTANCE The manuscript investigates and reports for the first time utilizing a non-GMO approach to alter the fermentation process of Pinot Noir wines. We have experimentally demonstrated that certain dietary compounds possess histone deacetylase (HDAC) inhibiting activity and can alter the wine characteristics by potentially altering yeast gene transcription, which was resulted from epigenetic effects. We have previously proposed the term "nutrifermentics" to represent this newly proposed field of research that provides insights on the effect of certain dietary compounds on microbial strains and their potential application in fermentation. This technological approach is a novel way to manipulate microorganisms for innovative food and beverage production with quality attributes catering for consumer's needs. Using a multidisciplinary approach with an emphasis on food fermentation and biotechnology, this study will be substantially useful and of broad interest to food microbiologists and biotechnologists who seek for innovative concepts with real-world application potential.

Salivaricin E and abundant dextranase activity may contribute to the anti-cariogenic potential of the probiotic candidate Streptococcus salivarius JH.

Dental caries is an infectious disease that is continuing to increase in prevalence, reducing the quality of life for millions worldwide as well as causing considerable expense, with an estimated US$108 billion spent on dental care in the USA each year. Oral probiotics are now being investigated to determine whether they could play a role in the prevention and treatment of this disease. Streptococcus salivarius strain JH is a potential probiotic candidate that produces multiple proteinaceous antimicrobials (bacteriocins), the inhibitory spectrum of which includes Streptococcus mutans, one of the principal causative agents of dental caries. The genome of strain JH has previously been shown to contain the biosynthetic loci for the bacteriocins salivaricin A3, streptin and streptococcin SA-FF22. Here we show that strain JH also produces salivaricin E, a 32 aa lantibiotic with a mass of 3565.9 Da, which is responsible for the inhibition of S. mutans growth. In addition, strain JH was shown to produce dextranase, an enzyme that hydrolyses (1 → 6)-α-D-glucosidic linkages, at levels higher than any other S. salivarius tested. In vitro testing showed that partial hydrolysis of the exopolymeric substances of S. mutans, using strain JH dextranase, improved the anti-S. mutans inhibitory activity of the lytic bacteriocin, zoocin A. The multiple bacteriocin and dextranase activities of strain JH support its candidature for development as an oral probiotic.

Investigation of Streptococcus salivarius-mediated inhibition of pneumococcal adherence to pharyngeal epithelial cells.

BACKGROUND: Pneumococcal adherence to the nasopharyngeal epithelium is a critical step in colonisation and disease. The probiotic bacterium, Streptococcus salivarius, can inhibit pneumococcal adherence to epithelial cells in vitro. We investigated the mechanism(s) of inhibition using a human pharyngeal epithelial cell line (Detroit 562) following pre-administration of two different strains of S. salivarius. RESULTS: Whilst the bacteriocin-encoding megaplasmids of S. salivarius strains K12 and M18 were essential to prevent pneumococcal growth on solid media, they were not required to inhibit pneumococcal adherence. Experiments testing S. salivarius K12 and two pneumococcal isolates (serotypes 19F and 6A) showed that inhibition of 19F may involve S. salivarius-mediated blocking of pneumococcal binding sites: a negative correlation was observed between adherence of K12 and 19F, and no inhibition occurred when K12 was prevented from contacting epithelial cells. K12-mediated inhibition of adherence by 6A may involve additional mechanisms, since no correlation was observed between adherence of K12 and 6A, and K12 could inhibit 6A adherence in the absence of cell contact. CONCLUSIONS: These results suggest that S. salivarius employs several mechanisms, including blocking pneumococcal binding sites, to reduce pneumococcal adherence to pharyngeal epithelial cells. These findings extend our understanding of how probiotics may inhibit pneumococcal adherence and could assist with the development of novel strategies to prevent pneumococcal colonisation in the future.

Streptococcus salivarius K12 Limits Group B Streptococcus Vaginal Colonization.

Streptococcus agalactiae (group B streptococcus [GBS]) colonizes the rectovaginal tract in 20% to 30% of women and during pregnancy can be transmitted to the newborn, causing severe invasive disease. Current routine screening and antibiotic prophylaxis have fallen short of complete prevention of GBS transmission, and GBS remains a leading cause of neonatal infection. We have investigated the ability of Streptococcus salivarius, a predominant member of the native human oral microbiota, to control GBS colonization. Comparison of the antibacterial activities of multiple S. salivarius strains by use of a deferred-antagonism test showed that S. salivarius strain K12 exhibited the broadest spectrum of activity against GBS. K12 effectively inhibited all GBS strains tested, including disease-implicated isolates from newborns and colonizing isolates from the vaginal tract of pregnant women. Inhibition was dependent on the presence of megaplasmid pSsal-K12, which encodes the bacteriocins salivaricin A and salivaricin B; however, in coculture experiments, GBS growth was impeded by K12 independently of the megaplasmid. We also demonstrated that K12 adheres to and invades human vaginal epithelial cells at levels comparable to GBS. Inhibitory activity of K12 was examined in vivo using a mouse model of GBS vaginal colonization. Mice colonized with GBS were treated vaginally with K12. K12 administration significantly reduced GBS vaginal colonization in comparison to nontreated controls, and this effect was partially dependent on the K12 megaplasmid. Our results suggest that K12 may have potential as a preventative therapy to control GBS vaginal colonization and thereby prevent its transmission to the neonate during pregnancy.

Preferential Binding of Polyphenols in Blackcurrant Extracts with Milk Proteins and the Effects on the Bioaccessibility and Antioxidant Activity of Polyphenols.

Milk proteins are well-known delivery agents; however, there is no clear understanding of the competitive interactions of milk proteins with polyphenols in mixed complex systems. Here, we investigate the preferential competitive interactions of different polyphenols present in blackcurrant extract with milk proteins by quantifying the protein-bound polyphenols and comparing the factors affecting these interactions. In addition, bioaccessibility and antioxidant activity were studied after in vitro gastric digestion. Our results indicated that polyphenols from blackcurrant extracts were preferentially bound to caseins more than whey proteins, with noncovalent interactions causing secondary structural changes in the protein. The hydrophobicity and the charge of the polyphenols were negatively and positively related to the number of polyphenols bound to casein and whey proteins, respectively. Moreover, the bioaccessibility and antioxidant activity of polyphenols were enhanced in the presence of milk proteins in milk-based blackcurrant samples when compared to polyphenol and protein-alone samples in the in vitro gastric phase. These findings underscore the critical role of milk proteins in encapsulating or delivering polyphenols. This will pave the way for boosting the bioavailability of polyphenols by complexing them with milk proteins and formulating functional dairy foods, integrating the beneficial effects of these compounds.

Amino acid requirements of the infant: the amino acid composition of human breast milk.

The recommended amino acid requirements of the infant are based on the amino acid composition of mature human breast milk. The amino acid composition of breast milk is usually determined following either acid or alkaline (for tryptophan) hydrolysis. For accuracy, however, the known effect of hydrolysis time on amino acid composition should be accounted for. Also, ideally the amino acid composition of breast milk should be given in units of digested (assumed to be absorbed) amino acids. A review of the literature is presented which gives mean total amino acid concentrations in mature human milk (n = 26 studies), mean hydrolysis correction factors (n = 3 studies) and mean true ileal amino acid digestibility coefficients (n = 3 studies, suckling piglet). There were differences between the estimates of amino acid concentration corrected for hydrolysis time and digestibility, and current FAO (2013) recommendations that were not corrected for these factors. The values based on the published literature up until 2023 (mg/g true protein) corrected for hydrolysis time and digestibility gave higher values (more than 16% higher) for leucine, lysine and threonine, and considerably higher values (greater than 30%) for histidine and tryptophan. Current recommendations may need revision.

Genome sequence of the bacteriocin-producing oral probiotic Streptococcus salivarius strain M18.

Streptococcus salivarius is a Gram-positive bacterial commensal and pioneer colonizer of the human oral cavity. Many strains produce ribosomally synthesized proteinaceous antibiotics (bacteriocins), and some strains have been developed for use as oral probiotics. Here, we present the draft genome sequence of the bacteriocin-producing oral probiotic S. salivarius strain M18.

Novel viscoelastic gelling agent with unique physico-chemical properties.

A novel innovative viscoelastic gelling agent (novel gel, NG) has been developed by combining citric acid (CA) and disodium 5-guanylate (DG). NG has the potential to replace other gelling agents such as gelatine, which has been commonly used in foods, dietary supplements, pharmaceutical and cosmetic products including ointments and sprays. NG has unique physico-chemical properties, including a wide range of concentration-dependent, temperature-sensitive gel strengths. Based on the rheological measurement results, NG depicted similar shear thinning behaviour to gelatine, within shear rates ranging from 25.8 to 129 (s-1). NG also significantly increased the shelf-life (by 21 days) of minced beef, as well as inhibited the growth of major spoilage pathogens, such as E. coli, S. aureus, Salmonella sp., Listeria sp., yeast and moulds, making it an ideal candidate for gelatine replacement.

Production of the Bsa lantibiotic by community-acquired Staphylococcus aureus strains.

Lantibiotics are antimicrobial peptides that have been the focus of much attention in recent years with a view to clinical, veterinary, and food applications. Although many lantibiotics are produced by food-grade bacteria or bacteria generally regarded as safe, some lantibiotics are produced by pathogens and, rather than contributing to food safety and/or health, add to the virulence potential of the producing strains. Indeed, genome sequencing has revealed the presence of genes apparently encoding a lantibiotic, designated Bsa (bacteriocin of Staphylococcus aureus), among clinical isolates of S. aureus and those associated with community-acquired methicillin-resistant S. aureus (MRSA) infections in particular. Here, we establish for the first time, through a combination of reverse genetics, mass spectrometry, and mutagenesis, that these genes encode a functional lantibiotic. We also reveal that Bsa is identical to the previously identified bacteriocin staphylococcin Au-26, produced by an S. aureus strain of vaginal origin. Our examination of MRSA isolates that produce the Panton-Valentine leukocidin demonstrates that many community-acquired S. aureus strains, and representatives of ST8 and ST80 in particular, are producers of Bsa. While possession of Bsa immunity genes does not significantly enhance resistance to the related lantibiotic gallidermin, the broad antimicrobial spectrum of Bsa strongly indicates that production of this bacteriocin confers a competitive ecological advantage on community-acquired S. aureus.

Evolution of Lantibiotic Salivaricins: New Weapons to Fight Infectious Diseases.

Lantibiotic salivaricins are polycyclic peptides containing lanthionine and/or ß-methyllanthionine residues produced by certain strains of Streptococcus salivarius, which almost exclusively reside in the human oral cavity. The importance of these molecules stems from their antimicrobial activity towards relevant oral pathogens which has so far been applied through the development of salivaricin-producing probiotic strains. However, salivaricins may also prove to be of great value in the development of new and novel antibacterial therapies in this era of emerging antibiotic resistance. In this review, we describe the biosynthesis, antimicrobial activity, structure, and mode of action of the lantibiotic salivaricins characterized to date. Moreover, we also provide an expert opinion and suggestions for future development of this important field of microbiology.

The effect of ingestion of milk supplemented with salivaricin A-producing Streptococcus salivarius on the bacteriocin-like inhibitory activity of streptococcal populations on the tongue.

The colonization efficacies of salivaricin A (SalA)-producing Streptococcus salivarius strains 20P3 and 5 were compared when given in milk to 219 children, using either 2-day or 9-day dosing regimens. Colonization levels overall were superior for strain 5, and the 9-day dosing schedule resulted in higher levels of both initial colonization and strain persistence. The indigenous streptococcal tongue populations of 20 (10.9%) of the 189 children in the 2-day trial showed markedly increased SalA-like inhibitory activity following use of the S. salivarius-supplemented milk. All 20 of these children were found to have had relatively small (<5% of total S. salivarius) indigenous tongue populations of SalA-producing S. salivarius, and the relative proportions and/or inhibitory activity of these SalA producers on the childrens' tongues increased following ingestion of the S. salivarius-supplemented milk. Because SalA is known to be strongly inhibitory to Streptococcus pyogenes, an important implication of this study is that the consumption of SalA-producing probiotic S. salivarius could potentially help to effect a sustained increase in SalA-mediated protection against S. pyogenes infection.

Influence of the probiotic Streptococcus salivarius strain M18 on indices of dental health in children: a randomized double-blind, placebo-controlled trial.

The prevalence of dental caries continues to increase, and novel strategies to reverse this trend appear necessary. The probiotic Streptococcus salivarius strain M18 offers the potential to confer oral health benefits as it produces bacteriocins targeting the important cariogenic species Streptococcus mutans, as well as the enzymes dextranase and urease, which could help reduce dental plaque accumulation and acidification, respectively. In a randomized double-blind, placebo-controlled study of 100 dental caries-active children, treatment with M18 was administered for 3 months and the participants were assessed for changes to their plaque score and gingival and soft-tissue health and to their salivary levels of S. salivarius, S. mutans, lactobacilli, ß-haemolytic streptococci and Candida species. At treatment end, the plaque scores were significantly (P = 0.05) lower for children in the M18-treated group, especially in subjects having high initial plaque scores. The absence of any significant adverse events supported the safety of the probiotic treatment. Cell-culture analyses of sequential saliva samples showed no differences between the probiotic and placebo groups in counts of the specifically enumerated oral micro-organisms, with the exception of the subgroup of the M18-treated children who appeared to have been colonized most effectively with M18. This subgroup exhibited reduced S. mutans counts, indicating that the anti-caries activity of M18 probiotic treatments may be enhanced if the efficiency of colonization is increased. It was concluded that S. salivarius M18 can provide oral health benefits when taken regularly.

Persistence of the oral probiotic Streptococcus salivarius M18 is dose dependent and megaplasmid transfer can augment their bacteriocin production and adhesion characteristics.

Bacteriocin-producing probiotic Streptococcus salivarius M18 offers beneficial modulatory capabilities within the oral microbiome, apparently through potent inhibitory activity against potentially deleterious bacteria, such as Streptococcus pyogenes. The oral cavity persistence of S. salivarius M18 was investigated in 75 subjects receiving four different doses for 28 days. Sixty per cent of the subjects already had some inhibitor-producing S. salivarius in their saliva prior to probiotic intervention. Strain M18's persistence was dependent upon the dose, but not the period of administration. Culture analysis indicated that in some individuals the introduced strain had almost entirely replaced the indigenous S. salivarius, though the total numbers of the species did not increase. Selected subjects showing either high or low probiotic persistence had their salivary populations profiled using Illumina sequencing of the V6 region of the 16S rRNA gene. Analysis indicated that while certain bacterial phenotypes were markedly modulated, the overall composition of the oral microbiome was not modified by the probiotic treatment. Megaplasmids encoding bacteriocins and adhesion factors were transferred in vitro to generate a transconjugant S. salivarius exhibiting enhanced antimicrobial production and binding capabilities to HEp-2 cells. Since no widespread perturbation of the existing indigenous microbiota was associated with oral instillation and given its antimicrobial activity against potentially pathogenic streptococci, it appears that application of probiotic strain M18 offers potential low impact alternative to classical antibiotic prophylaxis. For candidate probiotic strains having relatively poor antimicrobial or adhesive properties, unique derivatives displaying improved probiotic performance may be engineered in vitro by megaplasmid transfer.

Developing oral probiotics from Streptococcus salivarius.

Considerable human illness can be linked to the development of oral microbiota disequilibria. The predominant oral cavity commensal, Streptococcus salivarius has emerged as an important source of safe and efficacious probiotics, capable of fostering more balanced, health-associated oral microbiota. Strain K12, the prototype S. salivarius probiotic, originally introduced to counter Streptococcus pyogenes infections, now has an expanded repertoire of health-promoting applications. K12 and several more recently proposed S. salivarius probiotics are now being applied to control diverse bacterial consortia infections including otitis media, halitosis and dental caries. Other potential applications include upregulation of immunological defenses against respiratory viral infections and treatment of oral candidosis. An overview of the key steps required for probiotic development is also presented.

Salivaricin G32, a Homolog of the Prototype Streptococcus pyogenes Nisin-Like Lantibiotic SA-FF22, Produced by the Commensal Species Streptococcus salivarius.

Salivaricin G32, a 2667 Da novel member of the SA-FF22 cluster of lantibiotics, has been purified and characterized from Streptococcus salivarius strain G32. The inhibitory peptide differs from the Streptococcus pyogenes-produced SA-FF22 in the absence of lysine in position 2. The salivaricin G32 locus was widely distributed in BLIS-producing S. salivarius, with 6 (23%) of 26 strains PCR-positive for the structural gene, slnA. As for most other lantibiotics produced by S. salivarius, the salivaricin G32 locus can be megaplasmid encoded. Another member of the SA-FF22 family was detected in two Streptococcus dysgalactiae of bovine origin, an observation supportive of widespread distribution of this lantibiotic within the genus Streptococcus. Since the inhibitory spectrum of salivaricin G32 includes Streptococcus pyogenes, its production by S. salivarius, either as a member of the normal oral microflora or as a commercial probiotic, could serve to enhance protection of the human host against S. pyogenes infection.

Metabolism of L-selenomethionine and selenite by probiotic bacteria: in vitro and in vivo studies.

Since selenium supplements have been shown to undergo biotransformation in the gut, probiotic treatment in combination with selenium supplements may change selenium disposition. We investigated the metabolism of L-selenomethionine (SeMet) and selenite by probiotic bacteria in vitro and the disposition of selenium after probiotic treatment followed by oral dosing with SeMet and selenite in rats. When SeMet was incubated anaerobically with individual antibiotic-resistant probiotic strains (Streptococcus salivarius K12, Lactobacillus rhamnosus 67B, Lactobacillus acidophilus L10, and Bifidobacterium lactis LAFTI® B94) at 37°C for 24 h, 11-18% was metabolized with 44-80% of SeMet lost being converted to dimethyldiselenide (DMDSe) and dimethylselenide (DMSe). In similar incubations with selenite, metabolism was more extensive (26-100%) particularly by the lactobacilli with 0-4.8% of selenite lost being converted to DMSe and DMDSe accompanied by the formation of elemental selenium. Four groups of rats (n = 5/group) received a single oral dose of either SeMet or selenite (2 mg selenium/kg) at the time of the last dose of a probiotic mixture or its vehicle (lyoprotectant mixture used to maintain cell viability) administered every 12 h for 3 days. Another three groups of rats (n = 3/group) received a single oral dose of saline or SeMet or selenite at the same dose (untreated rats). Serum selenium concentrations over the subsequent 24 h were not significantly different between probiotic and vehicle treated rats but appeared to be more sustained (SeMet) or higher (selenite) than in the corresponding groups of untreated rats. Probiotic treated rats given SeMet also had selenium concentrations at 24 h that were significantly higher in liver and lower in kidney than untreated rats given SeMet. Thus, treatment with probiotics followed by SeMet significantly affects tissue levels of selenium.

Something Old and Something New: An Update on the Amazing Repertoire of Bacteriocins Produced by Streptococcus salivarius.

Streptococcus salivarius has an exclusive and intimate association with humans. We are its sole natural host, and its contribution to the relationship appears overwhelmingly benevolent. Beautifully adapted to its preferred habitat, the human tongue, it only rarely ventures far from this location in the healthy host and indeed appears ill-equipped to become invasive due to a scarcity of virulence attributes. We consider that its strategically advantageous lingual location and numerical predominance allow S. salivarius to carry out a population surveillance and modulation role within the oral microbiota. Some strains are armed with complex arrays of targeted antibiotic weaponry, much of which belongs to the lantibiotic class of bacteriocins and a key to their ability to assemble and utilize this armament is their possession of transmissible multi-bacteriocin-encoding megaplasmid DNA. This review traces the origins of research into S. salivarius bacteriocins and bacteriocin-like inhibitory substances, showcases some of the inhibitory activities that we currently have knowledge of, and speculates about potential directions for ongoing investigation and probiotic application of this previously under-rated human commensal.

Streptococcal bacteriocins and the case for Streptococcus salivarius as model oral probiotics.

Members of the Gram-positive bacterial genus Streptococcus are a diverse collection of species inhabiting many body sites and range from benign, nonpathogenic species to those causing life-threatening infections. The streptococci are also prolific producers of bacteriocins, which are ribosomally synthesized proteinaceous antibiotics that kill or inhibit species closely related to the producer bacterium. With the emergence of bacterial resistance to conventional antibiotics, there is an impetus to discover, and implement, new and preferably 'natural' antibiotics to treat or prevent bacterial infections, a niche that bacterial interference therapy mediated by bacteriocins could easily fill. This review focuses on describing the diversity of bacteriocins produced by streptococci and also puts forth a case for Streptococcus salivarius, a nonpathogenic and numerically predominant oral species, as an ideal candidate for development as the model probiotic for the oral cavity. S. salivarius is a safe species that not only produces broad-spectrum bacteriocins but harbors bacteriocin-encoding (and bacteriocin-inducing) transmissible DNA entities (megaplasmids).