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. Author manuscript; available in PMC: 2020 Sep 1.
Published in final edited form as: Arch Oral Biol. 2019 Jun 26;105:52–58. doi: 10.1016/j.archoralbio.2019.05.033

Analysis of Cariogenic Potential of Alternative Milk Beverages by In Vitro Streptococcus mutans Biofilm Model and Ex Vivo Caries Model

Yan Huang 1, Tatyana Thompson 2, Yapin Wang 2, Qingzhao Yu 3, Lin Zhu 3, Xiaoming Xu 2, Zezhang T Wen 2,4,*, Janice A Townsend 5,*
PMCID: PMC6629491  NIHMSID: NIHMS1035736  PMID: 31276938

Abstract

Objective:

To evaluate the potential of various alternative milk beverages to support bacterial biofilm formation and acid production and cause unbalanced demineralization.

Design:

In vitro assays were used to examine the ability of the beverages to support Streptococcus mutans’ biofilm formation and acid production from sugar fermentation and the capacity of the beverages to buffer pH changes. Biofilm formation was done using 96-well plate model. Acid production was measured using L-Lactate assay kit, and the buffering capacity was assessed by pH titration. For ex vivo caries model, enamel and dentine slabs and S. mutans biofilms were exposed to selected alternative milk beverages three times a day, 30 minutes each, and by the end of the experiments, slab’s demineralization was assessed by loss of surface microhardness.

Results:

Of the alternative milk beverages tested in this study, Original Almond consistently supported the most S. mutans biofilms, followed by Chocolate Cashew Milk, while the least biofilms were measured with Unsweetened Flax Milk. The most acids and the lowest culture pH were measured with Toasted Coconut Almond Milk, while the least buffering capacity was measured with Unsweetened Coconut Milk. The results of ex vivo caries model showed that like Bovine Whole Milk, repeated exposure to Original Almond led to significant enamel and dentine slab demineralization, when compared to those exposed to saline as a control (P<0.001).

Conclusions:

These results further provide support that popular alternative milk beverages, especially those with supplemental sugars, are potentially cariogenic.

Keywords: alternative milk beverages, dental caries, demineralization potential, Streptococcus mutans, ex vivo caries model

INTRODUCTION

Dental caries is still one the most prevalent infectious diseases and caries rates in preschool children are increasing in the United States (Tinanoff & Reisine, 2009). Caries is the dissolution of tooth structure by acidic metabolites of bacterial sugar fermentation in the plaque (Loesche, 1986). It is a multi-factorial disease and major contributing factors include key groups of bacteria in the plaque microbiota, saliva flow and composition, oral hygiene practice, and the nature and frequency of sugar intake. The constant consumption of a sucrose-rich diet under conditions such as saliva deficiency can prompt an ecological shift in the plaque microbiota (Bowen, Burne, Wu, & Koo, 2017; Takahashi & Nyvad, 2011), leading to overgrowth of acidogenic and aciduric species, which include Streptococcus mutans. As a commensal of the plaque microbiota, S. mutans is known for its ability to form robust biofilms on the tooth surface, to rapidly metabolize various kind of dietary sugars and release acids, and to survive numerous and frequent environmental challenges such as low pH in the plaque (Bowen et al., 2017; Lemos et al., 2019; Moye, Zeng, & Burne, 2014). S. mutans’ cariogenicity relies on its acidogenic and aciduric properties, which enable the bacterium not only to produce acid from sugar fermentation but also to survive and proliferate under an acidic environment. Consequently, the dysbiotic plaque microbiota and its continuous acid production breaks the balance of the demineralization-remineralization on the tooth surface, resulting in loss of tooth mineral content and the development of dental caries (Dashper et al., 2012; Loesche, 1986; Takahashi & Nyvad, 2011).

Bovine milk is one of the more widely consumed foods worldwide, although its consumption in the Unites States has decreased, with a total volume of sale reduced by >8% from 2013–2018 (Dun&Bradstreet, 2018). On the other hand, alternative milk beverages are on the rise over the last several years, which include those from soy, flax, rice, almond, coconut, cashew, pecan, and macadamia, with soy milk, almond milk and rice milk among the most prevalent in the United States (Dewan, 2016). It is estimated that the sales of the plant-based alternative milk beverages have grown from $5.1 billion in 2013 to a projected $10.9 billion in 2019 (Dewan, 2016). Various reasons can be attributed to this surge, including dietary restrictions like lactose intolerance, weight loss diets, hormones, antibiotics, genetic engineering, ethical concerns for animal rights, popularity of plant-based diets, and the perception that milk alternatives are healthier than bovine milk (Dewan, 2016; Dun&Bradstreet, 2018; Ellis & Lieb, 2015; IBISWorld, 2017; Kulis, Wright, Jones, & Burks, 2015; McCarthy, Parker, Ameerally, Drake, & Drake, 2017; Sethi, Tyagi, & Anurag, 2016). Another contributing factor is rising awareness of food allergies to bovine-based products. Food allergies have increased in prevalence during the past two decades and it is estimated that 4–6% of the US population is allergic to foods (Kulis et al., 2015). One of the most common sources of food allergies in the United States is bovine milk (Kulis et al., 2015). These allergies have consequences that are both physiological and psychological that result from social isolation which include anxiety and increased risk for bullying (Lieberman, Weiss, Furlong, Sicherer, & Sicherer, 2010). For all these reasons, alternative milk beverages are perceived by consumers to be a desirable replacement for bovine milk.

Bovine milk contains various antimicrobial peptides like lactoferrin, lysozyme, and peroxide, which may contribute to anticariogenicity (Skinner, Ziegler, & Ponza, 2004; Wang, Bleich, & Gortmaker, 2008). However, there is also strong evidence that bovine milk can cause caries if consumed in high frequencies. In an ex vivo caries model, plain and sweetened bovine milk have been found to have a high buffering capacity but also support the formation of S. mutans biofilms and cause significant reduction of hardness, indicative of caries potential (Bowen, Pearson, Rosalen, Miguel, & Shih, 1997; Giacaman & Munoz-Sandoval, 2014; Lee et al., 2018; Munoz-Sandoval, Munoz-Cifuentes, Giacaman, Ccahuana-Vasquez, & Cury, 2012; Prabhakar, Kurthukoti, & Gupta, 2010; Sheik & Erickson, 1996). In studies of dairy milk alternatives, Daspher et al. also found that soy milk supported significantly higher yield of S. mutans and higher quantities of acid production from fermentation. In addition, it also had a lower buffering capacity as compared to bovine milk, which suggests soy milk has a higher cariogenic potential than bovine milk (Dashper et al., 2012).

We have also recently shown that when analyzed using an in vitro S. mutans biofilm model, the almond milk beverages, especially the beverages containing supplemental cane sugar, displayed strong capacity to support S. mutans biofilm formation and acid production from sugar fermentation (Lee et al., 2018). In this study, an ex vivo caries model was used to further analyze the ability of selected alternative milk beverages to cause tooth enamel and dentine demineralization and thus, reduction of the hardness of the tooth structure. In addition, selected brands of flax, rice, coconut, cashew, pecan, and macadamia alternative milk beverages were also analyzed for their cariogenic potential using in vitro S. mutans biofilm model, including their ability to support biofilm formation and acid production by S. mutans and their capacity to buffer pH changes, with a bovine whole milk serving as a control.

MATERIALS AND METHODS

Institutional approvals.

All procedures utilized in this study conformed to the applicable ethical guidelines and regulations (Institutional Review Board # 8758 and Institutional Biosafety Committee #14379) of the Louisiana State University Health Sciences Center, New Orleans, Louisiana, USA.

Alternative milk beverages.

A total of fourteen beverages were analyzed in this study. They represented seven different types of alternative milk beverages and included three coconut milks, four cashew milks, two flax milks, two macadamia milks, one pecan milk, one almond and one bovine whole milk. The brand names, manufacturers of, and the abbreviations used for these milk beverages, along with their major nutrient content, are listed in Table 1.

Table 1.

Alternative milk beverages tested and the major nutrient facts

Name CODE Total CHO (g) † Sugars (g) † Sugars added‡ Proteins (g) † Total fat (g) †
Forager Project® Unsweetened Plain Cashew Milk FUC 4 1 None 1 3.5
Forager Project® Chocolate Cashew Milk FCC 10 6 None 2 5
Forager Project® Original Cashew Milk FOC 7 4 None 1 3.5
Califia Farms® Toasted Coconut Almond Milk CCA 2 1 None 0 4
So Delicious® Coconut Vanilla Milk SCV 9 8 O-Cane Sugar <1 4.5
So Delicious® Unsweetened Coconut Milk SCU 1 <1 None 0 4.5
So Delicious® Original Coconut Milk SCO 8 7 O-Cane Sugar <1 4.5
Good Karma® Unsweetened Flax Milk KFU 1 0 None 0 2.5
Good Karma® Flax Vanilla Milk KFV 11 11 Cane Sugar 0 2.5
Royal Hawaiian Orchards® Original Macadamia Milk OOM 1 0 None 0 4.5
Royal Hawaiian Orchards® Vanilla Macadamia Milk OVM 8 8 Cane Sugar 0 3.5
Malk® Maple Pecan Milk MMP 9 7 Maple Syrup 1 11
Horizon® Whole Milk COW 13 12 None 8 8
Almond Breeze® Almond Original AO 8 7 Cane juice 1 2.5
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Note:

†

indicates the amount of total carbohydrates (CHO), sugars, proteins or fat per 8 oz servings;

‡

indicates the major source of the sugars added, Forager Project®, Forager Project, San Francisco, CA; Califia Farms®, Califia Farms, Los Angeles, CA; So Delicious®, So Delicious Dairy Free, Springfield, OR; Good Karma®, Good Karma Foods, Inc., Boulder, CO; Royal Hawaiian Orchards®, MacFarms, LLC, Dana Point, CA; Malk®, Malk Organics, LLC, Houston, TX; Horizon®, The WhiteWave Food Company, Denver, CO, and Almond Breeze®, Blue Diamond Growers, Sacramento, CA.

Bacterial cultivation and biofilm formation.

S. mutans UA159, a representative acidogenic and aciduric bacterium commonly used in in vitro and in vivo caries model studies, was grown and maintained using Brain Heart Infusion (Difco Laboratories, Detroit, MI) medium at 37°C in an aerobic chamber with 5% carbon dioxide (Wen & Burne, 2002). For biofilm formation (Loo, Corliss, & Ganeshkumar, 2000; Wen & Burne, 2002), overnight cultures of S. mutans UA159 were transferred to fresh Brain Heart Infusion broth and allowed to grow until the mid-exponential phase (OD600nm ~0.5). Then, the actively growing S. mutans cultures were diluted 1:100 with various alternative milk beverages, and 200 μL aliquots were transferred to wells of 96 well culture plates (Corning, New York, NY) in triplicate. To evaluate the dilution effects, a set of beverages was diluted by 1:2 ratio with sterile deionized water and inoculated with actively growing S. mutans similarly as described above. For controls, milk beverages with no bacterial inoculum were also used. Following inoculation, the plates were incubated in an aerobic chamber with 5% carbon dioxide at 37°C to allow biofilms to develop. After 24 hours, the 96-well plates were agitated at 200 revolutions per minute for 5 minutes on a shaker (Shaker 20E; Labner International, Edison, NJ), and the non-adherent cells were removed by gentle rinsing three times in a water-bath. The adherent biofilm cells were stained with 0.1% crystal violet for 30 minutes, washed three times in water to remove the free dye, and then, the bound dye was extracted using an ethanol-acetone mix (4:1). Biofilm formation was then quantified by measuring the absorbance of the extracted solution using a spectrophotometer at 575 nm (Lee et al., 2018; Loo et al., 2000; Wen & Burne, 2002).

pH analysis and lactic acid estimation.

The pH of all alternative milk beverages was measured before and after S. mutans fermentation using a micro-pH probe (Hanna Instruments, Woonsocket, RI). For post-fermentation pH measurement and lactic acid estimation, a set of cultures were grown in 5 ml Falcon tubes similarly as described above. After 24 hours, the pH of the culture medium was recorded, and following centrifugation at 4°C at 3,000xg for 10 minutes, the concentration of lactic acid in the cell-free culture medium was measured using the colorimetric EnzyChrome™ L-Lactate Assay Kit (BioAssay Systems, Hayward, CA) according to the manufacturer’s instructions (Lee et al., 2018).

Buffering capacity.

The buffering capacity of the beverages was examined by pH titration using a micro-pH probe (Hanna Instruments, Woonsocket, RI), and the amount of 1 M hydrogen chloride required for a pH change of one unit was recorded as a correlate of buffering capacity (Lee et al., 2018).

Enamel and dentine demineralization assays.

For ex vivo caries model studies, enamel and dentine slabs (4 × 7 × 1 mm) were prepared using extracted third molars (Giacaman & Munoz-Sandoval, 2014). The initial surface microhardness was assessed by three Knoop indentations (Fan et al., 2012; Giacaman & Munoz-Sandoval, 2014), and the slabs were then randomly assigned into one of the following treatment groups, 10 each (N=10): Bovine Whole Milk (COW), Original Almond (AO), and saline (SA). For negative control, a set (N=4) of enamel and dentine slabs received similar treatment but no S. mutans inoculum. Following ultraviolet sterilization, slabs were first coated with pooled human whole saliva (Ahn, Wen, Brady, & Burne, 2008), and then placed in wells of 48-well plates. To initiate S. mutans biofilms, mid-exponential phase (OD600nm ~0.5) cultures were diluted by 1:100 with semi-defined biofilm medium with glucose (18 mM) and sucrose (2 mM) as the main carbon and energy sources (Loo et al., 2000; Wen & Burne, 2002), and 2 mL of the diluted cultures was transferred to each well and allowed to grow at 37°C in an aerobic chamber with 5% carbon dioxide for 16 hours. Slabs with S. mutans biofilms were then treated three times a day with either COW, AO or A, 30 minutes each, which reflects the approximate duration of each meal, from start to end. This was followed by SA rinse, twice, and then transferred back to wells containing biofilm medium with glucose (0.1 mM) and sucrose (0.05 mM), simulating the saliva. After 4 hours, the slabs were transferred again to either COW, AO or SA and incubated for 30 minutes as described above. Following brief rinses in saline, the slabs were incubated in biofilm medium with glucose (0.1 mM) and sucrose (0.05 mM) overnight as described above. Based on preliminary data, the duration of the experiment was 5 days for enamel slabs and 4 days for dentine slabs. By the end of the incubation, slabs were briefly rinsed in phosphate buffered saline, pH 7.0 and the biofilms were dispatched by brief sonication (Fan et al., 2012). Final surface microhardness of the slabs was measured similarly as described above and elsewhere. The percent surface microhardness loss was calculated as follows: (initial surface microhardness – final surface microhardness) x100 ÷ initial surface microhardness (Fan et al., 2012; Giacaman & Munoz-Sandoval, 2014).

Statistical analysis.

Analysis of Variance (ANOVA) was first used to determine if any significant differences existed among different milk products in the different aspects analyzed, and Tukey’s Studentized Range (HSD) test was then used to analyze the differences between different alternative milk products. Besides, Normality tests were also conducted to determine if the data were normally distributed, and the Brown-Forsythe’s test was used to analyze homogeneity of variance. All statistical analyses were conducted using SAS 9.0 (SAS Institute Inc., Cary, NC). A difference at P≤0.05 is considered statistically significant.

RESULTS

Biofilm growth in alternative milk beverages.

Analysis of Variance revealed significant differences (P<0.001) among the different alternative milk products in their ability to support S. mutans biofilm formation. Normality tests showed that the data were normally distributed, and the Brown-Forsythe’s test showed the milk product group variances were equal. HSD tests were then used to further examine the differences between different types of the alternative milk products, and the results showed that of the alternative milk beverages analyzed, Chocolate Cashew milk (FCC) supported the most biofilm formation by S. mutans (Fig. 1). It was followed by Vanilla Macadamia milk (OVM) and Coconut Vanilla milk (SCV), and the least biofilms were measured when S. mutans was grown in Flax Vanilla milk (KFV) and Unsweetened Flax milk (KFU) (Fig. 1). When the major types of alternative milk beverages were compared, the Macadamia milk products (Original Macadamia milk, OOM and OVM) yielded the most biofilms with an averaging absorbance of 0.99 (±0.842), followed by the Coconut milk products, including SCV, Unsweetened Coconut milk (SCU), Original Coconut milk (SCO) and Coconut Almond milk (CCA), averaging 0.967 (±0.408), and again, the least was observed with the Flaxmilk products (KFU and KFV), averaging 0.026 (±0.003) (Fig. 1). However, when compared to COW, none had comparable S. mutans biofilm overnight (Fig. 1). When the beverages were diluted by 1:2 ratio with distilled water, biofilm formation by S. mutans was shown to be reduced for all beverages except OVM, which displayed >3-fold increases instead, as compared to when grown in the original beverage alone (Data not shown).

Figure 1. Biofilm formation in milk beverages.

Figure 1.

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S. mutans was grown in 96-well polystyrene plates in milk beverages for 24 h, and biofilms were assessed by absorbance measurement following crystal violet staining. Data represent the averages (±SD in error bars) of at least three sets of separate experiments (N=9). *P<0.01 vs FUC, FOC, MMP, KFU, KFV and CCA, <0.05 vs SCU and SCO; #P<0.01 vs FCC, SCV, SCU, SCO, OVM and CCA. As a control, COW consistently yielded more than 10 units of biofilms (P<0.001 vs all others, data not shown).

pH of the alternative milk beverages and acid production following S. mutans growth.

When analyzed similarly as described above, SCU had the highest original pH at an average of 8.42 (Fig. 2). It was followed by FCC and KFU, at pH 8.38 and 8.35, respectively. The lowest pH was measured with FCC, at 6.24, followed by Original Cashew milk (FOC) at 6.70 and Unsweetened Plain Cashew milk (FUC) at 6.93. When the different types of milk beverages were compared with each other, significantly lower pH was found with the Cashew nut milk products with average of 6.63, and the highest pH was observed with the Coconut milk products, averaging pH of 8.26 (Fig. 2). Following bacterial fermentation, the lowest cultural pH was measured with FUC at 4.06 and the highest was with KFV at pH 7.44 (Fig. 3). When compared between the different groups, Cashew milk products displayed the lowest pH with an average of 4.12, while the highest cultural pH was measured with the Flax milk beverages at 6.96 (Fig. 3).

Figure 2. Analysis of initial pH.

Figure 2.

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The initial pHs of the milk beverages were measured using a micro-pH prob. Data represent averages (±SD in error bars) of two sets of separate experiments. The highest initial pH was measured with SCU and the lowest with FCC (P<0.01). #P<0.05 vs FUC, FOC, and MMP, <0.01 vs the others; *P<0.01 vs FCC, 0.05 vs FUC, FOC, and MMP, respectively.

Figure 3. pH analysis of culture medium.

Figure 3.

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The pH of the culture medium after 24 hours of bacterial fermentation was measured using a micro-pH prob. Data represent averages (±SD in error bars) of three sets of separate experiments (N=3). The highest pH was measured with KFV and the lowest with FUC (P<0.001). 1P<0.001 vs KFU, KFV, OOM, OVM and CCA; 2P<0.001 vs FUC, FCC, FOC, SCV, SCU, SCO, and MMP.

When the lactic acid in the cultural medium was analyzed, the highest amount was measured with FOC, with an average of 6.74 mM, and the least amount of lactic acid was shown in KFV averaging 4.49 mM (Fig. 4). When the different types of beverages were compared, the Cashew milk beverages produced more lactic acid than all others studied, averaging 5.93 mM. It was followed by the Coconut milk beverages, with an average of lactic acid of 4.93 mM.

Figure 4. Lactic acid analysis.

Figure 4.

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Lactic acid in the 24 hour culture medium was analyzed using a colorimetric assay. Data represent averages (±SD in error bars) of three separate sets (N=3) of separate experiments. *P<0.05 vs KFU, KFV, OVM and MMP.

Buffering capacity of the alternative milk beverages.

When analyzed similarly as described above, pH titration assays showed that the highest buffering capacity was measured with CCA, with the average amount of HCl required for reduction of pH for 1 unit at 112 μmoles (Fig. 5). The weakest buffering capacity was shown with SCU at 3.5 μmoles. As a group, the Macadamia milk beverages displayed the strongest buffering capacity, averaging 65.75 μmoles. Of the Coconut milk beverages analyzed, all, except CCA, showed the least amount of HCl needed to reduce the pH, with an average of 6.25 μmoles.

Figure 5. Buffering capacity analysis.

Figure 5.

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The buffering capacity of the milk beverages was measured by pH titration and is expressed as μmoles of HCl needed for titrating the pH by one unit. Data represent averages (±SD in error bars) of three sets of separate experiments (N=3). The highest buffering capacity was measured with CCA and the lowest with SCU. #P<0.05 vs FCC, <0.01 vs SCV, SCU, SCO, KFU, KFV, OOM, OVM, and MMP <0.001 vs CCA; *P<0.01 vs OOM, <0.001 vs all others.

Ex vivo analysis of the demineralization potential of Original Almond milk beverage.

Loss of SH (SHL) has been extensively used as a reliable method to evaluate demineralization mediated by bacterial sugar fermentation throughout the experimental period. Dentine has lower mineral content than the enamel, making it more prone to acid dissolution. As shown in Figure 5, both dentine and enamel demineralization were significantly higher when S. mutans biofilms were exposed to COW and AO, as compared to the control biofilms that were exposed to sterile saline alone (P<0.001) (Fig. 5). However, no significant differences in SHL were observed between the dentine slabs and enamel slabs exposed to COW and those exposed to AO (P>0.05). No significant differences in SHL were measured between the biofilms exposed to saline and those received no S. mutans (Data not shown).

DISCUSSION

The acidic metabolites of bacteria in the carious site is directly associated with dental caries. The ability to support cariogenic bacterium to colonize and accumulate on the tooth surface and to produce acids from sugar fermentation and the buffering capacity are indicators commonly used to analyze the cariogenic potential of various foods and beverages (Bowen et al., 1997; Dashper et al., 2012; Giacaman & Munoz-Sandoval, 2014; Lee et al., 2018; Munoz-Sandoval et al., 2012; Prabhakar et al., 2010). In a continuing effort, we used in vitro S. mutans biofilm model to analyze the potential of several popular alternative milk beverages, including three Coconut milk, three Cashew milk, two Flax milk, two Macadamia milk and one pecan, to support bacterial biofilm formation and cause acidic metabolites-mediated demineralization (Table 1). The results showed that consistent with our recent studies (Lee et al., 2018), S. mutans in COW yielded substantial biofilms. When compared, major differences exist between the major types of alternative milk beverages in their ability to support cariogenic S. mutans biofilm formation and acid production from sugar fermentation, with the Macadamia milk supporting the most biofilms. Of the two Macadamia Milk beverages analyzed, OVM was vanilla flavored and supplemented with cane sugar and contains eight grams per serving. Among the alternative milk beverages analyzed, OMV had the second most biofilm accumulation overnight, and it allowed > 3-fold more biofilm accumulation when diluted with distilled water. Interestingly, it yielded only limited lactic acid, especially when compared to OOM which on the other hand supported only limited biofilms. Similar observations were also made previously (Lee et al., 2018) and with FCC and SCV, which also produced a lot more biofilms as compared to the counterparts in the respective groups.

S. mutans is capable of utilizing various sugars at micro-concentrations, although the metabolic pathways vary in response to the environmental conditions. Under the conditions of glucose in excess or during aerobic growth, S. mutans catabolizes glucose through homofermentation pathways, producing primarily lactate, the most acidic of all organic acids. However, when grown under anaerobic conditions, the bacterium undergoes heterofermentation pathways, which also yield formate, acetate, acetone and ethanol (Bitoun, Liao, Yao, Xie, & Wen, 2012; Loesche, 1986; Yamada, Takahashi-Abbe, & Abbe, 1985). When the biofilm accumulates, the oxygen availability reduces, which could lead to a shift of metabolic pathway of bacterial fermentation pathway to heterofermentation, partly contributing to the reduced lactic acid production.

Coconut milk is known to possess antimicrobial properties, among other health benefits when used (Sethi et al., 2016). Interestingly, all coconut milk beverages tested showed strong support of S. mutans biofilm formation. On the other hand, all coconut milk beverages, except CCA, had poor buffering capacity (Fig. 5), and consistently, had the second lowest cultural pH following bacterial fermentation (Fig. 4). On the other hand, CCA, a blend of coconut and almond milk, possessed the strongest buffering capacity of the alternative milk beverages analyzed in this study. While it generated considerable lactic acid following bacterial fermentation, the cultural pH reduced only a little over one unit, which is different from the other coconut milk beverages. It remains unclear what contributed to the strong buffering capacity with the blender.

Of the cashew milk beverages analyzed, FCC had the most biofilms not only within the same type but also among all alternative milk beverages analyzed in the study (Fig. 1). While none containing supplemental sugar, the cashew milk beverages significant amount of sugars/carbohydrates with FCC containing the most within the group at 6 grams per serving (Table 1). It is therefore not totally surprising that the cashew milk beverages had the lowest cultural pH following overnight S. mutans fermentation.

Of the two flax milk beverages analyzed in the study, KFU was unsweetened with no sugar per serving, while KFV was vanilla flavored and supplemented with Cane sugar with a sugar level of eleven grams per serving, which is the highest among the alternative milk beverages studied. Interestingly, neither KFU nor KFV supported any significant S. mutans biofilms (Fig. 1). At the meantime, the flax milk beverages also demonstrated the second weakest buffering capacity among the beverages analyzed. This and the results that it produced limited lactic acids and especially, the limited pH reduction following overnight growth all suggest that the flax milk beverages did not support much S. mutans growth. Flax seeds are known to be rich in omega-3 fatty acids and phytochemicals that are known to possess strong antimicrobial effects (Joshi, Garg, & Juyal, 2014; Kaithwas, Mukerjee, Kumar, & Majumdar, 2011; Son & Song, 2016), although the exact factors that underlie the limited S. mutans biofilm formation and acid production in the flax milk beverages await further investigation.

The pecan milk beverage, MMP, analyzed in this study also contained maple syrup and had seven grams of sugar per serving. It did not yield any significant S. mutans biofilms overnight, but as indicated by the reduction of cultural pH of more than 2.7 units, it did support some substantial S. mutans growth. Maple syrup is a commonly used sweetener that is also claimed to contain higher levels of beneficial gradients, including phytochemicals that has been shown to drastically improve the action of antibiotics (Maisuria, Hosseinidoust, & Tufenkji, 2015; Sandoiu, 2017). However, it awaits further investigation if the limited biofilm formation can be attributed to the maple syrup or other components of the pecan extracts.

Of the alternative milk beverages tested, including those analyzed previously (Lee et al., 2018), AO consistently supported the most S. mutans biofilm formation, especially when it was diluted with deionized water (Lee et al., 2018). To further assess the cariogenic potential, the Original Almond milk beverage was subjected to ex vivo caries model analysis along with bovine whole milk (COW) serving as a control. While possessing strong buffering capacity and some antimicrobial components such as lactoferrin, lysozyme and peroxide, bovine whole milk has been shown to support substantial S. mutans’ biofilm formation (Bowen et al., 1997; Giacaman & Munoz-Sandoval, 2014; Lee et al., 2018; Munoz-Sandoval et al., 2012; Prabhakar et al., 2010; Sheik & Erickson, 1996). In ex vivo caries model, frequent exposure to bovine whole milk has also been shown to lead to significant demineralization, indicative of cariogenic potential (Giacaman & Munoz-Sandoval, 2014; Munoz-Sandoval et al., 2012). Consistently, the results presented here also showed that when S. mutans biofilms were expressed to COW, three times a day, 30 minutes each, both the enamel and dentine slabs displayed significantly more demineralization, as compared to those exposed to saline as a negative control. Similar results were also obtained when S. mutans biofilms were exposed to AO, and no significant differences were measured between the ones exposed to COW and those to AO. These results further demonstrated that Original Almond is cariogenic as bovine whole milk.

Conclusions

The results of both in vitro S. mutans biofilm model and ex vivo caries model have shown that like bovine whole milk, most of the alternative non-dairy milk beverages tested, especially those supplemented with sugars, are potentially cariogenic, although differences exist between different brands and types. Based on the results of this and our previous studies, Soy milk, the almond products, especially the Original Almond, Macadamia milk, the cashew products and the coconut products appear to support more bacterial biofilms and have greater potential of causing unbalanced demineralization and carious lesions, while the Flax milk beverages seem to have the least demineralization potential. This information should be taken into consideration by dentists when they counsel patients on diet selection and preventive care strategies. However, further studies using in vivo caries models and clinical trials will be needed for more definitive conclusions on the potential of the alternative milk beverages to cause unbalanced demineralization and dental caries. These future studies should evaluate the clinical implications of intake, frequency, volume, age, and dentition of the consumer in caries-like lesion models of deciduous and permanent teeth.

Supplementary Material

Highlights

Figure 6. Enamel and dentine demineralization.

Figure 6.

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Bar graph illustrates reduction of surface Knoop microhardness (%SHL in average, ±SD in error bars, N=10) following repeated exposures of the enamel and dentine slabs to whole milk (COW), Original Almond (AO), or saline (SA). *indicates significant differences when compared to the saline control (SA) at P<0.001.

Acknowledgements

This study was supported in part by National Institutes of Health /National Institute of Dental, Craniofacial Research (DE19452 to ZTW and DE26782 to XXU).

Footnotes

Declaration of conflict of interest

The authors report no conflicts of interest with the this manuscript and have no disclosures.

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