Determination of the effect of dairy powders on adherence of Streptococcus sobrinus and Streptococcus salivarius to hydroxylapatite and growth of these bacteria

Dental caries is a highly prevalent disease caused by colonisation of tooth surfaces by cariogenic bacteria, such as Streptococcus sobrinus and Streptococcus salivarius. Reducing initial adherence of such bacteria to teeth may delay onset of caries. Many foods, such as milk, can inhibit microbial adherence. In this investigation, the effect of untreated (UT) and enzyme-treated (ET) dairy powders on adherence of S. sobrinus and S. salivarius to hydroxylapatite (HA), an analogue of tooth enamel, was examined. Untreated (UT) acid whey protein concentrate (AWPC) 80 inhibited streptococcal adherence to phosphate-buffered saline-coated HA (PBS-HA) and saliva-coated HA (S-HA) by >80% at ?31.25 μg mL⁻¹. UT sweet WPC80, buttermilk powder and cream powder also significantly reduced adherence (P < 0.05). Enzyme-treatment of all dairy powders reduced their anti-adhesion activity. However, ET sweet WPC80 significantly inhibited growth of these streptococci (P < 0.05) at ?0.6 mg mL⁻¹. Therefore, dairy powders may reduce progression of dental caries by their anti-adhesion and/or antibacterial activity.     

Inhibition of adhesion of <Emphasis Type="Italic">Streptococcus mutans</Emphasis> to hydroxylapatite by commercial dairy powders and individual milk proteins

The aim of this study was to investigate the inhibitory effect of various dairy powders and milk constituents on the adhesion of a clinical isolate of Streptococcus mutans to hydroxylapatite (HA), an analogue of tooth enamel. Adhesion of a microorganism to a cell surface such as epithelial cells or tooth enamel is considered to be the first step in pathogenesis. Inhibiting this process may have therapeutic effects in vivo. The adherence assays were performed by incubating S. mutans with HA in the presence of each test material for 45 min, followed by centrifugal separation of the HA. Unbound bacteria were then quantified using a fluorescent dye. Sweet and Acid WPC80, buttermilk powder and cream powder were found to very effectively inhibit adherence of S. mutans to phosphate-buffered saline coated HA (PBS-HA). Sodium caseinate and the casein fractions α-,β- and κ-casein were also found to show high levels of anti-adhesive activity. A selection of test materials were assessed using saliva-coated HA (S-HA), and similar trends were observed. The results suggest commercial dairy powders, and certain milk proteins, can inhibit adhesion of S. mutans to HA and may have potential to control dental caries.     

THE EFFECT OF CASEINOMACROPEPTIDE AND WHEY PROTEIN CONCENTRATE ON STREPTOCOCCUS MUTANS ADHESION TO POLYSTYRENE SURFACES AND CELL AGGREGATION

Caseinomacropeptide (CMP) from bovine, ovine and caprine milk and whey protein concentrate (WPC) were both evaluated for adhesion inhibition of Streptococcus mutans to polystyrene surface and cell aggregation. Adhesion inhibition by the CMPs and WPC at 1 mg/mL was higher than 84%. The CMPs and WPC caused aggregation of S. mutans cells at low concentrations. These findings suggest the application of these milk derived proteins as potential dental caries inhibitors and for possible food application uses.     

Separation of an effective fraction from turmeric against Streptococcus mutans biofilms by the comparison of curcuminoid content and anti-acidogenic activity

Turmeric has long been used as a colouring and flavouring agent for foods. Curcuminoids are the main component of turmeric and have a range of pharmacological activities. In this study, a fraction that could show anti-biofilm activity was separated from turmeric, based on a comparison of curcuminoid content and anti-acidogenic activity against Streptococcus mutans, and the effects of the separated fraction and curcuminoids on the adherence ability of S. mutans and the physiological ability of S. mutans biofilms were examined at sub-minimum inhibitory concentration (MIC) levels. The separated fraction and curcuminoids had inhibitory effects on the sucrose-dependant adherence of S. mutans to saliva-coated hydroxyapatite (sHA) discs and the acidogenicity and aciduricity of S. mutans biofilms. These results suggest that the separated turmeric fraction and its components, curcuminoids, may be useful for controlling dental biofilms and subsequent dental caries formation.     

Effects of spray drying conditions and the addition of surfactants on the foaming properties of a whey protein concentrate

Whey is the main waste by-product from dairy industry and at the same time is the major source of globular proteins. These proteins are concentrated mainly through spray drying, but high temperatures affect the foaming properties of globular protein. The addition of surfactants can have a protective role against thermal effects. The aim of this work was to optimize the spray-drying condition and surfactant concentration to obtain a whey protein concentrate (WPC) to be used in hot beverages according to the industry criteria for foaming stability. Three temperatures and three surfactant concentrations were applied, and the optimization was conducted using response surface analysis. Sensory analysis was applied to the WPC obtained under optimal conditions. The results showed that the foaming stability according to industrial criteria was attained when the spray drying was performed at 210 °C with surfactant concentration of 1.50 g/100 g. This resulted in foaming capacity of 3.80 mL, moisture content of 1.82 g/100 g and apparent density of 0.181 g/cm³. The sensory analysis suggested that aroma was related to dairy, cooked and whey and taste was related to sweet and dairy notes. In conclusion, temperature and surfactant concentration played an important role in the foaming capacity and stability of WPC.     

Inhibition of Streptococcus mutans binding to hydroxylapatite using partially digested whey protein concentrate and individual whey proteins

The effect of a bovine whey protein concentrate partially digested with lipase to release free fatty acids (WPD) on growth and viability of Streptococcus mutans, and its adherence to hydroxylapatite (HA) in phosphate-buffered saline were examined. The effects of other whey mixtures and individual whey proteins, β-lactoglobulin (BLG), lactoferrin (LF), caseinoglycomacropeptide (CGMP) and α-lactalbumin were also investigated. Bacterial viability was assessed by incubation with whey protein and measuring colony-forming units. S. mutans binding was quantified using Syto-13 nucleic-acid binding dye and Alamar Blue, which measures cellular metabolic activity. WPD was bactericidal, and inhibited S. mutans growth and adhesion to HA. Other wheys inhibited S. mutans adhesion, particularly acid whey, as well as the parent compound for WPD, Carbelac 80, and a whey protein isolate. BLG, CGMP and LF inhibited S. mutans adhesion. A mixture containing 56% BLG and 20% CGMP was as effective an inhibitor of bacterial adhesion as WPD.     

Accurate quantification of thermophilic spores in dairy powders

Internationally, there are no official guidelines for the quantification of thermophilic spores in dairy products, which leads to variations in applied methodology. In this study, we assess the heat sensitivity of thermophilic spores, vegetative cells grown under laboratory conditions and spores in German dairy powders to determine appropriate heating conditions for accurate quantification of total thermophilic spores. The heat inactivation effect (80–95 °C) is limited for spores of Anoxybacillus flavithermus and Geobacillus stearothermophilus grown under laboratory conditions. However, for spores originating from whey, whey powder and skimmed milk powder (mostly identified as A. flavithermus), a different trend was observed; spore counts continuously reduced when heating time and temperature increased (80–98 °C, 10–30 min). The results indicate that data obtained using laboratory cultures cannot be extrapolated to commercial powders, and in this case, applying temperatures above 80 °C leads to an underestimation of spore counts in dairy powders.     

Novel Streptococcus infantarius subsp. infantarius variants harboring lactose metabolism genes homologous to Streptococcus thermophilus

Streptococcus infantarius subsp. infantarius belongs to the Streptococcus bovis/Streptococcus equinus complex (SBSEC) commonly associated with human and animal infections. We elucidated the lactose metabolism of S. infantarius subsp. infantarius predominant in African fermented milk products. S. infantarius subsp. infantarius isolates (n = 192) were identified in 88% of spontaneously fermented camel milk suusac samples (n = 24) from Kenya and Somalia at log₁₀ 8.2–8.5 CFU mL⁻¹. African S. infantarius isolates excreted stoichiometric amounts of galactose when grown on lactose, exhibiting a metabolism similar to Streptococcusthermophilus and distinct from their type strain. African S. infantarius subsp. infantarius CJ18 harbors a regular gal operon with 99.7–100% sequence identity to S. infantarius subsp. infantarius ATCC BAA-102^T and a gal-lac operon with 91.7–97.6% sequence identity to S. thermophilus, absent in all sequenced SBSEC strains analyzed. The expression and functionality of lacZ was demonstrated in a β-galactosidase assay. The gal-lac operon was identified in 100% of investigated S. infantarius isolates (n = 46) from suusac samples and confirmed in Malian fermented cow milk isolates. The African S. infantarius variant potentially evolved through horizontal gene transfer of an S. thermophilus-homologous lactose pathway. Safety assessments are needed to identify any putative health risks of this novel S. infantarius variant.     

Lactic acid bacteria diversity of African raw and fermented camel milk products reveals a highly competitive,potentially health-threatening predominant microflora

The microflora of 59 East African camel milk samples of unfermented raw milk supply chains and spontaneously fermented milk (suusac) was analyzed to describe the diversity of predominant lactic acid bacteria (LAB), identify potential health risks and study bacteriocin-like inhibitory substance (BLIS) production. Bacterial isolates (n = 532) were identified using a genotypic approach incorporating rep-PCR, 16S rRNA gene sequencing, and species-specific PCR assays. The bacterial numbers on MRS and M17 agar exceeded log₁₀ 6.5 colony forming units (CFU) mL^?1 for raw milk supply chain samples and log₁₀ 8 CFU mL^?1 for suusac. Streptococcus agalactiae and Staphylococcus spp. were predominant in unfermented products, while suusac contained predominantly Streptococcus infantarius subsp. infantarius followed by Lactococcus lactis subsp. lactis, Streptococcus thermophilus, and lactobacilli. Fifty per cent of S. infantarius subsp. infantarius isolates originating from 15 out of 24 suusac samples produced a BLIS active against other LAB or Listeria and representing a potential selective advantage during fermentation. This study provides a detailed insight at the genotypic level into the LAB diversity of previously unstudied dairy products. It indicates potential health risks for consumers and the need for hygienic and manufacturing interventions and reports a potentially novel BLIS produced by S. infantarius subsp. infantarius.     

Prebiotic fibre‐incorporated whey protein crisps processed by supercritical fluid extrusion

Prebiotic soluble fibre (fructooligosaccharides)‐incorporated whey protein crisps were produced by low‐shear supercritical fluid extrusion (SCFX), which utilises supercritical CO₂ as an expansion agent instead of steam. Protein crisps with desirable qualities were obtained with a formulation containing 8% prebiotic fibre and 60% whey protein concentrate (WPC‐80), which gave the final product with a protein content of 49.6% (w/w). A maximum of 70% WPC‐80 and 8% prebiotic fibre could be incorporated to produce expanded protein crisps; however, increasing WPC‐80 from 50% to 70% decreased the end‐product expansion ratio from 3.1 to 1.2 and increased the product hardness and piece density from 1.3 to 2.8 kN and 0.63 to 0.75 g mL^?1, respectively. Addition of 8% prebiotic fibre did not affect the textural qualities of final products. The process produced an expanded protein matrix with unique internal microstructure of uniformly distributed closed cells. Amino acid analysis indicated that 90% of the total lysine and 92% of the total essential amino acids were retained after SCFX processing and oven‐drying, indicating the preservation of protein nutritional quality during the process.     

Optimization of the rheological properties of probiotic yoghurts supplemented with milk proteins

This study aimed to optimize the rheological properties of probiotic yoghurts supplemented with skimmed milk powder (SMP), whey protein concentrate (WPC) and sodium caseinate (Na-Cn) by using an experimental design type simplex-centroid for mixture modeling. It included seven batches/trials: three were supplemented with each type of the dairy protein used, three corresponding to the binary mixtures and one to the ternary one in order to increase protein concentration in 1 g 100 g⁻¹ of final product. A control experiment was prepared without supplementing the milk base. Processed milk bases were fermented at 42 °C until pH 4.5 by using a starter culture blend that consisted of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Bifidobacterium animalis subsp. lactis. The kinetics of acidification was followed during the fermentation period as well the physico-chemical analyses, enumeration of viable bacteria and rheological characteristics of the yoghurts. Models were adjusted to the results (kinetic responses, counts of viable bacteria and rheological parameters) through three regression models (linear, quadratic and cubic special) applied to mixtures. The results showed that the addition of milk proteins affected slightly acidification profile and counts of S. thermophilus and B. animalis subsp. lactis, but it was significant for L. delbrueckii subsp. bulgaricus. Partially-replacing SMP (45 g/100 g) with WPC or Na-Cn simultaneously enhanced the rheological properties of probiotic yoghurts taking into account the kinetics of acidification and enumeration of viable bacteria.     

Diversity of Streptococcus thermophilus in bacteriocin production; inhibitory spectrum and occurrence of thermophilin genes

The bacteriocin-producing Streptococcus thermophilus strains that can dominate in natural dairy ecosystems, may also enhance safety in products obtained from natural cultures. In this study, we sought to identify bacteriocin production and bacteriocin genes in 75 strains of dairy and plant origin. The strains were tested for antimicrobial activity against pathogens or pathogen models, spoiling bacteria, and lactic acid bacteria associated with dairy products. All strains moderately inhibited Staphylococcus aureus P310, none inhibited Listeria innocua LMG 11387^T or Clostridium tyrobutyricum LMG 1285^T. In addition, 14 were active against one or more indicators in addition to S. aureus P310. Inhibition of other starter bacteria was more common than the inhibition of unwanted microorganisms. The involvement of a proteinaceous compound was ascertained in all cases. Results suggested that the selection of bacteriocinogenic S. thermophilus strains for use in biopreservation must take into account the effects exerted on other lactic acid bacteria.     

Immunoglobulins,growth factors and growth hormone in bovine colostrum and the effects of processing

In colostrum collected 0–80 h postpartum the contents of immunoglobulins (Igs), transforming growth factor beta-2 (TGF-β2), insulin-like growth factor-1 (IGF-1) and growth hormone (GH) were analysed. Colostrum initially contained 90 mg mL⁻¹ IgG1, 2.8 mg mL⁻¹ IgG2, 1.6 mg mL⁻¹ IgA, 4.5 mg mL⁻¹ IgM, and these concentrations declined by 92%, 87%, 93% and 84%, respectively, in the samples collected later. Of the growth factors, colostrum initially contained 289–310 ng mL⁻¹ TGF-β2 and the concentration diminished to 66 ng mL⁻¹. The content of IGF-1 and GH postpartum decreased from 870 to 150 ng mL⁻¹, and from 0.17 to <0.03 ng mL⁻¹, respectively. Heat treatment and freeze-drying of colostral whey decreased the content of Igs to 75%, while the contents of IGF-1 and TGF-β2 were unaffected. A similar processing, including filtration steps reduced also the IGF-1 and TGF-β2 by 25%. IgM seems to be the most sensitive of the Igs to processing.     

The inhibitory effect of Plectranthus barbatus and Plectranthus ecklonii leaves on the viability,glucosyltransferase activity and biofilm formation of Streptococcus sobrinus and Streptococcus mutans

Aqueous extracts of Plectranthus barbatus and Plectranthus ecklonii are traditionally used as anti-inflammatory and anti-fungal agents. The effect of these extracts and of its main component, rosmarinic acid, on the viability of the cariogenic bacteria, Streptococcus sobrinus and Streptococcus mutans, was determined by MIC and MBC. The influence of these extracts on the biofilm formation as well as on the inhibition of glucosyltransferase enzyme, produced by these species, was also analysed. Aqueous extracts of P. barbatus and P. ecklonii were stronger inhibitors than rosmarinic acid. MIC values of 3.8 and 4.7 mg/ml for S. sobrinus and 2.9 and 5.0 for S. mutans were obtained, while rosmarinic acid presented MIC values of 8.4 and 7.3 mg/ml. P. barbartus, P. ecklonii and rosmarinic acid presented MBC values of 9.5, 9.0 and 12.0 mg/ml for S. sobrinus, and 9.5, 10.0 and 12.5 mg/ml for S. mutans. The inhibition of biofilm formation by P. barbatus, P. ecklonii and rosmarinic acid presented IC₅₀ values of, respectively, 0.6, 1.0 and 3.1 mg/ml for S. sobrinus and 1.4 and 2.7 and 1.3 mg/ml for S. mutans. The glucosyltransferase inhibition activity by theses extracts and rosmarinic acid was calculated and IC₅₀ values presented were, respectively, 1.1, ca 1.2 and 2.1 mg/ml for S. sobrinus and 3.1, 1.6 and 3.9 mg/ml for S. mutans were obtained. These extracts may be useful in the prevention of dental carie.     

Antibacterial activity of peptides extracted from tryptic hydrolyzate of whey protein by nanofiltration

Ultrafiltration permeate of whey protein tryptic hydrolyzate was processed by nanofiltration (NF) to obtain retentate (NFR) and permeate (NFP) that were then tested as inhibitors of Listeria, Staphylococcus aureus and Escherichia coli. NFR at 20 mg mL^?1 was most effective as an inhibitor (P < 0.001); whereas E. coli was relatively resistant, the effect on Listeria and S. aureus was greater at 20 mg mL^?1 than at 10 mg mL^?1 (P < 0.01). Peptide analysis revealed that NFR was rich in anionic peptides over eight amino acid residues in length. The antibacterial activity of two anionic peptides (84–91 and 125–135) and a cationic peptide (36–42) derived from β-lactoglobulin was tested. Peptide 125–135 was more inhibitory (P < 0.05) than peptide 84–91 against Listeria monocytogenes and S. aureus; peptide 36–42 was not inhibitory. NFR appears to have potential as a natural bio-preservative.     

Lactose triggers biofilm formation by Streptococcus mutans

The most cariogenic bacterium, Streptococcus mutans, often adopts a sessile lifestyle in response to carbohydrates. Biofilm formation represents one of the most successful strategies for survival by S. mutans in a dental environment. This study reports induced biofilm formation by S. mutans in the presence of lactose, the primary sugar in milk. Importantly, no major difference was observed in S. mutans growth in the presence of different concentrations of lactose in growth medium, indicating that lactose has a specific effect on biofilm formation. Moreover, extracellular polysaccharides produced by S. mutans in response to lactose were found to be different from the polysaccharides produced in the presence of sucrose. It is further reported that several biofilm-related genes of S. mutans were significantly up-regulated in response to lactose. These results lead to the conclusion that lactose may promote biofilm formation by S. mutans, the most important bacterium involved in dental diseases.     

Evaluation of diacetyl encapsulated alginate–whey protein microspheres release kinetics and mechanism at simulated mouth conditions

In this paper, diacetyl encapsulated alginate–whey protein concentrate (AL–WPC) microcapsule were prepared based on the emulsification/internal gelation method; and diacetyl release was investigated at the simulated mouth condition in different ratios of artificial saliva (0, 1:4 and 1:8) and three various oral shear rates (0, 50 and 100 s-1). The gotten diacetyl release data were fitted to first-order, Korsmeyer–Peppas, Kopcha, and Makoid–Banakar models to evaluate release mechanisms and kinetics. We showed that the shear rate of release media had a significant (p < 0.05) effect on the release of diacetyl from AL–WPC microspheres, but the saliva ratio had no any significant effect. The diacetyl release data fit well to the all kinetic expression with R² values greater than 0.93. It was found that the release kinetics of diacetyl from AL–WPC microspheres followed a classical Fickian diffusion.     

Microbiological and physicochemical quality of fresh-cut apple enriched with the probiotic strain Lactobacillus rhamnosus GG

The effectiveness as protective culture of the probiotic Lactobacillus rhamonosus GG (L. rham. GG) against Salmonella and Listeria monocytogenes on minimally-processed apples throughout storage as well as its effect on apple quality and natural microflora was evaluated. Survival to subsequent exposure to gastric stress was also reported. Apples were cut into wedges and dipped in a solution containing Salmonella and L. monocytogenes (10⁵ cfu mL⁻¹) and/or L. rham. GG (10⁸ cfu mL⁻¹). Apple wedges were packed and stored at 5 and 10 °C. Periodically, microbial population, bacterial survival to gastric stress and quality of apple wedges were evaluated. Although Salmonella was not affected by co-inoculation with L. rham. GG, L. monocytogenes population was 1-log units lower in the presence of L. rham. GG. L. rham. GG population maintained over recommended levels for probiotic action (10⁶ cfu g⁻¹) along storage, however, viable cells after gastric stress were only above this level during the first 14 days. Pathogen survival after gastric stress was <1% after 7 days at 5 °C. Moreover, apple wedges quality was not affected by L. rham. GG addition. Thus, L. rham. GG could be a suitable probiotic for minimally-processed apples capable to reduce L. monocytogenes growth; nevertheless shelf life should not be higher to 14 days to guarantee the probiotic effect.     

Isolation and characterisation of a novel antibacterial peptide from bovine αS1-casein

Bovine casein was hydrolysed with a range of proteolytic enzymes including pepsin, trypsin, α-chymotrypsin and β-chymotrypsin, and assessed for antibacterial activity. The pepsin digest of bovine casein, which showed antibacterial activity, was fractionated using reverse phase high performance liquid chromatography and the antibacterial peptides isolated were characterised using electrospray ionisation mass spectrometry. Two antibacterial peptides were identified, a novel peptide (Cp1) which corresponded to residues 99–109 of bovine α_S1-casein and a previously reported peptide (Cp2) which corresponded to residues 183–207 of bovine α_S2-casein. The minimum inhibitory concentration (MIC) of Cp1 and Cp2 were determined against a range of bacterial cultures. Cp1 exhibited an MIC of 125 μg mL⁻¹ against all Gram-positive bacteria tested, and MIC ranging between 125 and >1000 μg mL⁻¹ against the Gram-negative bacteria tested. Cp2 was generally far more potent against the Gram-positive bacteria, exhibiting an MIC of 21 μg mL⁻¹, compared to MICs ranging from 332 to >664 μg mL⁻¹ against most of the Gram-negative bacteria tested.     

Prevalence and comparison of Streptococcus infantarius subsp. infantarius and Streptococcus gallolyticus subsp. macedonicus in raw and fermented dairy products from East and West Africa

Streptococcus infantarius subsp. infantarius (Sii) and Streptococcus gallolyticus subsp. macedonicus are members of the Streptococcus bovis/Streptococcus equinus complex (SBSEC) associated with human infections. SBSEC-related endocarditis was furthermore associated with rural residency in Southern Europe. SBSEC members are increasingly isolated as predominant species from fermented dairy products in Europe, Asia and Africa. African variants of Sii displayed dairy adaptations to lactose metabolism paralleling those of Streptococcus thermophilus including genome decay. In this study, the aim was to assess the prevalence of Sii and possibly other SBSEC members in dairy products of East and West Africa in order to identify their habitat, estimate their importance in dairy fermentation processes and determine geographic areas affected by this potential health risk. Presumptive SBSEC members were isolated on semi-selective M17 and SM agar media. Subsequent genotypic identification of isolates was based on rep-PCR fingerprinting and SBSEC-specific16S rRNA gene PCR assay. Detailed identification was achieved through application of novel primers enhancing the binding stringency in partial groES/groEL gene amplification and subsequent DNA sequencing. The presence of S. thermophilus-like lacS and lacZ genes in the SBSEC isolates was determined to elucidate the prevalence of this dairy adaptation. Isolates (n = 754) were obtained from 72 raw and 95 fermented milk samples from Côte d'Ivoire and Kenya on semi-selective agar media. Colonies of Sii were not detected from raw milk despite high microbial titers of approximately 10⁶ CFU/mL on M17 agar medium. However, after spontaneous milk fermentation Sii was genotypically identified in 94.1% of Kenyan samples and 60.8% of Kenyan isolates. Sii prevalence in Côte d'Ivoire displayed seasonal variations in samples from 32.3% (June) to 40.0% (Dec/Jan) and isolates from 20.5% (June) to 27.7% (Dec/Jan) present at titers of 10⁶–10⁸ CFU/mL. lacS and lacZ genes were detected in all Kenyan and 25.8% (June) to 65.4% (Dec/Jan) of Ivorian Sii isolates. Regional differences in prevalence of Sii and dairy adaptations were observed, but no clear effect of dairy animal, fermentation procedure and climate was revealed. Conclusively, the high prevalence of Sii in Kenya, Côte d'Ivoire in addition to Somalia, Sudan and Mali strongly indicates a pivotal role of Sii in traditional African dairy fermentations potentially paralleling that of typical western dairy species S. thermophilus. Putative health risks associated with the consumption of high amounts of live Sii and potential different degrees of evolutionary adaptation or ecological colonization require further epidemiologic and genomic investigations, particularly in Africa.