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

Bovine milk alternative beverages are on the rise. Among those available to consumers in the USA are soy, almond, cashew, and coconut milk-based beverages. Various market data report an increase in overall dairy alternative beverage sales from 2011 to 2014 in comparison to cow's milk with USD 1 billion in annual sales in the USA [Wang, 2016b]. Likely reasons for this surge include dietary restrictions like lactose intolerance, ethical concerns, interest in plant-based eating patterns, and other perceived health benefits [Ellis and Lieb, 2015]. While soy milk possesses nutritional benefits, concerns exist regarding its inherent phytoestrogen [Jefferson, 2010; Patisaul and Jefferson, 2010] and phytate effects [Gibson et al., 2010]. In recent years almond milk sales exceeded those of soy milk, generating over double the revenue of all other competitors in the dairy alternative category [Wang, 2016a].

One reason for the recent growth in alternative milk products is the concomitant awareness of food allergy to bovine-based products. Generally, food allergies have increased in the past 2 decades, affecting 4-6% of children in the USA [Kulis et al., 2015]. The foods most commonly associated with allergies in the USA are milk, eggs, peanuts, tree nuts, wheat, soy, fish, and shellfish [Kulis et al., 2015]. Consequently, the impact of food allergy extends beyond physiological effects, often leading to decreased quality of life including social isolation, bullying, and higher levels of anxiety [Lieberman et al., 2010]. According to the Guidelines for the Diagnosis and Management of Food Allergies in the USA, “the only prevention option for the patient is to avoid the food allergen, and treatment involves the management of symptoms as they appear” [Boyce et al., 2010]. Thus, alternative milk products serve as nutritional and palatable alternatives for such individuals. Despite increasing consumption rates, the cariogenic potential of alternative milk products is currently unknown.

Caries remains the primary concern of dental healthcare professionals because it is still the most prevalent infectious disease worldwide. Moreover, caries rates in children aged 2-5 years are on the rise in the USA [Tinanoff and Reisine, 2009]. Caries is initiated in dental plaque, a microbial biofilm grown on the tooth surfaces [Loesche, 1986]. Under conditions such as continuous consumption of a high sucrose-containing diet, an ecological shift in the plaque biofilm microbiota can take place, leading to overgrowth of acidogenic species resulting in eventual net loss of tooth mineral [Loesche, 1986; Dashper et al., 2012].

Several studies have analyzed the cariogenic properties of bovine milk [Sheik and Erickson, 1996; Bowen et al., 1997; Prabhakar et al., 2010; Munoz-Sandoval et al., 2012; Giacaman and Munoz-Sandoval, 2014]. Using in vitro caries and biofilm models, plain and sweetened bovine milks have been found to have a high buffering capacity and support the formation of Streptococcus mutans biofilms, which are indicative of caries potential; sugar supplementation further enhances such potential [Prabhakar et al., 2010; Giacaman and Munoz-Sandoval, 2014]. In studies of dairy milk alternatives, Dashper et al. [2012] reported that soy milk supported significantly higher quantities of organic acid production by S. mutans fermentation and had a lower buffering capacity in comparison to bovine milk, which indicates that soy milk has a higher cariogenic potential than bovine milk.

Despite recent increases in the consumption of almond milks, currently there is no information available concerning the cariogenic potential of these beverages. The aim of this study was to analyze the cariogenic potential of 6 different almond milk beverages in regard to biofilm formation, acid production by S. mutans, and buffering capacity, with soy milk and bovine whole milk serving as controls.

Six almond milk beverages were analyzed in this study, as shown in Table 1. They included 2 original unsweetened almond milks from Diamond Breeze® products (AU) and Silk® products (SU), 2 original sweetened almond milks from Diamond Breeze products (AO) and Silk products (SO), 1 Silk Vanilla Almond (VAN), and 1 Silk Chocolate Almond (CHOC). They were analyzed along with bovine whole milk (COW) and soy milk (SOY) as controls.

S. mutans UA159, a major cariogenic bacterial strain commonly used in in vitro and in vivo caries models, was maintained on brain heart infusion (BHI) (Difco) agar plates and transferred in BHI broth at 37°C in an aerobic chamber with 5% CO₂ [Wen and Burne, 2002]. For biofilm formation, Todd Hewitt broth (Difco) plus 0.2% (w/v) yeast extract (THY) was also used either together with milk beverages in a 1:2 ratio or alone as controls.

S. mutans UA159 cultures were transferred to fresh BHI medium and allowed to grow until the mid-exponential phase (OD_600nm ∼0.5). For biofilm formation [Loo et al., 2000; Wen and Burne, 2002], actively growing S. mutans cultures were diluted 1:100 with various milk beverages, and 200-µL aliquots were then transferred to wells of 96-well culture plates (Corning, NY, USA) in triplicate. To evaluate the dilution effects by saliva and/or other beverages in the oral cavity, a set of milk beverages were diluted in a 1:2 ratio with sterile deionized water or THY broth and inoculated with actively growing S. mutans similarly to that described above. For controls, milk beverages with no bacterial inoculum, as well as THY with and without inoculum, were used. Plates were incubated at 37°C in an aerobic chamber with 5% CO₂ to cultivate biofilms. After 24 h, the 96-well plates were agitated at 200 rpm for 5 min on a shaker (Shaker 20E; Labner International, Edison, NJ, USA), and the nonadherent cells were removed by gentle rinsing 3 times in water. The adherent biofilm cells were stained with 0.1% crystal violet for 30 min, then washed properly in water, and dye was extracted using an ethanol-acetone mix (4:1). Biofilm formation was then quantified by measuring the absorbance of the extracted solution with a spectrophotometer at 575 nm. The absorbance was further normalized using proper dilution factors, when needed [Loo et al., 2000; Wen and Burne, 2002].

The pH of all milk beverages before and after S. mutans fermentation was assessed using a micro-pH probe (Thermo Fisher Scientific, Waltham, MA, USA). For postfermentation pH reduction and lactic acid estimation, a set of cultures was set up using 5 mL Falcon tubes similarly to that described above. After 24 h, the pH of the culture medium was recorded, and following centrifugation at 4°C at 3,000 g for 10 min, the concentration of lactic acid in the cell-free culture medium was estimated using the colorimetric EnzyChrome™ L-lactate assay kit (BioAssay Systems, Hayward, CA, USA) according to the manufacturer's instructions.

Buffering capacity of the milk beverages was analyzed by pH titration with a micro-pH probe and calculating the amount of 1 M HCl required for a pH change of 1 unit, which was recorded as a correlate of buffering capacity.

Analysis of variance (ANOVA) was used first to determine whether significant differences exist among different milk products, and then the pairwise Tukey test and sometimes the t test were used to analyze the differences between different milk products. All statistical analyses were conducted using SAS 9.0 (SAS Institute Inc., Cary, NC, USA). A difference at p≤ 0.05 was considered statistically significant.

S. mutans exhibited the most biofilms when grown in SOY, with an average absorbance of 10.88 ± 1.73 (p <0.001 vs. all except COW) (Fig. 1). COW also yielded significantly more biofilms than the other milk beverages except SOY (p <0.001), and the least amount of biofilm was observed when S. mutans was grown in unsweetened AU and SU, with an average absorbance of 0.33 ± 0.05 and 0.53 ± 0.15, respectively. When compared among the almond milk beverages, AO supported significantly more biofilm growth than the others, with an average of 5.29 ± 1.63 (p< 0.001), while the unsweetened AU and SU yielded significantly less biofilm (p≤ 0.05). Similar results were obtained with the milk and milk alternatives that were diluted with deionized water, except the diluted SOY (SOYd) and the diluted AO (AOd) (online suppl. Fig. S1; see www.karger.com/doi/10.1159/000479936 for all online suppl. material). When grown in SOYd, S. mutans reduced biofilm formation by 1.98-fold (p< 0.01) compared to growth in undiluted SOY. On the other hand, S. mutans increased biofilm formation by 1.87-fold in AOd (p< 0.05), when compared to the undiluted milk. As a control, S. mutans generated only limited biofilms, with an average A_575nm of 0.39 ± 0.17, when grown on THY alone.

Almond milk beverages and soy milk all showed high initial pH, with the highest recorded being AO at 8.44 ± 0.07 (p≤ 0.01). COW had the lowest initial pH of 6.80 ± 0.02 (p< 0.001) (Fig. 2). Following bacterial fermentation for 24 h, AO had the lowest culture medium pH at 4.56 ± 0.66 (p< 0.001 vs. SU and AU only), and the biggest pH reduction by 3.88 units (p< 0.001). Similar results were also observed with SOY and COW.

In the cell-free culture medium, the highest amount of lactic acid was measured with COW, with an average of 16.00 ± 0.91 mM (p< 0.001 vs. SU and AU, p > 0.05 vs. the rest), and the least amount of lactic acid was shown in SU, averaging 4.12 ± 1.63 mM (p< 0.001 vs. COW) (Fig. 3). Of the almond milks analyzed, VAN supported the most lactic acid production, followed by SO, CHOC, and AO, and the least amount of lactic acid was measured with SU and AU. Similar trends, but in slightly reduced levels (p> 0.05), were measured with milk and milk alternatives diluted with deionized water (online suppl. Fig. S2).

pH titration results demonstrated that COW had the highest buffering capacity, with the average amount of HCl required for reduction of pH for 1 unit at 52.67 ± 15.04 µmol (p< 0.001) (Fig. 4). AU displayed the weakest capacity of buffering, with just over 6.67 ± 2.08 mmol of HCl needed to alter the pH by a similar amount (p< 0.05).

Dental caries is a disease that is directly associated with the acidic metabolites produced by microbes within plaque biofilm communities [Burne, 1998]. The ability to support cariogenic bacterium to form biofilms, to produce acids from sugar fermentation, and the buffering capacity are common indicators used to analyze the cariogenic potential of various foods and beverages [Bowen et al., 1997; Prabhakar et al., 2010; Dashper et al., 2012; Munoz-Sandoval et al., 2012; Giacaman and Munoz-Sandoval, 2014]. In this study, the cariogenic properties of 6 popular almond milk beverages, along with bovine whole milk and soy milk, were examined for the first time using cariogenic S. mutans grown in a commonly used in vitro biofilm model [Wen and Burne, 2002]. Our results demonstrate that, of the milk beverages analyzed, soy milk (SOY) and bovine whole milk (COW) supported the most biofilm formation by S. mutans and displayed some of the lowest culture pH after 24 h. Consistent with recent findings by Dashper et al. [2012], soy milk had one of the biggest reductions in culture medium pH, as also reflected by its ability to promote cariogenic biofilm formation and its poor buffering capacity. These results suggest that soy milk is a highly cariogenic beverage [Dashper et al., 2012]. Bovine milk, a major food product for humans of all age, is rich in calcium, phosphate, and casein, which are known to be beneficial to healthy tooth enamel and exhibit anticariogenic activity [Bowen et al., 1997; Munoz-Sandoval et al., 2012]. Bovine milk has strong buffering capacity, actually the highest among the milk beverages tested, but the significant amount of bacterial biofilm accumulated after 24 h also suggests major acid production from sugar fermentation, as supported by the results of lactic acid and pH analysis. These results also suggest that bovine milk is potentially cariogenic, consistent with recent findings by Prabhakar et al. [2010] and several others [Dashper et al., 2012; Munoz-Sandoval et al., 2012; Giacaman and Munoz-Sandoval, 2014].

Of the almond-based products, AO supported significantly more biofilm than all the others, and had the lowest culture pH and the biggest pH reduction after 24 h of bacterial fermentation. Relative to the unsweetened almond milk (AU), this sweetened almond brand is supplemented with evaporated cane juice (mainly sucrose) and contains more than 7 g of sugars per serving (23.33%, w/v) (Table 1). As a highly cariogenic sugar, sucrose is known to stimulate S. mutans biofilm formation. Therefore, it is not surprising that the sweetened almond AO produced more biofilms and resulted in more culture pH reduction than the unsweetened AU. Interestingly, the amount of lactic acid detected in the culture medium of AO was not as much as in some other almond milks, such as VAN and CHOC, which also contain lots of sugar and cane sugars but did not yield as much biofilm as AO. S. mutans is known for its ability to utilize various sugars at microconcentrations, although the metabolic pathways of glucose by this bacterium vary depending on the environmental conditions, such as glucose availability, oxygen tension, and pH. When glucose is available in excess or during aerobic growth, S. mutans catabolizes glucose through homofermentation pathways, yielding primarily lactate, the most acidic of all organic acids. However, under anaerobic conditions it undergoes heterofermentation, generating also formate, acetate, acetone, and ethanol [Yamada et al., 1985; Loesche, 1986; Bitoun et al., 2012]. When grown in this sucrose-sweetened AO, S. mutans generated significantly more biofilm, which probably results in a reduction of oxygen availability, especially for the bacterial cells deep in the biofilm clusters. The reduced oxygen availability could lead to a shift of metabolic pathway to heterofermentation by S. mutans, partly contributing to the reduced lactic acid production. However, other not yet identified factors in these beverages could also have played a role in influencing the metabolic pathways and acid production. The increased biofilm by S. mutans formation following dilutions in a 1:2 ratio of AO with deionized water (AOd; online suppl. Fig. S1) also support such a notion, although the nature of the factors and the underlying mechanisms await further investigation.

As shown in Table 1, the Silk brand Original Almond (SO) contains a similar amount of carbohydrates/sugars as AO, but interestingly, it yielded a lot less biofilm than AO, and unlike AO, had no significant reduction of culture pH following 24 h of bacterial fermentation. As indicated on the labels, one major difference between these 2 almond milks is the nature of the supplemental sugars, with SO being cane sugar and AO as evaporated cane juice. According to a US patent awarded in 2001, the “evaporated cane juice” is “not a syrup or a sugar but actually a juice concentrate” from sugar cane sticks. However, it awaits further investigation whether the evaporated cane juice or other factor(s) in this brand of almond milk contribute to the differences observed between these 2 milk alternatives.

Silk Vanilla Almond (VAN) contains the most carbohydrates among the milk beverages analyzed, and is also supplemented with cane sugar. Not surprisingly, it supported more acids from bacterial fermentation than the other milk beverages. In the 96-well biofilm model, however, VAN did not support much S. mutans biofilm formation compared to some of the other almond milks tested.

It is apparent that the sweetened almond brands led to enhanced biofilm formation by S.mutans and more acid production, leading to lower culture medium pH than the unsweetened brands. While a direct comparison cannot be made, similar patterns in biofilm formation, acid production, and pH reduction in sweetened and unsweetened cow's milk have been noted. For example, our results are consistent with findings of Giacaman and Munoz-Sandoval [2014], which showed that the addition of 10% sucrose to whole milk greatly enhances S. mutans biofilms and acid production, making it as cariogenic as a 10% sucrose solution [Prabhakar et al., 2010]. On the other hand, both unsweetened almond brands (SU and AU) had a pH above 5.5 after 24 h of fermentation, the critical pH for demineralization. Both SU and AU brands have no sugar according to the manufacturer's labeling. However, cognizance of the consumption frequency of milk alternatives with no added sugar compared to sucrose-laden drinks is warranted.

Almond as well as coconut contains phytochemicals, such as lauric acid, capric acid, and polyphenols, which have been shown to possess antimicrobial activities [Bergsson et al., 2001; Mandalari et al., 2010a, b]. Although no detailed information is available concerning the content of these phytochemicals in the almond milk products, their presence is likely to have an impact on the ability of S. mutans to grow and/or form biofilms. This may partly explain why, overall, almond milk alternatives produced a lot less biofilm than soy milk. The increased biofilm formation by S. mutans when grown in diluted almond milks, especially AOd, compared to the undiluted counterparts could also be in part attributed to the reduced concentrations of antimicrobials.

These results suggest that almond milk beverages have cariogenic potential, especially when supplemented with sugars, but are not as cariogenic as soy milk. The information generated from this study will be useful for dentists when they counsel patients and their parents on diet selection and preventive care strategies. However, a more definitive conclusion on the comparative cariogenicity of milk alternatives to traditional milk awaits further studies using in vivo caries models and clinical trials.

Almond milk beverages, especially the sweetened almond milks, support a significant amount of S. mutans biofilms, indicative of cariogenic properties, although they do not support as much cariogenic biofilm or acid production as soy milk. When choosing an almond milk product, patients should be advised that sweetened almond milks have increased cariogenic properties compared to the unsweetened varieties. As these products are increasingly consumed, dentists should include this information in their dietary counseling discussions.

This study was supported in part by the LSU School of Dentistry Residents' Research Fund.

All authors acknowledge that there is no conflict of interest with the content of the manuscript, and therefore all authors have nothing to disclose.

J.L., J.A.T., Z.T.W., and B.P. conceived the project and wrote the manuscript. J.L. did most of the experiments and some of the writing. T.T., A.D., and T.G. helped with some of the experiments. Q.Y. carried out the data analysis.