Journal of Indian Society of Pedodontics and Preventive Dentistry
Journal of Indian Society of Pedodontics and Preventive Dentistry
                                                   Official journal of the Indian Society of Pedodontics and Preventive Dentistry                           
Year : 2020  |  Volume : 38  |  Issue : 4  |  Page : 350--354

In vitro evaluation of milk-based, soy-based, and amino acid-based infant formulas on Streptococcus mutans biofilm formation

Hira Sadan, BM Shanthala, MA Zareena, George Babu, Vidhya Vijayan 
 Department of Pediatric and Preventive Dentistry, Coorg Institute of Dental Sciences, Kudagu, Karnataka, India

Correspondence Address:
Dr. Hira Sadan
Department of Pediatric and Preventive Dentistry, Coorg Institute of Dental Sciences, K.K. Campus, Maggula Village, Virajpet, Kudagu - 571 218, Karnataka


Background and Objective: Infant formulas are based on milk, and the addition of simple carbohydrates as a caloric source, for infants. The carbohydrates added in infant formulas can cause a significant increase of Streptococcus mutans in the oral cavity of infants adding to their cariogenicity. Aim: The aim of the study was to assess and compare the biofilm formation in three commercially available infant formulas;which are based on milk, soy and amino acid. Settings and Design: In vitro microbiological assay of Streptococcus mutans biofilm formation in milk based, soy based and amino acid based infant formulas. Materials and Methods: Twenty-four hour-cultured S. mutans and microtiter plates were used for analysis. At microtiter plate, 190 μL of modified TSB broth containing SBF, MBF, amino acid-based infant formulas, and dairy whitener as a positive control in five dilutions (1:05, 1:10, 1:20, 1:40, and 1:80) was added into respective wells. 10 μL of cultured S. mutans was inoculated into the wells and incubated at 37°C for 24 h. Biofilm was washed, fixed, and stained with crystal violet. The absorbance was measured to evaluate biofilm growth, which was read as optical densities in a spectrophotometer at 490 nm and was tabulated. Results: Three infant formulas tested showed S. mutans biofilm growth. Minimal biofilm growth was observed in amino acid-based formula at 1:80 dilution, followed by MBF at 1:10 dilution and SBF at 1:80 dilution. Conclusion: Commercially available infant formulas favor S. mutans biofilm growth and can be cariogenic. Amino acid-based infant formula was found to have less S. mutans biofilm growth than MBF and SBF.

How to cite this article:
Sadan H, Shanthala B M, Zareena M A, Babu G, Vijayan V. In vitro evaluation of milk-based, soy-based, and amino acid-based infant formulas on Streptococcus mutans biofilm formation.J Indian Soc Pedod Prev Dent 2020;38:350-354

How to cite this URL:
Sadan H, Shanthala B M, Zareena M A, Babu G, Vijayan V. In vitro evaluation of milk-based, soy-based, and amino acid-based infant formulas on Streptococcus mutans biofilm formation. J Indian Soc Pedod Prev Dent [serial online] 2020 [cited 2021 Feb 28 ];38:350-354
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The gold standard for nutrition in infants is always breastfeeding. The American Academy of Pediatrics (AAP) recommends exclusive breastfeeding for the first 6 months of life.[1] Infant formulas are suggested in situations where mothers are unable to breast feed, due to their medical problems or inability for infants to latch on.[2]

Infant formulas are those alternative sources that ensure sufficient calories and nutrient supplementations.[3] An amount of nutrients in the formula are adjusted to make them comparable to breast milk.[4] On manufacturing milk-based infant formulas, cow's milk is altered, and fluoridated water is usually added.[5] Some of those alterations include dilution of the protein content to accommodate the newborn's immature renal tubular system, the substitution of animal fat with vegetable oils, the substitution of animal protein (e.g., casein) for proteins found in breast milk (e.g., whey), and the modification of minerals (e.g., adding iron, adjusting the calcium: phosphorus ratio) in the infant formulas.[1]

Infant formulas can be classified by their content into the following groups: milk-based formula (MBF), soy-based formula (SBF), and protein-based formula (PBF). Protein based formulas are further divided into protein hydrolysate based formulas(PHF's) and amino-acid based formulas. Amino-acid based infant formulas are used in infants with severe allergies to cow's milk.[7]

SBFs are considered in cases of cow's milk allergy or lactose intolerance.[3] These formulas are lactose-free and utilize sucrose and/or corn syrup as the main carbohydrate. The AAP recommends SBFs for children who do not take animal protein and for infants with galactosemia or congenital lactase deficiency.[1]

PHFs are with proteins hydrolyzed into fragments of proteins and amino acids. The amino acid-based formulas provide protein in the form of free amino acids with no peptides[7] and are suitable for infants with protein sensitivity including galactosemia.[6]

Fermentable carbohydrates (e.g., lactose, corn syrup solids, sucrose, maltodextrins, and glucose polymers), in infant formulas, have been implicated in the development of early childhood caries (ECC).[8],[9] All SBF, PBF, and amino acid-based formula contain nonmilk extrinsic sugars (NMES) such as sucrose and glucose syrup as carbohydrate resources.[6] The etiology of ECC is multifactorial and is mainly attributed to a time-specific interaction of microorganisms with sugars on a tooth surface.[10]

Nowadays, infants are being constantly fed on infant formulas beyond their infancy and ECC is one of the major concerns in infants. The relationship between infant formula and ECC has demonstrated high caries inducing a potential following reduction in pH in vivo after rinsing infant formula and varied in vitro buffering capacity.[11] A study conducted to evaluate the knowledge about the dental caries experience and feeding habits in 3–5-year-old children in varying socioeconomic status in Indore, India, reported 61.1% of children fed with infant formula experienced ECC.[12]

Research hypothesis stated was that infant formulas can be cariogenic to an infant's primary dentition and consequent ECC. This study was designed to evaluate the effects of commercially available infant formulas on Streptococcus mutans biofilm formation.

 Materials and Methods

The present in vitro study was conducted after obtaining ethical clearance from the institutional review board.

Commercially available infant formulas selected are given below:

Similac Isomil Soy infant formula (lactose-free infant formula)Nutricia Neocate LCP amino acid-based hypoallergenic infant formula (amino acid-based formula)Nestle Lactogen infant formula (MBFs).

Amulya dairy whitener was selected as a positive control since it is generally prepared from dried milk fats with added sugar. Dairy whitener is generally used in making tea and coffee without having to add sugar and has fewer nutrients.

Replete concentration and source of carbohydrates in infant formulas and dairy whitener

The carbohydrate source in Amulya dairy whitener is as follows: total carbohydrate = 6.25 g/100 ml of standard solution and sucrose = 2.25 g/100 ml of standard solution, and in infant formulas: Lactogen constitutes total carbohydrate of 8.16 g/100 ml of prepared formula, dried glucose, and maltodextrin as the vehicle; Isomil SBF has total carbohydrate of 6.58 g/100 ml of prepared formula and sucrose of 1.3 g/100 ml of standard solution; and Neocate LCP formula constitutes total carbohydrate of 7.2 g/100 ml of prepared formula and dried glucose of 0.65 g/100 ml of prepared formula as per manufacturers.

The selected infant formulas and dairy whitener were purchased from a local grocery store and were used within 3 months from purchase.

Media preparation

Trypticase soy broth (TSB) constitutes tryptone 10 g, sodium chloride 10 g, yeast extract 6 g, and distilled water 1000 m; 30 ml of TSB broth was prepared and autoclaved at 121°C.

Dilution of samples

Samples of Isomil (soy-based), Lactogen (milk-based), Neocate LCP (amino acid-based) formulas and Amulya (dairy whitener) were prepared, respectively, in a series of dilutions (1:5, 1:10, 1:20, 1:40, and 1:80) in 1 ml TSB.

Four samples (SBF, amino acid-based formula, MBF, and dairy whitener) in five different concentrations (1:5, 1:10, 1:20, 1:40, and 1:80) were examined for the S. mutans biofilm formation.

Sample size and design

Since this study involved microbiological assay in controlled laboratory environment, it was decided to repeat the microbiological assay three times, to rule out the procedural error, if any were to occur.

Biofilm formation

Ninety-six well microtiter plates were used for analyzing the formation of S. mutans biofilm. The analysis was carried out in triplicates and 24 h-cultured S. mutans organisms were used for the examination.

In a microtiter plate, 190 μL of modified TSB broth containing SBF, MBF, amino acid-based formulas, and dairy whitener as a positive control was added into respective wells. Ten microliters of 24 h-cultured S. mutans was inoculated into the wells, respectively, and incubated the plates at 37°C for 24 h.

After incubation, 200 μL of the formulated inoculum was made up to 1000 μL by adding 0.9% saline (0.09 g w/v of sodium chloride in 10 ml of distilled water) and 1 ml normal TSB broth and 200 μL normal TSB broth mixed with 800 μL of saline. The growth was measured spectrophotometrically at 595 nm, respectively, by placing distilled water as blank.

Spectrophotometric value for 1 mL TSB broth = 0.095 and 200 μL TSB broth with 800 μL saline = 0.018 was considered.

The formulations were recollected and washed twice by centrifuging at 6000 rpm for 10 min; the supernatant was discarded. Biofilm was fixed by adding 1 mL of 10% formaldehyde and incubated for 30 min at room temperature. After incubation, the biofilm Eppendorf was centrifuged at 6000 rpm for 10 min. The supernatant was discarded and the pellet was washed twice with saline by centrifuging at 6000 rpm for 10 min. The pellets were re-suspended with 0.5% crystal violet and incubated for 30 min at room temperature. Later, the biofilm was washed three times with saline by centrifuging at 6000 rpm for 10 min. Crystal violet was extracted from the biofilm cells by adding 200 μL of isopropanol mixed by vortex and incubated for 1 h at room temperature. Later, the extract was diluted 1:5 (800 μL) with iso-propanol and read at 490 nm with isopropanol used as a blank.

The biofilm growth was read as optical densities in a spectrophotometer at 490 nm by a blinded investigator and was tabulated.


The mean of three measurements was taken, and the statistical analysis of two-way ANOVA for comparing the mean of different infant formulas with its dilutions and of Tukey's post hoc test for pairwise comparison between the infant formulas and dilutions was applied.

A box plot was drawn to show the shape of the distribution of the central value of biofilm formation and its variability at different dilutions in infant formulas and dairy whitener. Variability in SBF and amino acid-based was at 0.5 and 0.2, respectively, and was at 1.5–2.5 in MBF and 1.2–2.5 in dairy whitener. This is suggestive that SBF and amino acid-based formula to be more consistent with its values obtained, whereas MBF and dairy whitener were skewed and inconsistent [Graph 1].[INLINE:1]

Mean and standard deviation of biofilm formation in infant formulas (soy-based, milk-based, and amino acid-based) and dairy whitener at different dilutions (1:5, 1:10, 1:20, 1:40, and 1:80) were tabulated in [Table 1].{Table 1}

Two way ANOVA from [Table 2] shows that the comparison between different infants formulas in its different dilutions tested was statistically significant.{Table 2}

Post hoc test for interaction effect between the dilution of minimum and maximum biofilm formation within the infant formulas is tabulated in [Table 3], and it was found to be not significant between the dilutions tested with amino acid-based and soy-based infant formulas, whereas significant in milk-based infant formula and dairy whitener. This means that dilutions did not affect Streptococcus biofilm formation in soy-based and amino acid-based infant formulas.{Table 3}

Post hoc test for interaction between infant formulas at dilutions with minimal biofilm formation was tested to identify the less cariogenic potential infant formula dilution [Table 4], and it was found that ,1:80 dilution of amino acid infant formula was found to have least biofilm formation and was not statistically significant, when compared to milk and soy based infant formulas. Whereas, it was found to be significant when compared to dairy whitener.{Table 4}

Amino acid-based formula has shown the least biofilm formation at 1:80 dilution.


Despite the predominant evidence that human milk has a huge variety of nutritional and nonnutritional ascendancy, many children are fed on infant formulas even before 6 months and later. ECC is a major concern for a pediatric dentist because it infects any time from the emergence of primary dentition in infants.

The cariogenic potential of milk, infant formulas, and sugar solutions was studied, in a desalivated rat model, and it was found that infant formulas had increased cariogenic potential when compared to plain milk due to its higher carbohydrate variability.[13] Sheikh and Erickson have demonstrated that certain infant formulas support significant bacterial growth and play an important role in the establishment of cariogenic organisms in the oral cavity.[8]

The carbohydrate content of infant formula is either sucrose (NMES) or lactose based. Sucrose is considered the most cariogenic dietary carbohydrate because it is fermentable by oral bacteria.[4] A literature review of the cariogenic potential of infant formulas reported that (1) SBFs are significantly more cariogenic than MBFs; (2) infant formulas containing only NMES and those containing lactose with NMES are found to be significantly more cariogenic than formulas containing only lactose; (3) no significant correlation was found between cariogenicity and casein content in infant formulas; and (4) the cariogenicity of various types of infant formulas was inconclusive and recommended further studies.[1]

All three infant formulas (soy-based, amino-acid based, and milk-based) including dairy whitener tested have shown significant S. mutans biofilm formation and were similar, with the findings of the studies by Bowen et al.,[13] Chaudhary et al.,[6] and Hinds et al.[4]

Review on cariogenic potential of infant formulas based on its carbohydrate composition has reported that sucrose based infant formulas favored increase biofilm formation followed by those containing NMES with lactose or without lactose. The infant formulas based on lactose carbohydrate alone , were found to have less cariogenic potential.[1],[4] However, in the present study, dried glucose and maltodextrin containing milk-based infant formula has shown increased S. mutans biofilm formation than sucrose content soy-based infant formula. The probable reason is vowing to total carbohydrate concentration in infant formulas. The total carbohydrate concentration in milk-based infant formula is 8.16 g/100 ml and in soy-based is 6.58/100 ml. In the dilutions tested, amino acid-based infant formula in 1:80 dilution has shown minimal S. mutans biofilm growth compared to soy-based in 1:10 dilution and milk-based in 1:80 dilution.

The findings from this study can be discussed with the pediatrician to consider the dilutions of infant formulas, and their nutritional requirement, as it would not be a concern especially after 6 months of age for the reason; infants would have been introduced to semi-solid food by 6 months of age. Whereas in first six months of infants, the nutritional demands are met solely by breast feeding or infant formulas. Therefore the recommended dilutions for infant formulas from this study are : for Soy- based formula, at 10 g/100 ml of distilled water, for amino acid-based formula and milk-based formula at 12 g/100 ml of distilled water. However, further studies on understanding of acquisition of S. mutans and biofilm formation with the emergence of primary dentition at an early age of 6 months and consequent effect in developing ECC are recommended.


Dilution of the commercially available infant formula may be recommended with the concern of developing ECC, due to its cariogenic potential in infants after the emergence of primary dentition.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


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