Iran J Pediatr. In Press(In Press):e85978.
Published online 2019 June 11.
Impact of Green Vegetables and Green Smoothies on Enamel Mineral
Composition In Vitro
Ines Willershausen 1, Daniel Schulte 2, Brita Willershausen 3,* and Adriano Azaripour4
1Department of Orthodontics and Orofacial Orthopedics, University Hospital of Erlangen, Erlangen, Germany
2Institute of Applied Geosciences, Darmstadt, Germany
3Private Practice, Mediplus, Mainz, Germany
4Department of Operative Dentistry, Johannes Gutenberg University Hospital of Mainz, Mainz, Germany
*Corresponding author: Private Practice, Mediplus, Mainz, Germany. Email: firstname.lastname@example.org
Received 2018 November 01; Revised 2019 March 12; Accepted 2019 March 22.
Background: Recently, smoothies with a high vegetable content have become very popular, especially among young adults.
Objectives: This in vitro study was conducted to determine the erosive potential of selected green vegetables and green smoothies
on human dental enamel.
Methods: A total of ten green smoothies and eight freshly mixed vegetables were chosen, and pH, calcium and oxalic acid contents
were measured. The enamel specimens were obtained from wisdom teeth and incubated with 3 selected foods (spinach, parsley, or
green smoothie) for 12 and 24 hours (N = 6). Control samples were incubated with a physiological salt solution. An electron probe
microanalyzer (Jeol JXA 8900RL) was utilized to analyze the concentrations of calcium, phosphorus, magnesium and ﬂuorine at
various depths ranging from 5 to 50 µm within the enamel samples. A visual examination of the specimen was carried out with a
Results: The green smoothies showed pH values ranging from 3.6 to 4.2, and the values of the mixed vegetables ranged from 5.6 to
6.3. Only in the green smoothie group could a time-dependent release of diﬀerent minerals be observed. After 12 and 24 hours of
incubation, a pronounced loss of calcium and phosphorus was found to a depth of 15 µm. Digital microscopy of the enamel surfaces
conﬁrmed these ﬁndings.
Conclusions: In our in vitro study, incubation with the spinach and parsley showed no damage to the enamel specimens; in the
case of the investigated smoothies, a mild loss of minerals to a depth of 25 µm was observed. It must be considered that numerous
factors inﬂuence the enamel surface in vivo, translating these results to in vitro conditions must be done with great caution.
Keywords: Green Vegetables, Green Smoothie, Human Dental Enamel
A well-balanced modern diet includes cereals, seeds,
vegetables, fruits, dairy products, ﬁsh, meat, eggs, oils and
beverages, in particular, water. Over the last 30 years, fast
food cuisine has become established in the diet due to
lifestyle changes. Frequent consumption of this type of
nutrition with high amounts of fat and carbohydrate to-
gether with acidic soft drinks leads to the well-known ef-
fects of overweight and dental problems (1-3). Lifestyle
habits arising from the permanent availability of fast food
products and soft drinks in combination with intensiﬁed
oral hygiene measures, chewing habits and bruxism, have
led to an increased prevalence in dental erosion (4-9). In
an in vitro study, Lussi et al. (10) showed that the erosive ef-
fect of modern popular beverages is characterized by a sig-
niﬁcant reduction of the surface hardness of enamel spec-
imens and that in addition to the pH value, the titratable
acidity and other components of the beverages, such as Ca
or F, are also important. Ancient foodstuﬀs and nutritional
habits vary to a large extent from today’s dietary habits.
In prehistoric times, humans were hunter-gatherers and
had irregular food intake as well as limited food supply. An
explanation for the dental damage was the diet of plant
ﬁbers, hard seeds and foods containing grinding stone
grit. The major plant foods (e.g., agave, yucca, stool, prickly
pear,wild onion, mesquite) are rich in calcium oxalate phy-
toliths. In addition to the erosive eﬀects of oxalic acid,
the presence of phytoliths, which are harder than enamel,
may well have caused loss of dental hard tissue. Currently,
there is an increased awareness of healthy nourishment
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Willershausen I et al.
and a tendency toward a one-sided diet rich in vegetables.
Various types of green vegetables, such as spinach, broc-
coli and parsley, contain high amounts of acidic compo-
nents. Studies have reported that a vegetarian diet can
provoke dental damage, such as abrasion or erosion (11-
13). Consumption of acidic foods and soft drinks has been
linked with the development of dental erosion (3,5,10).
In general, dental erosion is primarily chemically induced
by organic or inorganic acids from extrinsic and intrinsic
sources. Tooth structures that are damaged and eroded by
acidic foods are more sensitive to further breakdown by
abrasion and attrition during tooth brushing (14). Recent
health promotion actions recommend consumption of 5
servings of fruit or vegetables per day. Smoothies are pro-
moted as an easy way to consume the necessary amounts
of fruits and vegetables (6). There are only a few publica-
tions that discuss the eﬀects of smoothies, in particular,
fruit smoothies, on human enamel (6,15).
The aim of our in vitro study was to investigate the
erosive potential of selected green vegetables and green
smoothies on human enamel samples. To quantify the loss
of essential minerals on the enamel surface, we selected
the electron probe microanalyzer, since it is the most sen-
sitive research method.
3.1. Green Vegetables and Green Smoothies
The experimental design was descriptive. For this
study, only green vegetables and green smoothies were se-
lected to determine the possible erosive eﬀect on enamel
surfaces. Green smoothies were selected since they repre-
sent a convenient way to increase children’s ﬁber and vita-
min intake. Green vegetables were further analyzed as con-
trols to investigate the eﬀect of the unprocessed foodstuﬀ.
Therefore, eight diﬀerent fresh green vegetables
(spinach, parsley, mangold, broccoli, arugula, kale, leek,
and romaine lettuce hearts) were each homogenized in
a blender for 5 minutes. Next, the vegetable supernatant
was separated from the residue with a centrifuge for 10
minutes (3000 U/min). For all examinations, only the
supernatants of the vegetables were used (Table 1). A
total of ten green smoothies were chosen, which were
mixtures of diﬀerent vegetables and fruits (Table 2). For
further analyses, the smoothies were also centrifuged for
10 minutes at 3000 U/min. The pH values and concentra-
tions of calcium and oxalic acid were determined from
the vegetable supernatants and the green smoothies.
Among all green vegetables, we selected spinach due to
its high concentration of oxalic acid and parsley because
of its high vitamin C content (Table 1). Within the green
smoothie group, we selected a smoothie with a relatively
high percentage of green vegetables and apple juice as
well as a low pH value (Table 2).
Table 1. Concentrations of Calcium and Oxalic Acid and pH Values in the Super-
natants of Eight Green Vegetables
Green Vegetables Oxalic Acid, mg/mL pH Ca, mg/mL
Spinach 391.3 6.3 0.014
Parsley 83.4 5.6 0.036
Mangold 225.4 6.2 0.025
Broccoli Not detectable 6.2 0.016
Arugula Not detectable 5.8 0.100
Kale Not detectable 6.2 0.018
Leek Not detectable 5.6 0.039
Romaine lettuce Not detectable 6.0 0.171
3.2. Human Enamel Specimen Preparation
For this investigation, twelve fully retained wisdom
teeth from young adult patients were selected. The pa-
tients were informed about the purpose of this investiga-
tion and gave consent for the use of their teeth. Further-
more, the protocol for the experimental analysis of hu-
man samples was approved by the Ethics Commission of
the University Medical Center, Mainz, Germany. The col-
lected teeth were visually examined with a digital micro-
scope, and all teeth with mineralization irregularities or
damage were excluded. The roots were separated from the
crowns, and the enamel surfaces were polished with sili-
con carbide and aluminum oxide discs and sliced horizon-
tally into pieces of approximately 3 mm thickness (16). All
crowns were then divided into up to six slices, resulting in
a total of 52 samples. All samples were washed with and
stored in physiological sodium chloride solution until fur-
3.3. Physiochemical Measurements
To select characteristic green vegetables, the pH of the
supernatant of the vegetables and of the green smooth-
ies were determined with a microelectrode (Novodirect,
Kehl, Germany). The calcium concentration of the samples
2Iran J Pediatr. In Press(In Press):e85978.
Willershausen I et al.
Table2. List of Ingredients of Ten Diﬀerent Green Smoothies, Which Are Available in German Supermarkets
Name of Green Smoothies Ingredients pH Ca, mg/mL
Saftig smoothie; Fresco y Zumos, S. A. Spain Apple juice 48%, apple puree 25%, cucumber puree 12%, banana puree
10%, lemon juice 4%, spinach and nettle extract
Bio-Alnavit Grüner Held, Alnavit GmbH; Bickenbach, Germany Pear puree 47%, kiwi puree 25%, acerola puree, broccoli puree 9.5%, bell
pepper puree, dandelion juice 2%, stinging juice 2%, spirulina 0.1%,
matcha powder 0.1%
Voelkel fair to go; 100% direct juice; Demeter e.V.; Germany Tomato juice 66%, carrot juice 13%, sauerkraut juice 6%, beetroot juice
5%, celery juice 4%, cucumber juice 2%, bell pepper paste 2%, onion
juice, bean juice, dill juice, herbal sea salt
Truefruits green smoothie; spinach; Bonn, Germany Apple juice 54.9%, banana puree 21%, organic spinach puree 11%, pear
puree 8%, organic kale puree 4%, ginger puree 1%, organic matcha tea
0.1%; percentage of organic ingredients: 15.1%
Truefruits green smoothie; arugula; Bonn, Germany Apple juice 48.9%, mango puree 19%, arrugula puree 15%, pear puree
11%, carrot puree 5%, mint 1%, chlorella 0.1%
Grüner Smoothie Voelkel; orange; Demeter Germany Apple juice 20%, orange juice 20%, pear juice 19%, banana puree 4%,
kale 8%, peach pulp 7%, spinach 5%, acerola pulp 5%, wheatgrass,
Grüner Smoothie Voelkela; Demeter Germany Apple juice 52.5%, mango pulp 17.5%, beetroot juice 16%, spinach 8%,
Grüner Smoothie Voelkel, mango; Höhbeck, Germany Apple juice 48%, mango pulp 15%, banana puree 13%, kale 6%, ginger
juice 6%, ginger, mineral water, lemon juice concentrate, acerola pulp
5%, spinach 8%,
Voelkel vegan to go; Kraftprotz gluten frei Demeter e.V.;
Cucumber juice 35%, apple juice 20.5%, mango pulp 10%, orange juice
10%, spinach 6.5%, kale 6.5%, banana puree 6%, quinoa 2.5%, wheatgrass
juice, lemon juice, acerola pulp, spirulina powder,matcha 0.1%
Innocent smoothie-; Innocent Alps; Salzburg, Austria Apple juice 48%, pineapple juice, kiwi puree 16%, grape juice, lime
juice 1.2%, spinach and stinging nettle extract
aThe marked smoothie was selected for the in vitro study.
was determined spectrophotometrically using a colori-
metric assay (Randox Laboratories, Krefeld, Germany). The
concentration of oxalic acid was measured spectrophoto-
metrically using a colorimetric assay test kit (Enzytec, R-
Biopharm AG, Darmstadt, Germany).
3.4. Exposure to Selected Green Vegetables and Green Smooth-
Exposure to selected green vegetables and green
smoothies for the speciﬁc and sensitive analysis of the
elements Ca, Mg, F, and P with the electron probe micro-
analyzer was repeated in six independent experiments.
For each test solution (spinach, parsley, green
smoothie), one sample per tooth served as the enamel
control and was incubated with 3 mL of the physiological
This in vitro study aimed to investigate the maximal
erosive potential of selected vegetables and smoothies on
dental enamel without further possibility of remineraliza-
tion. Therefore, we consciously incubated our samples for
up to 24 hours and abstained from employing a further in-
cubation period with artiﬁcial saliva solution.
The selected enamel slices (N = 48) were placed into
multiwell plates (Greiner, Frickenhausen, Germany) and
then incubated with 3 mL of the respective test super-
natants at 37ºC in a humidiﬁed atmosphere in a gas incu-
bator (Heraeus, type Function Line, Hanau, Germany) for
12 hours and 24 hours. After exposure, the supernatants
were removed, and the enamel samples were thoroughly
washed in sterile distilled water, air-dried and prepared for
electron probe microanalysis.
3.5. Electron Probe Microanalyses
The quantitative measurement of the elements Ca, F, P
and Mg in the enamel surfaces was conducted with an elec-
tron probe microanalyzer (JEOL JXA 8900 RL, JEOL, Eching,
Germany). For this analysis, the dental slices were embed-
ded in epoxy resin and processed according to a previously
published protocol (16).
3.6. Data Analysis
The statistical analysis was carried out with the SPSS
software (version 23.0; SPSS Inc., Chicago, IL, USA). For the
measurement of the concentrations of the various ele-
ments in the enamel slices, only descriptive analyses were
Iran J Pediatr. In Press(In Press):e85978. 3
Willershausen I et al.
performed, which are expressed as the means ±SD (stan-
The ten selected green smoothies showed pH values
ranging from 3.6 to 4.2 and calcium concentrations rang-
ing from 0.03 to 0.28 mg/mL (Table 2). For all smooth-
ies, the oxalic acid concentration was lower than the de-
tection limit. The pH values of the eight green vegetables
ranged from 5.6 (parsley) to 6.3 (spinach), and the con-
centration of free calcium in the supernatants was low for
all vegetables (Table 1). Oxalic acid was only detectable
in spinach (391.3 mg/L), mangold (255.4 mg/L) and parsley
(83.4 mg/L). Since qualitative and quantitative erosive dam-
ages can be detected by the Electron Probe Microanalyzer,
which allows the detection of characteristic elements, we
purposely abstained from the unspeciﬁc investigation of
the surface roughness.
With the electron beam microprobe analyzer, the el-
emental concentrations of Ca, P, Mg and F in the enamel
samples were measured at depths of up to 50 µm (Figure
1). The incubation of the enamel samples with spinach and
parsley showed no erosive potential over the incubation
time of 24 hours. In contrast, the incubation of the dental
samples with the green smoothie showed, in comparison
to the controls, a mild erosive potential. The incubation
time of 12 hours showed a loss of Ca and P at a depth of 20
µm, while the longer exposure time demonstrated a loss
of minerals up to 25 µm. The ﬂuorine content showed con-
siderable variation at all depths and in all specimens. The
visual examination of the enamel samples with the digital
microscope showed only a slight alteration of the surface
topography for the specimens incubated for 24 hours with
the green smoothie (Figure 2).
Nutritional habits can inﬂuence dental health. In par-
ticular, frequent consumption of diets rich in sugar or
acids can lead to dental decay or dental erosion (4,8,17). Re-
cently, increased health awareness has evoked increasing
popularity of fruit smoothies (6,15) and so-called super-
foods such as chia seeds or goji berries. Green smoothies, a
blended mixture of fruits and vegetables, are viscous fruit
juices with high ﬁber content [10% - 19%], and these bev-
erages are very popular for adolescents and young adults.
Our in vitro study aimed to elucidate the possible erosive
eﬀects of green smoothies and, in particular, green vegeta-
bles as a major component of these modern beverages.
The etiology of erosive tooth wear is a complex process
with diﬀerent causative and individual factors (18); it is im-
portant to recognize initial changes and to assess possi-
ble erosive processes with sensitive techniques. We investi-
gated parsley and spinach as representative green vegeta-
bles, which are also known for being especially healthy be-
cause they contain numerous minerals and are rich in vi-
tamin C. Spinach also has high oxalic acid content, which
binds especially eﬀectively to calcium as a chelator. How-
ever, Hannig et al. (19) showed in an in vitro study that in-
cubation of bovine enamel specimens with oxalic acid did
not have a pronounced erosive eﬀect. The present study
showed that a green smoothie with a high content of green
vegetables and apple juice led to a notable loss of the essen-
tial minerals calcium, phosphate and magnesium. Oxalic
acid was not detectable in this smoothie and therefore can-
not be responsible for the mineral loss. However, the low
pH and considerable titratable acidity reﬂect the content
of other acidic fruit components. In contrast, the super-
natants of spinach and parsley induced only a slight loss
of calcium and phosphate on the enamel surface.
When assessing the concentration of diﬀerent ele-
ments in human teeth, it must be taken into consideration
that even within the same tooth, greater variability in ele-
ments exists. The concentrations of magnesium and ﬂuo-
ride, e.g., as opposed to calcium and phosphate, are more
likely to be inﬂuenced by environmental factors such as
nutrition or ﬂuoride supplementation (20,21). Both the
pH value and other factors, such as acid type, buﬀer ca-
pacity and mineral components, must be taken into con-
sideration. For example, soft drinks contain considerable
amounts of fruit acids, whereas green vegetables very of-
ten contain high amounts of vitamin C and considerable
quantities of oxalic acid (22). Vegetarian or vegan diets that
are naturally rich in raw fruits and vegetables can partic-
ularly damage teeth by erosion or abrasion (11). Linkos-
alo and Markkanen (12) found in Finnish vegetarians a 77%
prevalence of erosion of teeth when compared to individ-
uals consuming a balanced diet. On the other hand, Her-
man et al. (11) observed erosive changes in teeth in 39% of
vegetarians compared to 24% in the control group. Similar
results were reported by Staufenbiel et al. (23), who exam-
ined the oral health of 100 vegetarians and used a question-
naire concerning nutritional and eating habits. The vege-
tarians showed a signiﬁcantly higher number of teeth with
dental erosion. To study the erosive eﬀects of green vegeta-
bles, we selected wisdom teeth of young adults, whereas
4Iran J Pediatr. In Press(In Press):e85978.
Willershausen I et al.
Figure 1. Concentrations of the minerals calcium, phosphorus, magnesium and ﬂuorine (determined as CaO, P2O5, MgO, and F) in human enamel specimens (N = 6; mean,
SD) after an incubation time of 12 and 24 hours with diﬀerent green vegetable or green smoothie supernatants (spinach: pH = 6.3; parsley: pH = 5.6 and green smoothie: pH =
previous studies were performed on bovine teeth, as they
are larger and easier to obtain but do not necessarily reﬂect
the human situation (3,19,24).
Our in vitro study demonstrated that green smoothies
containing a high percentage of acidic fruit induced a loss
of calcium and phosphate from enamel, whereas the se-
lected green vegetables alone had no substantial eﬀect on
Iran J Pediatr. In Press(In Press):e85978. 5
Willershausen I et al.
Figure 2. Digital microscopy of human enamel surfaces. A, control sample, B, enamel sample after incubation with a green smoothie for 24 hours (Magniﬁcation 500 ×)
the enamel surface.
Since multiple factors, such as remineralization, re-
duce the erosive eﬀect within the oral cavity, green smooth-
ies can be considered a tooth-friendly beverage when con-
sumed in moderation.
The authors wish to thank Claudia Darmstadt for excel-
lent technical assistance.
Authors’ Contribution: Conception or design: Ines
Willershausen and Adriano Azaripour. Acquisition and
analysis: Daniel Schulte and Brita Willershausen. Draft-
ing the work: Ines Willershausen, Daniel Schulte, Brita
Willershausen, and Adriano Azaripour. Final approval:
Ines Willershausen, Daniel Schulte, Brita Willershausen,
and Adriano Azaripour.
Conﬂict of Interests: The authors declare that there is no
conﬂict of interests.
Ethical Considerations: For this in vitro study, no ethical
considerations were required.
Financial Disclosure: The authors do not have any ﬁnan-
cial interest in the companies that produce the materials
included in this article.
Funding/Support: No funding from sources other than
the Department of Operative Dentistry and Periodontol-
ogy of the University Medical Center Mainz has to be re-
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