Activity of Streptococcus mutans VicR Is Modulated by Antisense RNA

The VicRK 2-component system of Streptococcus mutans regulates genes associated with cell wall biogenesis and biofilm formation. A putative RNase III–encoding gene ( rnc) is located downstream from the vicRKX operon. The goals of this study were to investigate the potential role of VicR in the regulation of adjacent downstream genes and evaluate transcription levels of vicR during planktonic and biofilm growth. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to investigate whether vicRKX and adjacent downstream genes were cotranscribed. Binding of purified recombinant VicR protein to promoter regions of vicR, rnc, and syfA genes was confirmed by electrophoretic mobility shift assay and by chromatin immunoprecipitation analyses. VicR antisense (AS vicR) RNA was detected by Northern blotting and qRT-PCR assays. AS vicR overexpression mutants were constructed, and the biofilm biomass was determined by crystal violet microtiter assay. Adjacent downstream genes rnc, smc, syfA, smu.1511, and syfB were cotranscribed with vicRKX. The predicted promoter regions of vicR, rnc, and syfA genes were directly regulated by VicR. An AS vicR RNA transcript was detected upstream of the rnc gene. Expression of the AS vicR RNA transcript was elevated in planktonic cultures and repressed during biofilm growth. In addition, Western blot data showed that expression of the VicR protein decreased by 35% in planktonic as compared with biofilm cultures. Furthermore, we show that overexpression of AS vicR led to a reduction in biofilm formation. The downstream genes rnc, smc, syfA, smu.1511, and syfB are cotranscribed with vicRKX. VicR is autophosphorylated, and rnc and syfA are directly regulated by VicR. Expression of VicR protein correlated inversely with different levels of AS vicR RNA transcript and growth conditions. The biofilm biomass decreased in the AS vicR overexpression mutant. These data suggest a role for the AS vicR RNA transcript in posttranscriptional regulation of VicR protein production in S. mutans.

Download Full-text

Role of the Streptococcus mutans irvA Gene in GbpC-Independent, Dextran-Dependent Aggregation and Biofilm Formation

Applied and Environmental Microbiology ◽

10.1128/aem.01015-09 ◽

2009 ◽

Vol 75 (22) ◽

pp. 7037-7043 ◽

Cited By ~ 15

Author(s):

Min Zhu ◽

Dragana Ajdić ◽

Yuan Liu ◽

David Lynch ◽

Justin Merritt ◽

...

Keyword(s):

Streptococcus Mutans ◽

Biofilm Formation ◽

Growth Conditions ◽

Parental Background ◽

A Cell ◽

Major Surface ◽

Biofilm Development ◽

Small Aggregation

ABSTRACT Dextran-dependent aggregation (DDAG) of Streptococcus mutans is an in vitro phenomenon that is believed to represent a property of the organism that is beneficial for sucrose-dependent biofilm development. GbpC, a cell surface glucan-binding protein, is responsible for DDAG in S. mutans when cultured under defined stressful conditions. Recent reports have described a putative transcriptional regulator gene, irvA, located just upstream of gbpC, that is normally repressed by the product of an adjacent gene, irvR. When repression of irvA is relieved, there is a resulting increase in the expression of GbpC and decreases in competence and synthesis of the antibiotic mutacin I. This study examined the role of irvA in DDAG and biofilm formation by engineering strains that overexpressed irvA (IrvA+) on an extrachromosomal plasmid. The IrvA+ strain displayed large aggregation particles that did not require stressful growth conditions. A novel finding was that overexpression of irvA in a gbpC mutant background retained a measure of DDAG, albeit very small aggregation particles. Biofilms formed by the IrvA+ strain in the parental background possessed larger-than-normal microcolonies. In a gbpC mutant background, the overexpression of irvA reversed the fragile biofilm phenotype normally associated with loss of GbpC. Real-time PCR and Northern blot analyses found that expression of gbpC did not change significantly in the IrvA+ strain but expression of spaP, encoding the major surface adhesin P1, increased significantly. Inactivation of spaP eliminated the small-particle DDAG. The results suggest that IrvA promotes DDAG not only by GbpC, but also via an increase in P1.

Download Full-text

Control of Enzyme IIscr and Sucrose-6-Phosphate Hydrolase Activities in Streptococcus mutans by Transcriptional Repressor ScrR Binding to the cis-Active Determinants of the scr Regulon

Journal of Bacteriology ◽

10.1128/jb.185.19.5791-5799.2003 ◽

2003 ◽

Vol 185 (19) ◽

pp. 5791-5799 ◽

Cited By ~ 12

Author(s):

Bing Wang ◽

Howard K. Kuramitsu

Keyword(s):

Streptococcus Mutans ◽

Transcriptional Repressor ◽

Direct Repeat ◽

Dnase I ◽

Repeat Sequence ◽

Promoter Regions ◽

Mobility Shift ◽

Promoter Sequences ◽

In The Wild ◽

Dna Binding Properties

ABSTRACT In Streptococcus mutans, enzyme IIscr and sucrose-6-phosphate hydrolase are two important enzymes in the transport and metabolism of dietary sucrose. The scr regulon of S. mutans is composed of three genes, scrA and scrB, which code for enzyme IIscr and sucrose-6-phosphate hydrolase, respectively, and scrR, which codes for a GalR-LacI-type transcription regulator. It was previously shown that expression of both scrA and scrB is similarly induced by sucrose. Mutation in the scrR gene resulted in increased expression of scrB relative to that in the wild-type strain. In this study, we employed DNA mobility shift and DNase I protection assays with a purified ScrR-histidine tag fusion protein to examine the DNA binding properties of ScrR to the promoter regions of the scrA and scrB genes. The results showed that ScrR bound specifically to the promoter regions of both scrA and scrB. Two regions with high affinity for ScrR in the promoter sequences of the scrA and scrB genes were identified by DNase I protection assays. One, O C, which includes a 20-bp imperfect inverted-repeat sequence, is located between the two promoters, and the other, O B, is located within the scrB promoter region containing a 37-bp imperfect direct-repeat sequence. Mutations of O B and O C resulted in constitutive transcription and expression of both the scrA and scrB genes. Our results indicated that S. mutans coordinates the activities of enzyme IIscr and sucrose-6-phosphate hydrolase by transcriptional repressor ScrR binding to the promoter regions of the scr regulon.

Download Full-text

Effect of Human Milk and its Components on Streptococcus Mutans Biofilm Formation

Journal of Clinical Pediatric Dentistry ◽

10.17796/1053-4628-39.3.255 ◽

2015 ◽

Vol 39 (3) ◽

pp. 255-261 ◽

Cited By ~ 11

Author(s):

LM Allison ◽

LA Walker ◽

BJ Sanders ◽

Z Yang ◽

G Eckert ◽

...

Keyword(s):

Breast Milk ◽

Streptococcus Mutans ◽

Biofilm Formation ◽

Current Knowledge ◽

Post Partum ◽

Human Breast Milk ◽

Human Breast ◽

Flat Bottom ◽

Biofilm Growth ◽

Microtiter Plates

Objective: This study investigated the effects of human breast milk and its components on the nutritional aspect of the caries process due to Streptococcus mutans UA159 biofilm formation. Study design: Human breast milk was collected from 11 mothers during 3-9 months postpartum. To test for the effect on biofilm formation, a 16-hour culture of S. mutans was treated with dilutions of human breast milk and several major components of human breast milk, lactose, lactoferrin, IgA, and bovine casein in sterile 96-well flat bottom microtiter plates for 24 hours. The biofilms were fixed, washed, stained with crystal violet, and extracted. Absorbance was measured to evaluate biofilm growth mass. Results: Dilutions 1:10-1:2,560 of the human breast milk samples increased biofilm formation by 1.5-3.8 fold compared to the control. Lactoferrin decreased biofilm formation significantly in all dilutions (average milk concentration of 3 mg/ml). Lactose had no effect at average breast milk concentrations (60 mg/ml) except at its lowest concentration (15 mg/ml) where it was increased. IgA significantly decreased biofilm formation at its highest concentration of 2,400 μg/ml (average milk concentration 600 μg/ml). Casein caused significantly increased biofilm formation at all concentrations tested above the average milk content (2.3 mg/ml). Conclusions: The results of this study demonstrate an increase in S. mutans biofilm formation by human breast milk 3-9 months post partum. Among its major components, only casein significantly increased biofilm formation among the concentrations analyzed. Lactose had no effect except at 15 mg/ml. Lactoferrin and IgA significantly decreased S. mutans biofilm formation at their highest concentrations. This information expands the current knowledge regarding the nutritional influence of breastfeeding and validates the necessity to begin an oral hygiene regimen once the first tooth erupts.

Download Full-text

Effects of Cola-Flavored Beverages and Caffeine on Streptococcus mutans Biofilm Formation and Metabolic Activity

Journal of Clinical Pediatric Dentistry ◽

10.17796/1053-4628-41.4.294 ◽

2017 ◽

Vol 41 (4) ◽

pp. 294-299 ◽

Cited By ~ 2

Author(s):

Roger P Dotsey ◽

Elizabeth A S Moser ◽

George J Eckert ◽

Richard L Gregory

Keyword(s):

Streptococcus Mutans ◽

Metabolic Activity ◽

Crystal Violet ◽

Biofilm Formation ◽

Bacterial Biofilm ◽

Water Soluble ◽

High Fructose Corn Syrup ◽

Biofilm Growth ◽

High Fructose

Objective: To examine the effects of cola-flavored beverages and caffeine on growth and metabolism of Streptococcus mutans biofilm. This study was designed to determine if carbonated beverages or caffeine can increase S. mutans growth and biofilm formation and metabolic activity in vitro, potentially leading to increased S. mutans-associated cariogenicity in children that consume them. Study Design: Six different cola-flavored products, plus pure caffeine, and pure high fructose corn syrup (HFCS), at different concentrations similar to those in the beverages were tested. A 16-hour culture of S. mutans was treated with different dilutions in bacteriological media. To test for the effect on biofilm formation, the biofilm was stained with crystal violet. The absorbance was determined to evaluate biofilm growth. Biofilm metabolic activity was measured based on biofilm having the ability to reduce XTT to a water-soluble orange compound. Results: The inclusion of HFCS in the beverages, as well as pure HFCS, significantly enhanced bacterial biofilm formation and metabolic activity. Pure caffeine and the presence of caffeine in beverages did not significantly increase biofilm formation, but pure caffeine significantly increased metabolism, and Diet Coke had significantly greater metabolic activity than Caffeine-Free Diet Coke. Conclusions: HFCS increases both the biofilm formation and metabolism of S. mutans, and caffeine in some cases increases metabolism of S. mutans.

Download Full-text

Antimicrobial Effect of a Peptide Containing Novel Oral Spray on Streptococcus mutans

BioMed Research International ◽

10.1155/2020/6853652 ◽

2020 ◽

Vol 2020 ◽

pp. 1-13 ◽

Cited By ~ 1

Author(s):

Kaixin Xiong ◽

Xuan Chen ◽

Hantao Hu ◽

Huihui Hou ◽

Peng Gao ◽

...

Keyword(s):

Antimicrobial Peptide ◽

Minimal Inhibitory Concentration ◽

Streptococcus Mutans ◽

Biofilm Formation ◽

Inhibitory Concentration ◽

Antimicrobial Effect ◽

Inhibitory Effect ◽

Diffusion Method ◽

Qrt Pcr ◽

Initial Adhesion

Objective. To investigate the antibacterial effect of a novel antimicrobial peptide containing oral spray GERM CLEAN on Streptococcus mutans (S. mutans) in vitro and further explore the related mechanisms at phenotypic and transcriptional levels. Methods. The disk diffusion method was used to preliminarily appraise the antimicrobial effect of GERM CLEAN. The minimal inhibitory concentration (MIC) of GREM CLEAN towards S. mutans was determined by the broth dilution method. S. mutans virulence-related phenotypic assays including initial adhesive assay, pH drop, exopolysaccharides (EPS), and biofilm formation measurements and quantitative real-time PCR (qRT-PCR) were further applied to detect the inhibitory mechanisms of GREM CLEAN at 1/2MIC. Results. The diameter (10.18 ± 1.744 mm) of inhibition zones formed by GERM CLEAN preliminarily indicated its inhibitory effect on the major cariogenic bacteria S. mutans. The minimal inhibitory concentration of GERM CLEAN on S. mutans was 100% mass fraction (the stock solution). The study of the antibacterial mechanism showed that GERM CLEAN had a certain inhibitory effect on the initial adhesion, acid production, extracellular polysaccharides (EPS) production, and biofilm formation of S. mutans. GERM CLEAN disturbed S. mutans biofilm physiology mainly through destruction of biofilm architecture and suppression of bacterial growth. The results of qRT-PCR further confirmed that the expression levels of EPS and lactic acid generation genes including gtfB, gtfC, gtfD, and ldh were significantly repressed by treating with GERM CLEAN, and this was consistent with our phenotypic results. Conclusion. The novel antimicrobial peptide containing oral spray GERM CLEAN has an anti-Streptococcus mutans effect and the inhibitory property may be due to suppression of the virulence factors of S. mutans including adhesive, acidogenicity, EPS, and biofilm formation.

Download Full-text

MgrA Negatively Regulates Biofilm Formation and Detachment by Repressing the Expression of psm Operons in Staphylococcus aureus

Applied and Environmental Microbiology ◽

10.1128/aem.01008-18 ◽

2018 ◽

Vol 84 (16) ◽

Cited By ~ 8

Author(s):

Qiu Jiang ◽

Zeyu Jin ◽

Baolin Sun

Keyword(s):

Staphylococcus Aureus ◽

Mutant Strain ◽

Biofilm Formation ◽

Negative Regulator ◽

Regulatory Mechanisms ◽

Promoter Regions ◽

Mobility Shift ◽

Content Type ◽

Multiple Functions ◽

Biofilm Development

ABSTRACT Phenol-soluble modulins (PSMs) are amphipathic peptides that are produced by staphylococci and play important roles in Staphylococcus aureus biofilm formation and dissemination. Although the multiple functions of PSMs have been recognized, the regulatory mechanisms controlling the expression of psm operons remain largely unknown. In this study, we identified MgrA in a DNA pulldown assay and further demonstrated, by electrophoretic mobility shift assays and DNase I footprinting assays, that MgrA could bind specifically to the promoter regions of psm operons. We then constructed an isogenic mgrA deletion strain and compared biofilm formation and detachment in the wild-type and isogenic mgrA deletion strains. Our results indicated that biofilm formation and detachment were significantly increased in the mgrA mutant strain. Real-time quantitative reverse transcription-PCR data indicated that MgrA repressed the transcription of psm operons in cultures and biofilms, suggesting that MgrA is a negative regulator of psm expression. Furthermore, we analyzed biofilm formation by the psm mutant strains, and we found that PSMs promoted biofilm structuring and development in the mgrA mutant strain. These findings reveal that MgrA negatively regulates biofilm formation and detachment by repressing the expression of psm operons through direct binding to the psm promoter regions.IMPORTANCE Staphylococcus aureus is a human and animal pathogen that can cause biofilm-associated infections. PSMs have multiple functions in biofilm development and virulence in staphylococcal pathogenesis. This study has revealed that MgrA can negatively regulate psm expression by binding directly to the promoter regions of psm operons. Furthermore, our results show that MgrA can modulate biofilm structuring and development by repressing the production of PSMs in S. aureus. Our findings provide novel insights into the regulatory mechanisms of S. aureus psm gene expression, biofilm development, and pathogenesis.

Download Full-text

Effect of a Tea Polyphenol on Different Levels of Exposure of Nicotine and Tobacco Extract on Streptococcus mutans Biofilm Formation

Frontiers in Oral Health ◽

10.3389/froh.2021.737378 ◽

2021 ◽

Vol 2 ◽

Author(s):

Emily S. Taylor ◽

Grace F. Gomez ◽

Elizabeth A. S. Moser ◽

Brian J. Sanders ◽

Richard L. Gregory

Keyword(s):

Streptococcus Mutans ◽

Biofilm Formation ◽

Inhibitory Effect ◽

Biofilm Growth ◽

Secondary Exposure ◽

Smoking Exposure ◽

Dye Staining ◽

Total Growth ◽

Tobacco Extract ◽

Different Levels

Objective: The purpose of this study was to compare the effects of different levels of nicotine and tobacco extract exposure on Streptococcus mutans biofilm formation and the inhibitory effect of the polyphenol epigallocatechin-3 gallate (EGCG) found in green tea. This study addressed the results of biofilm assays with EGCG and varying relative concentrations of nicotine and tobacco extract consistent with primary, secondary and tertiary levels of smoking exposure. Primary smoking exposure to nicotine has been demonstrated to significantly increase biofilm formation, while EGCG has been demonstrated to reduce S. mutans biofilm formation.Methods:S. mutans was treated with varying levels of nicotine or cigarette smoke condensate (CSC) concentrations (0–32 mg/ml and 0–2 mg/ml, respectively) in Tryptic Soy broth supplemented with 1% sucrose for different lengths of time simulating primary, secondary and tertiary smoking exposure with and without 0.25 mg/ml EGCG. The amount of total growth and biofilm formed was determined using a spectrophotometric crystal violet dye staining assay.Results: For both nicotine and CSC, primary exposure displayed overall significantly less growth compared to secondary exposure. For nicotine, secondary exposure demonstrated significantly greater growth than tertiary exposure levels. Overall, significantly greater total bacterial growth and biofilm formation in the presence of nicotine and CSC was observed in the absence of EGCG than in the presence of EGCG. However, biofilm growth was not significantly different among different concentrations of CSC.Conclusion: The results of this study help illustrate that nicotine-induced S. mutans biofilm formation is reduced by the presence of EGCG. This provides further evidence of the potential beneficial properties of polyphenols.

Download Full-text

Lauryl Gallate Activity and Streptococcus mutans: Its Effects on Biofilm Formation, Acidogenicity and Gene Expression

Molecules ◽

10.3390/molecules25163685 ◽

2020 ◽

Vol 25 (16) ◽

pp. 3685

Author(s):

Vika Gabe ◽

Mouhammad Zeidan ◽

Tomas Kacergius ◽

Maksim Bratchikov ◽

Mizied Falah ◽

...

Keyword(s):

Gene Expression ◽

Streptococcus Mutans ◽

Biofilm Formation ◽

Solid Surfaces ◽

Dependent Manner ◽

Ethyl Gallate ◽

Planktonic Cells ◽

Biofilm Growth ◽

Biofilm Development ◽

Lauryl Gallate

Streptococcus mutans bacterium is implicated in the pathogenesis of dental caries due to the production of biofilm and organic acids from dietary sucrose. Despite the availability of various means of prophylaxis, caries still has a high worldwide prevalence. Therefore, it is important to find new pharmaceuticals to inhibit S. mutans biofilm formation and acidogenicity. The aim of the current study was to evaluate the activity of lauryl gallate (dodecyl gallate) against S. mutans acidogenicity, the expression of biofilm-associated genes, and biofilm development on solid surfaces (polystyrene, glass). The biofilm quantities produced by S. mutans bacteria were assessed using colorimetric and optical profilometry techniques. Acidogenicity was evaluated by measuring the pH of the biofilm growth medium with microelectrode. Assessment of the expression of gene coding for glucan-binding protein B (gbpB), glucosyltranferases B, -C, -D (gtfB, -C, -D), and the F-ATPase β subunit of F1 protein (atpD) was carried out using a quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The results demonstrate the capacity of lauryl gallate to significantly inhibit S. mutans acidogenicity and biofilm development on solid surfaces, in a dose-dependent manner, compared to untreated bacteria (p < 0.05). The highest activity of lauryl gallate occurred at a concentration of 98.98 µM, at which it suppressed biofilm formation by 100% and lowered pH levels by 98%. The effect of lauryl gallate treatment on gene expression changes, as demonstrated by our RT-qPCR data, was limited to the gtfD gene only, was a significant (48%) decrease in gene expression, obtained for the biofilm-producing bacteria, while a 300% increase in fold change for the same gene occurred in the planktonic cells. It is important to note that in previous studies we showed a broader effect of related derivatives. However, a similar magnitude of difference in effects between biofilm and planktonic cells for the atpD gene was obtained after treatment with octyl gallate and reverse magnitude for the same gene after treatment with ethyl gallate. Therefore, to ascertain the possible direct or indirect effects of lauryl gallate, as well as octyl gallate and ethyl gallate, more research is needed to examine the effects on the amount of enzymes and on the enzymatic activity of the products of the affected genes that are involved in the production and maintenance of biofilm by S. mutans.

Download Full-text

The effect of ArcA on the growth, motility, biofilm formation, and virulence of Plesiomonas shigelloides

BMC Microbiology ◽

10.1186/s12866-021-02322-y ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Junxiang Yan ◽

Yuehua Li ◽

Xueqian Guo ◽

Xiaochen Wang ◽

Fenxia Liu ◽

...

Keyword(s):

Mutant Strain ◽

Biofilm Formation ◽

Target Genes ◽

Glucose Utilization ◽

Anaerobic Metabolism ◽

Promoter Regions ◽

Mobility Shift ◽

Plesiomonas Shigelloides ◽

Mobility Shift Assay ◽

Aerobic And Anaerobic

Abstract Background The anoxic redox control binary system plays an important role in the response to oxygen as a signal in the environment. In particular, phosphorylated ArcA, as a global transcription factor, binds to the promoter regions of its target genes to regulate the expression of aerobic and anaerobic metabolism genes. However, the function of ArcA in Plesiomonas shigelloides is unknown. Results In the present study, P. shigelloides was used as the research object. The differences in growth, motility, biofilm formation, and virulence between the WT strain and the ΔarcA isogenic deletion mutant strain were compared. The data showed that the absence of arcA not only caused growth retardation of P. shigelloides in the log phase, but also greatly reduced the glucose utilization in M9 medium before the stationary phase. The motility of the ΔarcA mutant strain was either greatly reduced when grown in swim agar, or basically lost when grown in swarm agar. The electrophoretic mobility shift assay results showed that ArcA bound to the promoter regions of the flaK, rpoN, and cheV genes, indicating that ArcA directly regulates the expression of these three motility-related genes in P. shigelloides. Meanwhile, the ability of the ΔarcA strain to infect Caco-2 cells was reduced by 40%; on the contrary, its biofilm formation was enhanced. Furthermore, the complementation of the WT arcA gene from pBAD33-arcA+ was constructed and all of the above features of the pBAD33-arcA+ complemented strain were restored to the WT level. Conclusions We showed the effect of ArcA on the growth, motility, biofilm formation, and virulence of Plesiomonas shigelloides, and demonstrated that ArcA functions as a positive regulator controls the motility of P. shigelloides by directly regulating the expression of flaK, rpoN and cheV genes.

Download Full-text

Analysis of the Cariogenic Potential of Various Almond Milk Beverages using a Streptococcus mutans Biofilm Model in vitro

Caries Research ◽

10.1159/000479936 ◽

2017 ◽

Vol 52 (1-2) ◽

pp. 51-57 ◽

Cited By ~ 6

Author(s):

Janelle Lee ◽

Janice A. Townsend ◽

Tatyana Thompson ◽

Thomas Garitty ◽

Arpan De ◽

...

Keyword(s):

Streptococcus Mutans ◽

Biofilm Formation ◽

Acid Production ◽

Bovine Milk ◽

Buffering Capacity ◽

Soy Milk ◽

Ph Titration ◽

Biofilm Growth ◽

Titration Assay

To evaluate the cariogenic properties of almond milk beverages, 6 almond milks, along with soy and whole bovine milk, were analyzed for their abilities to support Streptococcus mutans biofilm formation and acid production, and their capacity to buffer changes in pH. Biofilm formation by S. mutans was analyzed using an in vitro 96-well plate model and measured by crystal violet staining. Acid production by S. mutans was evaluated by a colorimetric L-lactate assay and pH measurement of bacterial cultures. Buffering capacity was assessed by a pH titration assay. Soy milk supported the most biofilm growth, while the least was observed with unsweetened almond milk (both p < 0.001). Among almond milks, sucrose-sweetened milk led to the highest level of biofilm formation (p < 0.001), while the least was observed with unsweetened milk (p < 0.05). Sucrose-sweetened almond milk yielded the lowest pH (4.56 ± 0.66), followed by soy milk and bovine milk; the highest pH was with unsweetened almond milk (6.48 ± 0.5). When analyzed by pH titration, the unsweetened almond milk displayed the weakest buffering capacity while bovine milk showed the highest (p < 0.001). These results suggest that the almond milk beverages, except those that are sweetened with sucrose, possess limited cariogenic properties, while soy milk exhibits the most cariogenic potential. As milk alternatives become increasingly popular, dentists must counsel their patients that almond milks, especially sucrose-sweetened varieties, have cariogenic potential. For patients who are lactose-intolerant or suffer from milk allergy, almond milks may be a better alternative than soy-based products.

Download Full-text