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Article

Zinc Tolerance of Special Yeasts and Lactic Acid Bacteria for Use in the Food Industry

by
Samantha Rossi
1,
Maria Maares
2,
Helena Kieserling
3,
Sascha Rohn
3,
Oliver Schlüter
4,
Francesca Patrignani
1,5,
Rosalba Lanciotti
1,5,
Hajo Haase
2 and
Claudia Keil
2,*
1
Campus Food Science, Department of Agricultural and Food Sciences, Alma Mater Studiorum, University of Bologna, p.zza Goidanich 60, 47521 Cesena, Italy
2
Department of Food Chemistry and Toxicology, Institute of Food Technology and Food Chemistry, Technische Universität Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany
3
Department of Food Chemistry and Analysis, Institute of Food Technology and Food Chemistry, Technische Universität Berlin, Straße des 17. Juni 135l, 10623 Berlin, Germany
4
Leibniz Institute for Agricultural Engineering and Bioeconomy, Quality and Safety of Food and Feed, 14469 Potsdam, Germany
5
Interdepartmental Centre for Agri-Food Industrial Research, University of Bologna, Quinto Bucci 336, 47521 Bologna, Italy
*
Author to whom correspondence should be addressed.
Fermentation 2023, 9(6), 521; https://doi.org/10.3390/fermentation9060521
Submission received: 4 May 2023 / Revised: 25 May 2023 / Accepted: 26 May 2023 / Published: 27 May 2023
(This article belongs to the Special Issue Fermented Foods for Boosting Health)
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Abstract

:
In order to address human zinc deficiency, fortifying staple foods with zinc is a safe and cost-effective solution. To ensure the nutritional properties and quality of a final product, zinc tolerance of the microorganisms involved in the fermentation is necessary. Bread, which is widely consumed, occupies a substantial place in many people’s diets, and is often based on a sourdough making process; thus, it might be an important headlining product. This study investigated the zinc tolerance of yeasts and lactic acid bacteria that are specifically suited to produce sourdough bread made with cricket powder hydrolysate and wheat flour. Amongst the yeasts, Kazachstania servazzii KAZ2 and Kazachstania unispora FM2 were only slightly affected in regard to cell growth and colony-forming ability when cultured in YPD broth spiked with 0.5 or 1 mM ZnSO4, respectively. Yarrowia lipolytica RO25 showed a higher tolerance for up to 2.5 mM zinc (ZnSO4). All the yeast strains were capable of accumulating zinc in the range between 200 and 400 fg/cell. The heterofermentative lactic acid bacterium Fructilactobacillus sanfranciscensis DG1 appeared to have a moderate zinc requirement and was homeostatically balanced, even under a high 20 mM extracellular ZnSO4 load. A better understanding of zinc homeostasis in yeast and lactic acid bacteria for food industry applications may lead to improvements in zinc fortification, which might contribute to diminishing Zn deficiencies, especially in vulnerable population groups.

1. Introduction

More than one billion people worldwide (17% of the world’s population) suffer from zinc deficiency. In particular, people in Asia, Sub-Saharan Africa, and Central America were identified as being at the highest risk of inadequate zinc intake, based on the low zinc content in their regional nutrition [1]. Additionally, in Western countries, a considerable percentage of the population, mostly the elderly, is at risk of zinc deficiency, due to inadequate dietary intake and reduced absorption of this essential micronutrient [2,3]. Even moderate zinc deficiency can have substantial consequences; an impaired immune system and the resulting increased susceptibility to infections are of particular concern. Zinc deficiency is considered as a possible risk factor for increased susceptibility to and severe progression of COVID-19 [4]. People in the Global South are most affected by zinc deficiency-related problems such as diarrhea and increased mortality rates [5,6,7]. The prevention or reversal of a zinc deficit can be achieved by dietary diversification, the consumption of (bio)fortified foods, as well as the consumption of zinc supplements [8,9]. Potentially suitable staple foods for fortification with zinc are milk and milk powder, grains or flours thereof, and cereal products in general (wheat, rice, maize) [10,11,12,13]. The fortification of bread and other bakery goods with zinc salts offers the possibility of achieving a more favorable zinc/phytate ratio, and, thus, producing high-quality products with good zinc bioavailability for human consumption [14,15,16]. The use of sourdough to improve the texture and palatability of baked goods is becoming increasingly important and highly appreciated by consumers [17]. The process of sourdough fermentation is one of the oldest methods of leavening bread. Through the fermentation process, lactic acid bacteria (LAB) and yeasts turn grain flour into attractive, tasty, and more digestible bread. [18,19,20]. Moreover, longer fermentation processes in sourdough making even increase the degradation of phytic acid [21,22,23,24]. This is suggested to further improve the bioavailability of trace elements [25,26].
In addition to wheat flour, other raw materials are nowadays being discussed and investigated for their use in the production of sourdough-based bakery goods, e.g., hydrolyzed insect powder [27,28]. The fortification of insect-blended wheat flour-based products with zinc could be a useful and safe intervention, particularly for the most zinc-deficient populations in Sub-Saharan Africa and South Asia, where entomophagy is a long-standing tradition [29,30]. Micronutrient-enriched insect-based food products could also provide new market opportunities in the Western world, and promote the acceptance of insect-based foods in societies that are currently more reluctant [31,32]. However, fortification has to be integrated into the production process without interfering, for example, with fermentation during sourdough making. In this case, the tolerance of the microorganisms toward the compound of interest has to be tested. Despite Saccharomyces cerevisiae’s dominance in industrial fermentation, other nonconventional yeasts have also emerged as valuable contributors [33,34,35,36]. Recent studies, performed by Rossi et al. [37,38], have highlighted the use of Yarrowia lipolytica RO25 cricket-based hydrolysate as an ingredient for sourdough breads implementing Kazachstania unispora (basionym: Saccharomyces unisporus), Kazachstania servazzii (basionym: Saccharomyces servazzii), and the heterofermentative LAB Fructilactobacillus sanfranciscensis (formerly Lactobacillus sanfranciscensis) as fermentative organisms. In light of the potential health and economic benefits of micronutrient fortifications of wheat bakery products valorized with insect powders, this study examined how zinc affects these four microorganisms in order to gain an understanding of their tolerance and accumulation of zinc.

2. Materials and Methods

2.1. Materials

Agar bacteriological (Oxoid Deutschland GmbH, Wesel, Germany), 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Carl Roth GmbH + Co. KG, Karlsruhe, Germany), hydrogen peroxide (30%, ultrapure; Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany), MRS (Oxoid Deutschland GmbH, Wesel, Germany) with 2% w/v maltose (mMRS) broth, NaCl (Carl Roth GmbH + Co. KG, Karlsruhe, Germany), nitric acid (67%, ultrapure) (Carl Roth GmbH + Co. KG, Karlsruhe, Germany), TraceCERT®/transition metal mix for ICP-MS (100 mg/L each: V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ag, and Cd in 2% nitric acid) (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany), TraceCERT®/Rhodium standard for ICP-MS (1000 mg/L Rh in hydrochloric acid) (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany), Yeast Extract–Peptone–Dextrose (YPD) Broth (Oxoid Deutschland GmbH, Wesel, Germany), and ZnSO4·7H2O (Sigma-Aldrich, Steinheim, Germany) were used.

2.2. Preparation of Zinc-Enriched Microorganisms

Microorganisms used in the study were the yeast strains Yarrowia lipolytica RO25, Kazachstania unispora FM2, Kazachstania servazzii KAZ2, and the heterofermentative LAB Fructilactobacillus sanfranciscensis DG1 (obtained from the strain collection of the Department of Agricultural and Food Sciences, University of Bologna, Bologna, Italy) [38]. Yarrowia lipolytica RO25 was regularly cultivated in YPD broth at 25 °C with agitation for 48 h. The two yeast strains of Kazachstania (FM2, KAZ2) were subcultured in YPD broth at 30 °C. Fructilactobacillus sanfranciscensis DG1 was grown in MRS broth +2% w/v maltose (mMRS) at 30 °C for 24 h. Using these pre-cultures, the microbial strains were inoculated with a 1:100 dilution either in YPD for the yeasts or mMRS for the LAB. Microorganisms were incubated at the appropriate temperature and conditions, until the beginning of the logarithmic phase was reached. Subsequently, defined amounts of ZnSO4 (0.5–10 mM for yeasts and 5–20 mM for the LAB) were added and the cultivation continued until the stationary phase was reached. For each strain, a control sample was prepared with the addition of water instead of ZnSO4. At the end of the incubation, aliquots of cells were taken for the determination of the colony-forming ability and zinc quantification. The remaining cell suspension was collected in 50 mL tubes and cells were separated by centrifugation (3000× g for 5 min at room temperature) and freeze-dried.

2.3. OD600 Assay

Biomass accumulation was monitored by OD600 measurements [39]. For each sample, 1.5 mL was transferred into a cuvette and the OD was measured at 600 nm using a UV/vis spectrometer (Spectronic Unicam UV1, Thermo Fisher Scientific Inc., Waltham, MA, USA).

2.4. Determination of Cell Viability via Colony-Forming Units

The number of colony-forming units (cfu) was determined using a traditional plate counting method [40]. Samples were subjected to decimal dilutions in physiologic saline solution. Then, 100 µL of the appropriate dilution was plated on YPD-agar for the yeasts or mMRS-agar for LAB. Plates inoculated with Yarrowia lipolytica RO25 were aerobically incubated at 25 °C for 48–72 h, while the other plates were incubated at 30 °C for 24–48 h before counting cfu.

2.5. Determination of Cellular Zinc Concentration

To determine the concentration of zinc in the cells, fresh biomass was centrifuged (3000× g for 5 min at room temperature), supernatant was discarded, and cells were rinsed twice with HEPES buffer (50 mM HEPES in bidistilled water adjusted to pH 6.5 with sodium hydroxide solution; depleted of multivalent cations by Chelex® 100 resin pretreatment according to Alker et al. (2019)) [41]. Cell pellets were dissolved in 500 µL of a 1:1 mixture of ultrapure HNO3 (65%) and H2O2 and dried twice at 95 °C overnight using a thermoshaker. Following dilution in 0.65 % HNO3 containing 5 μg/L rhodium, samples were analyzed on an Elan DRC II inductively coupled plasma-mass spectrometer (ICP-MS; PerkinElmer Inc., Rodgau, Germany) [42]. The experimental conditions for the ICP-MS measurement are listed in Table 1.

2.6. FTIR Analysis

Infrared spectra of the dried yeast and bacteria samples were measured with a Bruker Tensor II spectrometer (Bruker Optic GmbH, Karlsruhe, Germany) equipped with a liquid nitrogen-cooled mercury cadmium telluride detector. The samples were measured in absorption mode with a Platinum ATR Diamond cell (Bruker Optic GmbH, Karlsruhe, Germany) against the ambient air as a background. Each spectrum resulted from an average of 120 scans recorded with a resolution of 2 cm−1 from 4000–800 cm−1 at 20 °C. Spectra were off-set-corrected in the region of 3800–4000 cm−1 using the software provided with the spectrometer (OPUS 7.5) [42].

2.7. Statistical Analyses

The statistical significance of the experimental results was calculated using GraphPad prism software (version 8.0, GraphPad Software, San Diego, CA, USA). All experiments were conducted in at least four independent replicates as indicated in the figure legends.

3. Results

The first experiments based on OD600 cell density measurements focused on recording the yeast and LAB growth in batch culture. Using this information, the time of zinc addition during the early logarithmic phase and the duration of fermentation were determined for the follow-up studies (Figure 1). The heterofermentative LAB Fructilactobacillus sanfranciscensis DG1 was treated with ZnSO4 seven hours after starting the fermentation, and it grew for an additional 17 h. Kazachstania unispora FM2 cultures received Zn treatment from 4 h after starting the fermentation until 29 h, while Kazachstania servazzii KAZ2 was incubated between 5 and 32 h. The incubation of Yarrowia lipolytica RO25 with ZnSO4 lasted from 8 to 30 h.
Different zinc concentrations between 0 and 20 mM were then applied in order to investigate the effects of zinc on the growth performance of the four microorganisms, analyzing biomass accumulation via OD600 measurements (Figure 2) and the enumeration of colony-forming units (Figure 3). For the yeast strain Kazachstania servazzii KAZ2, a statistically significant reduction in the final OD600 was observed when culturing in YPD containing additional 0.5 mM ZnSO4 (~20% reduced biomass). Increasing to 2.5 mM ZnSO4, the final OD600 absorbance of the yeast suspensions was reduced by about 75% compared to control yeasts (Figure 2). Still, the OD600 value was statistically higher than before zinc addition, suggesting ongoing yeast proliferation (Supplementary Table S1). The other Saccharomyces strain Kazachstania unispora FM2 was slightly better at tolerating zinc; OD600 values were almost comparable to controls in the presence of 1 mM ZnSO4. When adding 2.5 mM zinc into YPD broth, a 50% reduction in biomass outcome was obtained (Figure 2). Yarrowia lipolytica RO25 tolerated zinc treatment even better, with growth inhibition starting at 5 mM ZnSO4 (Figure 2).
A further increase in the extracellular zinc led to increased cytotoxicity in all three yeasts, as reduced viability was observed based on their cell density measurements (Figure 2) as well as colony-forming ability (Figure 3).
The LAB strain Fructilactobacillus sanfranciscensis DG1 proved to be much more resistant toward ZnSO4 with no changes in OD600 measurements and only minor effects in colony-forming ability with up to 20 mM zinc addition (Figure 2 and Figure 3).
Upon the completion of fermentation, the zinc content of all samples with a significant amount of cell yield was determined. The zinc levels in yeast cells grown in standard media ranged from 3.6 fg/cell to 8.0 fg/cell (equivalent to 3.3 to 7.8 × 107 atoms of zinc per cell) (Figure 4A). This value clearly exceeds the zinc content previously determined in yeast cells grown with the minimally required amount of zinc (~1 × 107 Zn atoms/ Saccharomyces cerevisiae cell; [43]) and is consistent with the good propagation of either Yarrowia lipolytica RO25, Kazachstania unispora FM2, or Kazachstania servazzii KAZ2, when grown in YPD broth (Figure 1 and Figure 2). Adding non-cytotoxic amounts of ZnSO4 to the media enhanced the zinc content in all of the three yeast strain cells with similar relative enrichment quota compared to basal zinc (Figure 4A,B). In the presence of an additional 1 mM ZnSO4 in the YPD medium, the cellular zinc content of Kazachstania unispora FM2 and Kazachstania servazzii KAZ2 increased to values up to 180–200 fg/cell. Yarrowia lipolytica RO25 was slightly less responsive; an equivalent zinc treatment led to ~75 fg zinc/ cell. A zinc surplus of 5 mM resulted in an 80-fold increase in zinc content compared to control cells grown in YPD (~400 fg zinc/Yarrowia lipolytica RO25 cell) (Figure 4A,B).
Regarding their total zinc content, Fructilactobacillus sanfranciscensis DG1 was one hundred times lower in basal zinc than the yeasts after incubation in the standard mMRS medium (approximately 0.032 fg Zn/cell). Even after fermentation at comparable zinc excess (5 mM ZnSO4), Yarrowia lipolytica RO25 and Fructilactobacillus sanfranciscensis DG1 differed by a factor of 150 (Figure 4A).
Potential changes in the general (bio)chemical composition of yeasts and LAB after incubation in zinc-enriched media were studied by FTIR of the cells. Figure 5 shows the total mid-IR range (4000–800 cm−1). Irrespective of zinc treatment, spectra were relatively similar in the range of 3100–2800 cm−1 and 1700–1500 cm−1, which were dominated by bands assigned to the stretching vibrations of membrane fatty acids and to the amide I and II bands of proteins or peptides. Minor changes in the spectra were observed in the 1200–900 cm−1 region. Band changes in this region were assigned to C–O/C–O–C vibrations of polysaccharides, nucleic acids, phospholipids, and pyrophosphates. The biosorption or intracellular binding of zinc in microorganisms is largely mediated by negatively charged molecules such as carboxyl, (poly)phosphates, or hydroxyl groups.

4. Discussion

Zinc is a trace element essential for numerous physiological functions in humans such as growth, immune defense, reproduction, cognition, collagen synthesis, bone mineralization, and neurobehavioral development [7,8]. As the human body has a limited zinc-storing capacity, zinc deficiency can develop rapidly when zinc intake is low. Several intervention strategies can be used to treat zinc deficiency. Postharvest food fortification with zinc salts is a cost-effective and sustainable option for increasing zinc intake, with a positive impact on selected indicators of zinc status [10,12,13]. Due to their widespread consumption and their inherent high nutritional value, wheat flour and wheat-based bakery goods are preferred candidates for zinc (bio)fortification [46,47,48]. The WHO has already given recommendations for zinc concentrations and compounds to be used for wheat flour fortification (30–100 ppm Zn; considering the phytate extraction quota and the estimated per capita availability of flour) [49,50]. The Global Fortification Data Exchange (GFDx) database provides an overview of the fortification status for countries with mandatory and voluntary zinc fortification standards with regard to wheat flour (fortification level 15–101 ppm Zn for wheat flour) [51]. Considering the imminent depletion of sustainable resources, insect flour is expected to play an increasingly important role as a food ingredient in the near future [38,52]. Although insects have an abundance of minerals and trace elements, their gastrointestinal solubility is limited. This makes (bio)fortification a good option for improving their micronutrient value [53]. A crucial topic in the preparation of zinc-fortified insect-powder-blended bakery goods, such as cricket powder hydrolysate valorized sourdough bread [37], is the effect of zinc on yeasts and LAB added as starter or adjunctive cultures. Knowledge about the effects of this microelement on non-conventional yeasts and LAB, which might become relevant in the production of (sourdough-based) bakery goods [35], is not yet comprehensive.
Amongst the three yeast strains studied here, Yarrowia lipolytica RO25 was slightly more zinc tolerable with unrestricted growth up to 2.5 mM ZnSO4 addition. This observation is consistent with reports for marine Yarrowia lipolytica strains under heavy metal stress [54,55]. Zinc tolerance and enrichment have so far mainly been studied in Saccharomyces cerevisiae and Saccharomyces pastorianus strains, which have zinc tolerance and an enrichment quota of the same magnitude as the yeasts in the present study [42]. A few studies have examined the zinc tolerance of yeast strains derived from environmental samples. García-Béjar et al. (2020) conducted a comprehensive screening of 213 wild yeast strains from various sources, including flowers, animals, water, and soils from the food industry environment, to assess zinc biodetoxification and resistance. The results revealed significant differences depending on the yeast genus or species surveyed [56]. According to De Nicola et al. (2009), Distiller’s M-type Saccharomyces cerevisae yeasts (Kerry Biosciences Ltd., Menstrie, Scotland) were found to have a basal content of 15 fg Zn/cell after incubation in YPM, which increased to 200 fg zinc/cell following 100 µM ZnSO4 treatment [57]. For Saccharomyces cerevisiae laboratory strain DY1457, zinc levels of 3.7 fg/cell were determined after 24 h incubation in SD media (Zn level in SD medium ~2.5 µM). A further increase in extracellular zinc supply was accompanied by an increase in cell-associated zinc (~ 84 fg Zn/cell upon 1 mM ZnCl2 treatment) [58]. Yet, extreme zinc surplus (4 mM ZnCl2) led to the collapse of zinc homeostasis and reduced the cell growth of Saccharomyces cerevisiae DY1457 [58,59]. For another laboratory strain Saccharomyces cerevisiae EB83, Garca-Béjar et al. (2020) determined a zinc content of ~30 pg/cell after incubation in YPD broth supplemented with 382 µM ZnSO4, with most of the zinc detected inside the cells (80%). Existing data regarding the compartmentation of zinc in Yarrowia lipolytica cells indicate that significant amounts of zinc are associated with cell wall components, in addition to zinc internalization [60]. Intracellular bio-enrichment, by now best investigated for Saccharomyces cerevisiae and Schizosaccharomyces pombe, involves various transporters and trafficking components. This includes membrane transport proteins of the solute carrier family (SLC)39 (Zrt- and Irt-like protein (ZIP)), a family responsible for the transport of zinc from the extracellular environment or intracellular membrane compartments into the cytosol. Zinc transport across the plasma membrane involves (at least in Saccharomyces cerevisiae) two systems: the high affinity Zrt1 (the apparent Km for zinc ions is 10 nM) and Zrt2 (apparent Km ∼100 nM) transporters. In Saccharomyces cerevisiae cells exposed to high zinc concentrations, regulated endocytosis of these plasma membrane proteins prevents intracellular zinc overaccumulation [61]. As counterparts, CDF transporters (Cation Diffusion Facilitator) move cytosolic zinc into organelles to be stored for an interim period or to be secreted. By sequestering excess zinc into the vacuole, the Saccharomyces cerevisiae ZRC1 vacuolar zinc transporter helps to handle a so called “cellular zinc shock” [58,59,61]. Vacuolar zinc accumulation in yeast is largely dependent on zinc-binding ligands. Polyphosphate, characterized by long chains of phosphate groups, accumulates in large amounts within the yeast vacuole and binds zinc with high affinity. Furthermore, organic anions such as glutamate and citrate are considered to contribute to temporary zinc storage in the vacuoles [58,62,63]. Aside from that, cytoplasmic proteins and peptides (metallothionein, glutathione, phytochelatins) also contribute to zinc storage in yeast cells [61,64,65,66]. However, these mechanisms have limitations to their capacity. Zinc dyshomeostasis due to extracellular zinc excess or disturbed cellular zinc distribution is toxic and impedes yeast growth and metabolism [58,67].
Similar to yeasts, LAB developed mechanisms to tolerate zinc by preventing its toxicity. Several lactobacilli and bifidobacteria have been reported to resist various metal ions at different concentration levels; some of them exhibited the capacity to tolerate up to 100–500 mM extracellular zinc [68,69]. To resist zinc toxicity, LAB can induce the expression of zinc efflux systems most likely encoded by czcD or cadA genes [70,71]. Moreover, they limit the amount of zinc available through surface biosorption and the extracellular formation of ZnO nanoparticles [69,72]. Fructilactobacillus sanfranciscensis DG1’s tolerance toward zinc may be due to one of these mechanisms or even a combination thereof. When being fermented in mMRS broth, Fructilactobacillus sanfranciscensis DG1 contains approximately 0.032 fg Zn/cell (~300.000 atoms of zinc per cell), which is very close to the experimentally determined “minimum zinc quota” in prokaryotes (105 atoms of zinc per Escherichia coli cell). According to the available evidence, the minimum level of zinc is likely to be determined by the number of zinc binding sites that are present on proteins essential for optimal cell function [43]. According to our data, Fructilactobacillus sanfranciscensis DG1 appears to have a moderate zinc requirement and to be homeostatically balanced despite a high extracellular zinc load.
Although the conditions under which the yeasts and bacteria exposed in the tests carried out here differ from those under which sourdough bread is made, it seems likely that the addition of ZnSO4 in the above-mentioned concentration range for wheat flour fortification (approximately 0.18–0.6 mM Zn; assuming a density of sourdough bread of ~0.4 [73]) should not negatively affect the growth and metabolic activity of the microorganisms. A thorough understanding of the role of zinc in the reprogramming of metabolic networks in conventional and biotechnologically improved yeast and lactic acid bacteria is crucial for the breeding of robust strains and their suitability for the production of trace element-enriched goods.

5. Conclusions

Food fortification has become a recognized measure to improve the micronutrient status of large populations in recent years. Cereal flours and bakery products are good candidates for zinc fortification. A future application of this technology to other sustainable resources would be of substantial benefit from a trace element supply perspective. The results regarding the zinc tolerance and zinc enrichment capacities of the three yeast strains Yarrowia lipolytica RO25, Kazachstania unispora FM2, Kazakhstania servazzii KAZ2, and the heterofermentative lactic acid bacterium Fructilactobacillus sanfranciscensis DG1 presented here provide valuable information and will be used soon to create zinc-enriched bakery goods with the inclusion of hydrolyzed cricket flour as a protein-rich nutritional resource. Besides providing micronutrients, zinc-enriched microorganisms may also improve health through their effects on human probiotics—either provided as part of baked goods or as supplements [74]. Bread is one of the most widely eaten staple foods in the world. Both traditional and modern aspects of bakery production offer a wide range of opportunities to integrate nutritional and health benefits. In low- and middle-income countries, where cheap staple foods are prevalent and dietary diversity is limited, the consumption of zinc-fortified (homemade) bakery goods can be a solution to address trace element deficiencies. Over the past few years, functional fortified foods have become increasingly popular in Western countries. There is great potential for financial growth in this market, particularly since the effect is also considered to be scientifically proven.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/fermentation9060521/s1, Table S1: Results of a two-way ANOVA on growth of yeast and lactic acid bacteria during the Zn-enrichment procedure analyzed by OD600 measurement.

Author Contributions

Conceptualization: C.K. and O.S.; data curation: C.K. and S.R. (Samantha Rossi); formal analysis: C.K., S.R. (Samantha Rossi), M.M. and H.K.; investigation: C.K., M.M. and S.R. (Samantha Rossi); project administration: C.K.; resources: C.K., M.M., O.S., S.R. (Sascha Rohn), and H.H.; supervision: C.K., O.S., F.P. and R.L.; writing—original draft: C.K.; writing—review and editing: C.K., S.R. (Samantha Rossi), M.M., H.K., S.R. (Sascha Rohn), F.P., O.S., R.L. and H.H. All authors have read and agreed to the published version of the manuscript.

Funding

We acknowledge support by the German Research Foundation and the Open Access Publication Fund of TU Berlin.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

The authors would like to thank Julia Keitel, Silvia Heim, and Timo Neubert for excellent technical assistance.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Growth curves of the lactic acid bacteria and yeasts. The microorganisms (A) Fructilactobacillus sanfranciscensis DG1, (B) Kazachstania unispora FM2, (C) Kazachstania servazzii KAZ2 and (D) Yarrowia lipolytica RO25 were grown in their respective incubation media without additional ZnSO4 supply. Growth was monitored over the incubation time by measuring OD600. Based on these experiments, the transition from resting to logarithmic growth phase was determined (marked with arrows), at which zinc was added in the subsequent experiments.
Figure 1. Growth curves of the lactic acid bacteria and yeasts. The microorganisms (A) Fructilactobacillus sanfranciscensis DG1, (B) Kazachstania unispora FM2, (C) Kazachstania servazzii KAZ2 and (D) Yarrowia lipolytica RO25 were grown in their respective incubation media without additional ZnSO4 supply. Growth was monitored over the incubation time by measuring OD600. Based on these experiments, the transition from resting to logarithmic growth phase was determined (marked with arrows), at which zinc was added in the subsequent experiments.
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Figure 2. Growth of yeast and lactic acid bacteria analyzed by OD600 measurement. The microorganisms were transferred from a pre-culture into the respective incubation medium. At the beginning of the log growth phase, defined amounts of ZnSO4 were added and the cultivation continued until the stationary phase was reached. The growth of (A) Yarrowia lipolytica RO25, (B) Kazachstania unispora FM2, (C) Kazachstania servazzii KAZ2 and (D) Fructilactobacillus sanfranciscensis DG1 was analyzed by measuring the optical density at 600 nm. Data are shown as means ± SEM of four independent replicates. Significant differences from 0 mM ZnSO4 at the respective time point are indicated *** p < 0.001; two-way ANOVA with Tukey’s multiple comparison test).
Figure 2. Growth of yeast and lactic acid bacteria analyzed by OD600 measurement. The microorganisms were transferred from a pre-culture into the respective incubation medium. At the beginning of the log growth phase, defined amounts of ZnSO4 were added and the cultivation continued until the stationary phase was reached. The growth of (A) Yarrowia lipolytica RO25, (B) Kazachstania unispora FM2, (C) Kazachstania servazzii KAZ2 and (D) Fructilactobacillus sanfranciscensis DG1 was analyzed by measuring the optical density at 600 nm. Data are shown as means ± SEM of four independent replicates. Significant differences from 0 mM ZnSO4 at the respective time point are indicated *** p < 0.001; two-way ANOVA with Tukey’s multiple comparison test).
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Figure 3. Viability of yeast and lactic acid bacteria following fermentation in Zn-enriched media analyzed by colony formation. An appropriate dilution of the microorganisms at the end of the enrichment procedure was plated on YPD or mMRS to allow the growth of yeasts and LAB colonies (A) Yarrowia lipolytica RO25, (B) Kazachstania unispora FM2, (C) Kazachstania servazzii KAZ2 and (D) Fructilactobacillus sanfranciscensis DG1, respectively. Data are shown as means ± SEM of four independent replicates. Significant differences to 0 mM ZnSO4 are indicated (* p < 0.05, ** p < 0.01, *** p < 0.001; one-way ANOVA with Kruskal–Wallis).
Figure 3. Viability of yeast and lactic acid bacteria following fermentation in Zn-enriched media analyzed by colony formation. An appropriate dilution of the microorganisms at the end of the enrichment procedure was plated on YPD or mMRS to allow the growth of yeasts and LAB colonies (A) Yarrowia lipolytica RO25, (B) Kazachstania unispora FM2, (C) Kazachstania servazzii KAZ2 and (D) Fructilactobacillus sanfranciscensis DG1, respectively. Data are shown as means ± SEM of four independent replicates. Significant differences to 0 mM ZnSO4 are indicated (* p < 0.05, ** p < 0.01, *** p < 0.001; one-way ANOVA with Kruskal–Wallis).
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Figure 4. Zinc quantification in yeast and lactic acid bacteria biomass following fermentation in Zn-enriched media. The zinc content of the microbial biomass at the end of the enrichment procedure was measured by ICP-MS. Cellular zinc levels (A) and relative zinc enrichment compared to cells incubated in basal medium (B) are shown. Data are shown as means ± SEM of four independent replicates. Significant differences from 0 mM ZnSO4 are indicated (** p < 0.01; one-way ANOVA with Kruskal–Wallis).
Figure 4. Zinc quantification in yeast and lactic acid bacteria biomass following fermentation in Zn-enriched media. The zinc content of the microbial biomass at the end of the enrichment procedure was measured by ICP-MS. Cellular zinc levels (A) and relative zinc enrichment compared to cells incubated in basal medium (B) are shown. Data are shown as means ± SEM of four independent replicates. Significant differences from 0 mM ZnSO4 are indicated (** p < 0.01; one-way ANOVA with Kruskal–Wallis).
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Figure 5. Fourier-transform infrared spectroscopy (FTIR) spectra of yeast and lactic acid bacteria fermented in Zn-enrichment media. Infrared spectra of dried yeast material measured in absorption mode against ambient air as background, recorded from 4000–800 cm−1 at 20 °C (N = 120 scans). (A) Yarrowia lipolytica RO25, (B) Kazachstania unispora FM2, (C) Kazachstania servazzii KAZ2 and (D) Fructilactobacillus sanfranciscensis DG1. Spectra are shown after offset-correction in the region of 2010–2000 cm−1. Interpretation of the spectra is based on the references [42,44,45].
Figure 5. Fourier-transform infrared spectroscopy (FTIR) spectra of yeast and lactic acid bacteria fermented in Zn-enrichment media. Infrared spectra of dried yeast material measured in absorption mode against ambient air as background, recorded from 4000–800 cm−1 at 20 °C (N = 120 scans). (A) Yarrowia lipolytica RO25, (B) Kazachstania unispora FM2, (C) Kazachstania servazzii KAZ2 and (D) Fructilactobacillus sanfranciscensis DG1. Spectra are shown after offset-correction in the region of 2010–2000 cm−1. Interpretation of the spectra is based on the references [42,44,45].
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Table
Table 1. Experimental conditions for ICP-MS measurements.
Table 1. Experimental conditions for ICP-MS measurements.
Forward power1550 W
Cool gas flow15 L min−1
Auxiliary gas flow0.9 L min−1 (Argon)
Nebulizer gas flow0.9 L min−1 (Argon)
Nebulizer typeMicroMist
Quadrupole (m/z)66 (Zn), 103 (Rh)
Limit of quantitation0.6 µg L−1 (Zn)
Calibration range1–100 µg L−1 (Zn)
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Rossi, S.; Maares, M.; Kieserling, H.; Rohn, S.; Schlüter, O.; Patrignani, F.; Lanciotti, R.; Haase, H.; Keil, C. Zinc Tolerance of Special Yeasts and Lactic Acid Bacteria for Use in the Food Industry. Fermentation 2023, 9, 521. https://doi.org/10.3390/fermentation9060521

AMA Style

Rossi S, Maares M, Kieserling H, Rohn S, Schlüter O, Patrignani F, Lanciotti R, Haase H, Keil C. Zinc Tolerance of Special Yeasts and Lactic Acid Bacteria for Use in the Food Industry. Fermentation. 2023; 9(6):521. https://doi.org/10.3390/fermentation9060521

Chicago/Turabian Style

Rossi, Samantha, Maria Maares, Helena Kieserling, Sascha Rohn, Oliver Schlüter, Francesca Patrignani, Rosalba Lanciotti, Hajo Haase, and Claudia Keil. 2023. "Zinc Tolerance of Special Yeasts and Lactic Acid Bacteria for Use in the Food Industry" Fermentation 9, no. 6: 521. https://doi.org/10.3390/fermentation9060521

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