Composition and bioaccessibility of inorganic elements in plant-based yogurts

Overview

Rebellato and colleagues at the Institute of Food Technology (ITAL, São Paulo) and Adolfo Lutz Institute measured 10 essential inorganic elements (Ca, Cu, Fe, K, Mg, Mn, Na, P, Zn, Se) by inductively coupled plasma optical emission spectrometry (ICP-OES) and 6 trace elements (Al, Ba, Co, chromium (Cr), Mo, Ni) by inductively coupled plasma mass spectrometry (ICP-MS) in 44 plant-based yogurt sample-lots and 1 cow-milk natural yogurt sample-lot purchased from retail establishments in Campinas (São Paulo, Brazil) between August and October 2022. Six plant-based sample-lots (samples 2, 5, 12, 13, 16) and the cow-milk comparator (sample 18) were additionally subjected to a static in vitro INFOGEST 2.0 simulated gastrointestinal digestion to estimate the bioaccessible fraction of each element. The selected subset spanned different brand sources and formulation compositions. Bioaccessibility of essential elements ranged widely across plant-based products: calcium bioaccessibility ranged 17.2 to 55.6%, magnesium 7.7 to 70.3%, phosphorus < LOQ to 76.2%, with the cow-milk comparator at 40.6% (Ca), 61.1% (Mg), and 76.2% (P). Among the trace elements, the bioaccessibility of Mo exceeded 70% in plant-based samples and reached 71% in the animal-based comparator, while nickel (Ni) bioaccessibility in plant-based samples ranged 7 to 90%, Ba 12 to 76%, and Co 11% and 89% in the two samples (2 and 13) where Co was detected. The authors note that despite the lower trace-element contents observed in plant-based versus animal-based yogurts, bioaccessibility fractions above 50% for several elements (Cr, Ni, Mo, Ba, Co) reinforce the importance of characterizing both total content and bioaccessible fraction when assessing exposure from this emerging product category.

Key numbers

All concentrations reported in micrograms per kilogram (µg/kg) wet weight of product as commercialized for trace elements, and in milligrams per 100 g (mg/100 g) wet weight for essential macro/micronutrient elements, per the source’s Sampling and Sample Preparation sections. Sample mineralization: 0.5 g of sample weighed into 50 mL graduated tube; 4 mL HNO3 + 2 mL H2O2 added; held at rest overnight; ultrasonic bath 80 °C for 35 min; cooled to room temperature; volume adjusted to 20 mL with ultrapure water; filtered through 0.45 µm PTFE filter. Triplicate analytical replicates per sample-lot (n = 3). Values reported as mean ± SD. Letters denote significant per-lot differences (one-way ANOVA + Tukey’s test, p < 0.05). “nd” = not detected (< LOQ); “<LOQ” = below limit of quantification. LOQs: 0.4 µg/100 g for Se; 0.04 mg/100 g for Cu, Mn, Zn; 0.58 mg/100 g for Mg; 1.64 mg/100 g for Ca and Na; 2.44 mg/100 g for K; 2.48 mg/100 g for P. For trace elements (Table 5/6): LOQ = 4 µg/kg for Ba, Co, Cr, Mo, Ni; 200 µg/kg for aluminum (Al).

Trace-element content across plant-based yogurts (Table 5, source p. 7)

Mean, minimum, and maximum total content (µg/kg wet weight) across the 44 plant-based yogurt sample-lots:

Per the authors’ narrative (Section 4.1, p. 7), the minimum-to-maximum increase factors across plant-based samples were 45-fold (Al), and substantial multi-hundred-fold increases for Cr, Ni, Mo, and Ba, with the lowest variability observed for Co. Total Cr, Ni, Mo, and Ba content figures for these same 44 plant-based sample-lots and the cow-milk comparator were previously reported in Inorganic Contaminants in Plant-Based Yogurts Commercialized in Brazil using ICP-MS measurements on the same digestate.

Essential-element bioaccessibility for plant-based and animal-based yogurts (Table 4, source p. 6)

Total content (mg/100 g), soluble fraction (mg/100 g), and percent bioaccessibility for the 5 plant-based sample-lots (samples 2, 5, 12, 13, 16) and the cow-milk comparator (sample 18) selected for in vitro INFOGEST 2.0 digestion. Selection criteria stated by the authors: different brand sources and different formulation compositions.

The bioaccessibility of Ca in plant-based samples that quantified the soluble fraction (samples 5, 13, 16) ranged 17.2 to 36.4%, all lower than the cow-milk-yogurt comparator (40.6%). The plant-based Mg bioaccessibility (7.66 to 70.3%) bracketed the animal-based value of 61.1%, with sample 16 (containing strawberry and red fruits, fortified with Ca, requiring reconstitution from powder) showing the highest plant-based Mg bioaccessibility at 70.3%. Plant-based P bioaccessibility ranged < LOQ (sample 16, where total P was below LOQ) to 75.4% (sample 5), and sample 18 (cow-milk) reached 76.2%. Zn bioaccessibility was only quantified in the cow-milk comparator (85%). Mn, Cu, and Fe bioaccessibility were only quantified in plant-based samples (Mn 28.6 to 30.4%, Cu 25.0 to 61.2%, Fe 9.17 to 32.7%); the cow-milk comparator was < LOQ for these three soluble fractions. Se total content was quantified across most samples but the soluble fraction was below LOQ in all samples; the authors report that the bioaccessibility percentage could not be calculated for Se.

Trace-element bioaccessibility (Table 6, source p. 7)

Total content (µg/kg), soluble fraction (µg/kg), and percent bioaccessibility for Al, Ba, Co, Cr, Mo, Ni for the same 6 sample-lots:

Sample 5 was the only plant-based sample-lot to show quantifiable Al bioaccessibility (11%); all other plant-based sample-lots in the bioaccessibility subset and the animal-based comparator showed Al below LOQ in the soluble fraction. Ba bioaccessibility ranged 11.7 to 76.1% across the 5 plant-based sample-lots; the cow-milk comparator was < LOQ. Co bioaccessibility was quantifiable in only 2 plant-based sample-lots (sample 2 at 11% and sample 13 at 89%). Cr bioaccessibility ranged 12.8 to 63.9% in plant-based samples (with the Section 4.1 narrative stating a 8 to 64% range across the broader sample set); the cow-milk comparator was < LOQ for Cr. Mo bioaccessibility was the only element quantified in the animal-based comparator (70.9%); plant-based Mo bioaccessibility ranged 77.9 to 87.9% in the two plant-based sample-lots where Mo was above LOQ. Ni bioaccessibility ranged 7.0 to 89.9% across the 5 plant-based sample-lots; the cow-milk comparator was < LOQ for Ni.

Contribution to daily reference intake values (Table 3, source p. 6)

The authors compute the % contribution of one 170 g serving of plant-based or animal-based yogurt to the Brazilian National Health Surveillance Agency (ANVISA, 2020) daily reference values (DRVs) for essential elements, presented as average, minimum, and maximum values across the survey:

The text additionally notes that Cr and Mo, although not in the table, also have DRVs established: when calculating the RDI contribution for a 170 g serving, values above 120% are observed for adult individuals, pregnant women, breastfeeding women, and children aged 4–8 years for molybdenum; for chromium, contributions of approximately 40% are observed for adults, pregnant women, and breastfeeding women, and 100% for children aged 4–8 years. The authors flag the Cr and Mo contributions, combined with bioaccessibility fractions of 8 to 64% for Cr and above 70% for Mo, as supporting their recommendation for moderate consumption of plant-based yogurts.

Principal Component Analysis (Section 5, source p. 8)

A 45-sample × 16-variable PCA matrix was constructed (essential and trace elements combined; 720 trial data points). After removing sample 4 (lots A, B, C) as an outlier, the rebuilt 42 × 16 PCA explained 62.4% of total variance on PC1 and PC2. Four groups emerged in the score plot: (1) pea/soy-protein veg-protein samples 1, 2, 3 grouped by higher Al, Fe, Mo, and Na; (2) sample 12 alone (the only red-fruit-containing sample) by higher Ba and Co; (3) samples 4, 5B, 5C, 5D, 6 (one brand, tricalcium-phosphate-fortified) by higher Ca and Cr; and (4) the remaining samples including the cow-milk yogurt (sample 18), classified by low amounts of all elements. Samples 16 and 17 did not cluster with any other group (high Ni and K, and high Mg and Mn, respectively).

Methods (brief)

Analytical method: essential elements (Ca, Cu, Fe, K, Mg, Mn, Na, P, Zn, Se) determined by inductively coupled plasma optical emission spectrometry on a 5100 VDV ICP-OES (Agilent Technologies, Tokyo, Japan), RF power 1200 W, double-pass cyclonic spray chamber, seaspray nebulizer at 0.70 L/min. Trace elements (Al, Ba, Cr, Co, Mo, Ni, Se — Se intended for both instruments but only quantified at LOQ on ICP-MS) determined by inductively coupled plasma mass spectrometry on a Thermo Scientific iCAP RQ ICP-MS (Bremen, Germany), RF power 1550 W, He collision-gas mode at 5.00 mL/min, micromist nebulizer at 0.98 L/min, dwell time 0.3 s (0.02 s for internal standard), monitored isotopes ²⁷Al, ⁵³Cr, ⁵⁹Co, ⁶⁰Ni, ⁷⁸Se, ⁹⁷Mo, ¹³⁷Ba, internal standards ⁴⁵Sc, ⁷²Ge, ¹¹⁵In, ¹⁰³Rh, ²⁰⁹Bi, ¹⁹⁵Pt at 50 µg/L.

Sample preparation: ultrasound-assisted acid digestion adapted from the authors’ prior protocol. Approximately 0.5 g of sample weighed into a 50 mL graduated tube; 4 mL sub-boiling-distilled HNO3 and 2 mL 30% H2O2 added; tube held overnight at rest; heated in Easy 180 H ultrasonic bath (Elma, Germany) at 80 °C for 35 min; cooled to room temperature; volume adjusted to 20 mL with ultrapure water (Gehaka reverse-osmosis system, > 18.2 MΩ·cm); filtered through 0.45 µm PTFE filter (Agilent Technologies, Tokyo, Japan). All mineralization performed in triplicate including analytical blank.

In vitro digestion: INFOGEST 2.0 protocol (Brodkorb et al., 2019) with one modification — the gastric lipase enzyme was not used due to unavailability. Enzyme activities verified prior to each assay: α-amylase 10080 uranium (U)/mg, porcine pepsin (P6887) 3843 U/mg, bovine bile (B3883), pancreatin (P7545) 7.2 U/mg. The protocol was adjusted to use 2.5 g of sample. At the end of the intestinal phase, samples were centrifuged at 3500 g at 4 °C for 30 min and the upper phase transferred to digestion tubes, which were then incubated at 100 °C overnight. The mineralization was performed on the soluble fraction according to the same protocol described above. All analyses were performed in 3 separate repetitions.

Analytical control: certified reference materials SRM 1547 Peach Leaves (National Institute of Standards and Technology, Gaithersburg, USA) and ERM-BD 151 skimmed milk powder (Joint Research Center, Geel, Belgium). Method validation according to INMETRO guide: recoveries from 99 to 111%; precision from 2 to 14%; LOQ ranging 0.4 µg/100 g (Se) to 2.5 mg/100 g (P) for essential elements, and 4 µg/kg (Ba, Co, Cr, Mo, Ni) to 200 µg/kg (Al) for trace elements. Certified standard solutions used for analytical curves were Specsol-Quimlab (Jacareí, Brazil), containing the minerals Ca, Cu, Fe, K, Mg, Mn, Na, P, Zn, and Se for essential elements, and Al, Ba, Cr, Co, Mo, and Ni for trace elements.

Statistical analysis: results reported as mean ± SD of n = 3 independent analytical replicates per sample-lot. F-test, one-way ANOVA, and Tukey’s test (p < 0.05) for among-lot comparisons within each sample, using Statistica 7.0 (StatSoft, Tulsa, OK, USA). Principal Component Analysis (PCA) using Pirouette 3.11 (Infometrix Inc., Bothell, WA, USA) on the 45 × 16 (later 42 × 16) matrix.

Limitations explicitly named by the authors: this is a novel food category with little published data on mineral composition or bioaccessibility; lipase was omitted from the INFOGEST 2.0 protocol due to unavailability of the enzyme, which may underestimate the bioaccessible fraction for lipid-soluble elements; bioaccessibility was evaluated on a deliberately small subset (5 plant-based + 1 cow-milk sample-lots) selected by formulation and brand-source diversity rather than by statistical power. Conflicts of Interest: none declared. Data Availability: “available on request.”

Implications

Certification: This is the first published evaluation of essential-element bioaccessibility in commercial plant-based yogurts using the INFOGEST 2.0 protocol. It contributes direct occurrence and bioaccessibility evidence for Ca, Mg, P, Zn, Mn, Cu, Fe, Se, Na, K (essential macro/micronutrients) and Al, Ba, Co, Cr, Mo, Ni (trace and potentially-toxic elements) in coconut-cream-based, coconut-milk-powder reconstituted, pea/soy-protein-isolate veg-protein, and soy-chocolate plant-yogurt formulations. The two most exposure-relevant findings for HMI scope are (a) soy-chocolate sample 13 showed 89% Co bioaccessibility and 33.9% Ba bioaccessibility from a 700 µg/kg Ba total content, indicating that the elevated trace-metal content already documented in this formulation type (Inorganic Contaminants in Plant-Based Yogurts Commercialized in Brazil) is largely bioaccessible from a single-meal exposure perspective, and (b) Ni bioaccessibility ranged 7 to 90% across the bioaccessibility subset, with the highest-Ni-content formulations (samples 12, 13, 16) showing intermediate bioaccessibility (29.9 to 51.3%) and lower-Ni-content formulations (samples 2, 5) showing the extremes (7.0 and 89.9%). The cow-milk yogurt comparator was below LOQ for the soluble fractions of Al, Ba, Co, Cr, Ni; Mo at 70.9% bioaccessibility was the only trace element above LOQ in the digested cow-milk sample, parallel to the high Mo bioaccessibility (77.9 to 87.9%) observed in plant-based samples.

App: Route to coconut, soy, soy-protein-isolate, cocoa, and milk-and-dairy ingredient pages, and to aluminum, chromium, cobalt, molybdenum, nickel, and barium metal pages. The matrices array includes yogurt to surface this paper to category-level synthesis once a plant-yogurt product slug is locked. Downstream synthesis on this source should preserve the bioaccessibility dimension as a distinct sub-block of the contamination profile; reporting total content alone without the bioaccessibility multiplier underestimates plant-yogurt-specific exposure relative to cow-milk yogurt for elements with high plant-yogurt soluble fractions (Mo, Ba, Ni in soy-chocolate; Co in soy-cocoa).

Courses: Useful for illustrating (a) the difference between total content and bioaccessibility as exposure metrics in fortification-driven foods (samples 5 and 13 fortified with Ca, Sample 16 fortified with Ca; the bioaccessibility percentage diverged from total content because tricalcium phosphate fortificants have different solubility from intrinsic ingredient calcium), (b) the application of the INFOGEST 2.0 protocol in a static three-compartment digestion model with quantification of the soluble fraction after centrifugation, (c) the interpretation of bioaccessibility data alongside RDI contribution to inform consumer-direct moderation recommendations (Cr above 100% RDI in children and 8 to 64% bioaccessible; Mo above 120% RDI in all populations and above 70% bioaccessible), and (d) the limitation of static in vitro models that omit the lipase step (as performed here).

Related evidence

• Coconut
• Soy
• Soy Protein Isolate
• Cocoa
• Milk and Dairy
• Aluminum
• Chromium
• Cobalt
• Nickel
• Molybdenum
• Barium

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