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Article

Succession of Bacterial and Fungal Communities during Fermentation of Medicinal Plants

by
Simon Sauer
1,2,
Leon Dlugosch
2,
Felix Milke
2,
Thorsten Brinkhoff
2,
Dietmar R. Kammerer
1,
Florian C. Stintzing
1,* and
Meinhard Simon
2,*
1
WALA Heilmittel GmbH, Dorfstrasse 1, 73087 Bad Boll, Germany
2
Institute for Chemistry and Biology of the Marine Environment, University of Oldenburg, Carl von Ossietzky Strasse 9-11, 26129 Oldenburg, Germany
*
Authors to whom correspondence should be addressed.
Fermentation 2022, 8(8), 383; https://doi.org/10.3390/fermentation8080383
Submission received: 18 July 2022 / Revised: 5 August 2022 / Accepted: 8 August 2022 / Published: 11 August 2022
(This article belongs to the Section Microbial Metabolism, Physiology & Genetics)
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Abstract

:
The fermentation of medicinal plants has been studied very little, as compared to the fermentation of food and beverages. One approach applies fermentation by single bacterial or fungal strains and targets the production of specific compounds or preservation of the fermented material. Spontaneous fermentation by an autochthonous starter community may lead to a more diverse blend of fermentation products because co-occurring microbes may activate the biosynthetic potentials and formation of compounds not produced in single strain approaches. We applied the community approach and studied the fermentation of four medicinal plants (Achillea millefolium, Taraxacum officinale, Mercurialis perennis, and Euphrasia officinalis), according to a standardized pharmaceutical fermentation method. It is based on the spontaneous fermentation by plant-specific bacterial and fungal communities under a distinct temperature regime, with a recurrent cooling during the first week and further fermentation for at least six months. The results revealed both general and plant-specific patterns in the composition and succession of microbial communities during fermentation. Lactic acid bacteria increasingly dominated in all preparations, whereas the fungal communities retained more plant-specific features. Three distinct fermentation phases with characteristic bacterial communities were identified, i.e., early, middle, and late phases. Co-occurrence network analyses revealed the plant-specific features of the microbial communities.

Graphical Abstract

1. Introduction

Microbial fermentation has been used for ages as a traditional method to preserve food, making otherwise indigestible food edible, improving its nutritional value, and providing medicinal preparations. Even though many applications have been hardly used during the last few decades, fermentation has experienced a recent revival [1,2,3]. Fermentation of medicinal plants (MPs) in pharmaceutical processes, however, has hardly been applied [4,5]. It is used in traditional Chinese [6,7], Ayurveda [8], and Kampo medicine [9], as well as for the production of mother tinctures of MPs in various medical applications [10,11,12,13]. However, very little is known about the microbial taxa involved in the fermentation of MPs.
The fermentation of plant material by bacteria and/or fungi, as applied for health-promoting and medicinal properties, leads to biochemical transformations and decomposition of various organic compounds. It also brings about the formation of novel and bioactive substances and yields a variety of organic acids and alcohols, which eventually preserve the fermentation products from further microbial degradation. Thus, primary MP compounds may be retained, modified, or supplemented by additional metabolites. Likewise, the microbial community mediates a bio-activation of herbal medicinal compounds, which may improve the therapeutic potential and efficacies and may decrease or remove toxic compounds [14,15,16,17]. A variety of bacteria and fungi have been reported to produce bioactive compounds with a wide potential of therapeutic applications [18,19]. Lactic acid bacteria (LAB), one of the key players in fermentation, were shown to exhibit beneficial features in various fermented plant preparations. Bacteria of this group have been successfully used as probiotics in pig husbandry, thus preventing negative effects of antibiotics [20]. Furthermore, LAB-fermented plants have been applied to treat diabetes and liver diseases in rats [21], reduce stress [22], and inhibit the proliferation of cancer cell lines better than non-fermented plant extracts [23]. Artemisia princeps Pamp., fermented by Lactobacillus plantarum SN13T, showed therapeutic potential by reducing the release of interleukin-8 in a human hepatoma cell line [24]. In these applications, predominantly single LAB strains were applied. Traditional fermentation of MPs, however, applies complex microbial communities, including bacteria and fungi. The complex interaction of co-occurring microorganisms may lead to the modification of plant-borne compounds or production of novel bioactive compounds [5,25,26,27,28,29]. These microbial communities originate from added starter communities or the plant-specific microbiome. Thus, the fermentation process may be affected by the environmental conditions of the MPs, season, and age of the used plant organs and can be modulated by added starting communities or by controlled fermentation conditions favoring distinct community members [9,30,31].
To broaden our knowledge on the fermentation of MPs and microbes involved, we carried out an in-depth survey of the succession of bacterial and fungal communities during fermentation of Achillea millefolium L., Mercurialis perennis L., Taraxacum officinale L., and two Euphrasia species (E. officinalis L. and E. rostkoviana Hayne), without further distinction during harvest. We applied amplicon sequencing of a fragment of the bacterial 16S rRNA gene and fungal ITS 2 region. Achillea millefolium is a perennial plant native to Asia, Europe, and North America. Pharmacological applications of this plant comprise the treatment of loss of appetite, gut complaints, wounds, and menstrual spasms [32]; additionally, even anti-inflammatory and anti-cancer potentials were recorded [33]. Mercurialis perennis, native to Europe, northern Africa, and the Middle East, is mainly used for the treatment of poorly healing wounds, mammilitis of lactating women, hemorrhoids, or conjunctivitis [34,35]. Taraxacum officinale has wide medicinal applications since ancient times, and it has been used in traditional and native American medicine [36]. Beside antimicrobial activity [37,38], Taraxacum spp. reveal protective action against hepatotoxicity, but also oxidative stress and the inhibition of proliferation of cancer cell lines were reported [39]. Euphrasia officinalis and E. rostkoviana, native to Europe, Turkey, and Georgia, are known for the treatment and prevention of conjunctivitis, other ocular diseases, and the treatment of colds [40].
The fermentation of all MPs was conducted for six to twelve months, in accordance with the German Homoeopathic Pharmacopoeia [40] (Figure 1), and we addressed the following questions. (i) Do MPs exhibit a general or a MP-specific temporal pattern of the microbial communities during fermentation? (ii) Does the succession of bacterial and fungal communities vary similarly during the fermentation process? (iii) How do MPs differ in the co-occurrence of bacterial and fungal taxa? (iv) Does the recurring cooling twice a day in the first week of fermentation, according to the GHP method, affect the composition of the microbial communities? (v) Does the use of a MP harvested in different years affect the composition and succession of microbial communities during fermentation?

2. Materials and Methods

2.1. Collection of Plants

Achillea millefolium was harvested on 22 July 2016 and 26 June 2018, in the MP garden of WALA Heilmittel GmbH (Bad Boll/Eckwälden, Germany). Open flowers, including the top 10 cm of the stem and leaves, were harvested and used for further processing. Taraxacum officinale was harvested as whole plants, including roots, in the MP garden of WALA Heilmittel GmbH on 27 April 2017. Flowering herbs of Mercurialis perennis (male and female 1:1) were harvested close to Bad Boll, Eckwälden (Germany), at the Turmberg (48°37′56″ N, 9°34′20″ E), and processed on three separate days (27, 28, and 30 March 2017). Euphrasia officinalis and E. rostkoviana were harvested as whole plants, including roots, on 1 August 2017 in Deilingen (Germany). No differentiation between E. officinalis and E. rostkoviana Hayne was made.

2.2. Fermentation According to the GHP Standard

We sampled the microbial communities of the four MPs during the fermentation performed, according to the official GHP standards (Figure 1 and Supplementary Table S1).
The process can be briefly summarized as follows. Freshly harvested plants were manually cleaned from brown, dead, infested, or infected tissue. When roots were present, they were cleaned with tap water and a brush. The clean plant material was chopped by knife and ground by mortar and pestle into a pulpy macerate. Achillea millefolium and E. officinalis were fermented, according to GHP method 33c, including macerated plant material, honey, deionized water, and lactose in a ratio of 100:0.75:125:0.75 (w:w:w:w). Taraxacum officinale and M. perennis were fermented, according to GHP method 34c, including macerated plant material, deionized water, and cow whey in a ratio of 4:3:2 (w:w:w) (Figure 1). The fermentation was conducted in 5 L containers, incubated at 37 °C for 2 days, and thereafter at 20 °C until day 7. Incubation of each MP was performed in triplicate, except for Euphrasia, which was fermented in only one replicate. During the first week, fermentation containers were cooled twice per day by placing each in the morning and evening for 2 h on crushed ice (for the schedule of cooling see Supplementary Table S1), and the content was stirred for 1 min in the beginning and at the end of the cooling phases. On day 3, the coarse plant material was removed by filtration through a sterile cotton cloth, dried, and incinerated in a porcelain crucible on a Bunsen burner. The obtained ash was added to the fermentation batch on day 7 at a concentration of 50 ppm. Thereafter, the fermenting filtrate was transferred to sterile 1 L glass bottles, processed for three weeks at 20 °C in the dark, and thereafter at 18 °C for the remaining phase of 6 to 14 months, according to the GHP standard [40].
To test for the recurring cooling effect of the GHP method on the composition of the microbial communities, Achillea plant material harvested in 2016 was also fermented without the cooling phases in the first week, but otherwise similarly to the GHP method (henceforth referred to as “warm” treatment).

2.3. Sampling

During the first seven days, 1 mL samples of the homogenous slurry were withdrawn every morning after starting the fermentation (day 0) at the beginning of the cooling phase (Figure 1). Starting with the second week, sampling intervals were increased (Table 1). Since then, 2 mL samples, including the filtrate and settled sludge, were withdrawn using a sterile 30 cm long steel cannula (Unimed, Lausanne, Switzerland) through the sterilized rubber lid without opening the bottle. The samples were transferred into 2 mL tubes (Eppendorf, Hamburg, Germany), immediately centrifuged at 12,900× g for 5 min at 4 °C, and stored at −80 °C until further processing. At each sampling, the pH was recorded (InLab Expert Pro-ISM-IP67, Mettler-Toledo AG, Greifensee, Switzerland). Fermentation additives (lactose monohydrate, whey, honey, and water), according to the GHP methods, were sampled as controls. As reference, freshly macerated Achillea plant tissue was sampled prior to fermentation start.

2.4. DNA/RNA Extraction

DNA was extracted using the DNeasy PowerSoil Kit (Qiagen, Hilden, Germany), according to the manufacturer’s manual. The final extracts were quantified using a Qubit 2.0 fluorometer (Invitrogen/ThermoFisher, Waltham, MA, USA) and diluted with DNA-free water or concentrated by using co-precipitant pink (Meridian Bioscience, Memphis, TN, USA), following the manual to adjust to a final concentration of 1–10 µg/µL DNA.
To assess the metabolically active bacterial community on the basis of 16S rRNA sequencing, RNA extraction was performed at days 5 and 7 during the warm and cool treatments of Achillea in 2016 by using a total RNA purification kit (Norgen, Thorold, ON, Canada), according to the manufacturer’s manual. Combined with an on-column DNA removal protocol (RNase-Free DNase I Kit, Product # 25710, Norgen) and additional DNA digestion (DNA-freeTM Kit, Invitrogen/ThermoFisher), we ensured that no DNA was left, as checked by PCR of the 16S rRNA-gene by the universal primers 341f/R806. The reverse transcriptase assay was carried out with the innuSCRIPT reverse transcriptase (Analytik Jena, Jena, Germany), according to the manual and by using random primers (100 ng/µL).

2.5. Sequencing

The internal transcribed spacer (ITS) region of fungi was amplified with primer f-ITS7 (5′- GTGARTCATCGAATCTTTG-3′)/ITS4 (5′-TCCTCGCCTTATTGATATGC-3′) targeting the ITS 2 region. The bacterial V5–V6 region of the 16S rRNA-gene was amplified with primer pair 799f (5′-AACMGGATTAGATACCCKG-3′)/1115r (5′-AGGGTTGCGCTCGTTG-3′) to reduce chloroplast-DNA signals. Controls were visually checked through an agarose gel. Samples were subsequently sequenced by LGC Genomics GmbH (Berlin, Germany) on an Illumina MiSeq platform (Illumina bcl2fastq 2.17.1.14 software, San Diego, CA, USA).

2.6. Bioinformatics

Sequence data from plant, whey, and fermentation samples of the bacterial 16S rRNA gene, 16S rRNA, and fungal ITS sequences were processed as follows. Trimmomatic 0.36 [41] was used to truncate low quality read ends if the average quality dropped below 15. Primer sequences were removed from amplicon sequences using bbduk (https://sourceforge.net/projects/bbmap/) accessed on 7 January 2018. All datasets were subsequently processed with USEARCH v.10.0.240 [42]. Sequences were merged, and low-quality sequences were discarded (shorter than 300 base pairs (bp), accumulative sequencing error rate ≥ 1%), thus resulting in 14,523,679 and 3,741,682 high-quality (HQ) amplicon sequences for the 799f-1115r primer set and fungal ITS, respectively. HQ sequences were pooled according to the used primer set. In addition, sequences from the 16S rRNA gene dataset were truncated to equal length of 300 bp. Due to high variance in ITS sequence length, this step was skipped for fungal ITS datasets. Subsequently, all sequences were dereplicated and sorted by abundance. Chimeric sequences were removed, and remaining sequences were denoised into zero-radius taxonomic units (ZOTUs) using the unoise3 algorithm, with a minimum unique sequence abundance of 8 across all samples. In total, 20,133 (bacterial 16S rRNA gene dataset) and 5932 (fungal ITS) ZOTUs were generated. All ZOTUs were taxonomically classified by alignment employing the USEARCH algorithm against the GTDB SSU database release 89 [43] for 16S rRNA gene sequences and the UNITE ITS database 8.0 [44], with an e-value cut-off of 1 × 10−10, minimum sequence identity of 90%, and maxaccepts/maxrejects option disabled. An abundance table was created by mapping HQ sequences of each sample to the ZOTUs. A subset of samples from fresh plants, comminuted plant material, and fermentations was used in this analysis (see Supplementary Table S2).

2.7. Statistical Analysis

Statistical analyses were performed using R v4.0.5 [45] with the packages vegan [46], ape [47], drc [48], picante [49], and igrpaph [50]. Only samples with more than 1000 mapped reads were considered for further analysis.
To account for varying sequencing depth, count tables of bacterial 16S rRNA gene, and fungal ITS datasets were repeatedly rarefied to 1000 sequences per sample (99 times). Subsequently, richness and Shannon entropy, as well as species coverage, were calculated for each iteration, and the mean value was used for further analyses. Effective number of taxa (EN) was calculated according to reference [51]. Linear model fitting was used to determine a relationship between richness and EN of the associated communities during fermentation.
Prior to further analysis, unrarefied DNA and RNA samples were converted to relative abundances by dividing individual ZOTU counts by the total number of reads per sample. Bray–Curtis distances [52] of bacterial and fungal abundances of each fermentation and sampling time were visualized by nonmetric multidimensional scaling (NMDS) (k = 2; 999 permutations).
A co-occurrence correlation matrix was inferred using SparCC [53] to account for compositional data. We analyzed the 50 most abundant bacteria and fungi, in total, 100 ZOTUs, for their co-occurrence structure. Both datasets were first normalized to proportions and then joined. A modified version of the SparCC algorithm to use fractions, instead of counts was applied on the combined bacterial and fungal dataset [54]. The data were shuffled 999 times by randomly assigning a ZOTU proportion within each sample from all the proportion values of these ZOTUs in all other samples with replacements. SparCC was run on all shuffled datasets and a pseudo p-value for all correlation values was computed as the proportion of shuffled datasets for which a correlation value at least as extreme as the one computed for the original data was obtained. Only positive correlations with a strength of r ≥ 0.5 and Benjamini–Hochberg [55] adjusted p-value ≤ 0.05 were used for further analysis. Clusters were calculated by using edge-betweenness between vertices of undirected and unweighted networks.

3. Results

We analyzed the microbiome of the MPs Achillea, Taraxacum, Mercurialis, and Euphrasia during fermentation in a time series of 6 to 14 months and collected 236 samples in total for sequencing the V5-V6 variable region of the 16S rRNA gene of bacterial and genomic ITS 2 region of fungal communities. After removing samples with <1000 counts, we obtained 195 bacterial and 164 fungal high-quality samples. A rarefaction analysis showed sufficient coverage of the fungal genera (88.0 ± 11.4%) and bacterial species (87.8 ± 11.1%) (Supplementary Figure S1). In total 21,417 bacterial and 6637 fungal ZOTUs were generated.
The initial pH values of the MP tissues macerated in distilled water were 6.2, 5.8, 5.2, and 4.7 for Mercurialis, Euphrasia, Achillea, and Taraxacum, respectively, and they decreased strongly in the initial fermentation phase, reaching values below 4.5 on day three (Figure 2). During the subsequent fermentation phase, the pH remained low in most MP treatments but increased slightly in the fermentations of Achillea 2016 and Taraxacum.

3.1. Richness and Diversity

Initially, the richness and EN of the bacterial communities were much higher in the preparations of Mercurialis, Taraxacum, and Euphrasia than in those of Achillea, but they decreased rapidly within three to seven days (Figure 3A,C,E,G). Both indices remained low until the fermentation end of all four MP preparations, except for that of Achillea 2018, in which they increased towards the end of the fermentation. In the preparations of Mercurialis and Taraxacum, the added whey contributed very little to their initially high bacterial richness (see below).
The initial fungal richness and EN were also higher in the preparations of Mercurialis, Taraxacum, and Euphrasia than in those of Achillea (Figure 3B,D,F,H). In the preparations of Taraxacum and Euphrasia, these indices decreased during day 7 to 25 of fermentation and were, thus, much slower than those of the respective bacterial communities. The preparation of Mercurialis exhibited the highest fungal richness and EN over almost the entire fermentation period, whereas in Euphrasia and Taraxacum, these indices continuously decreased until days 7 and 27, respectively. In the preparations of Euphrasia, they increased again, whereas in that of Taraxacum, they remained low thereafter. Richness and EN in the preparations of Achillea remained fairly constant over the initial seven days and decreased slightly during further fermentation time in the preparation of 2018 (Figure 3B,F).

3.2. Community Composition and Succession

The bacterial communities of freshly macerated Mercurialis, Taraxacum, and Euphrasia comprised different lineages of Alpha- and Gammaproteobacteria, as well as small proportions of Firmicutes, Deinococcota, Bacteroidota, and Actinobacteriota, whereas freshly macerated Achillea was largely dominated by Gammaproteobacteria (Figure 4). On the order level, Achillea was greatly dominated by Enterobacterale, and Euphrasia by Sphingomonadales and Rhizobiales. These orders were also present on Mercurialis and Taraxacum, but at lower proportions and accompanied by Lactobacillales, Brachyspirales, and several orders of Actinobacteria (Figure 4). The initially high proportions of Lactobacillales and Brachyspirales in the preparations of Mercurialis and Taraxacum may have resulted, at least partially, from the added whey (Supplementary Figure S2). Brachyspirales, however, were also detected in the preparation of Achillea (Figure 4, Supplementary Figure S3). In the preparations of Achillea, Taraxacum, and Euphrasia, Lactobacillales became rapidly dominant and, from day three onwards, constituted at least 85% of the bacterial community (Figure 4). The preparation of Mercurialis, Enterobacterales with the genera Pantoea, Klebsiella, and Kosakonia dominated from day three to seven and persisted throughout the further fermentation. Lactobacillus constituted high proportions from day 39 onwards, except in one replicate on day 226 (Figure 4 and Supplementary Figure S2). Pantoea was also present during fermentation of Achillea and Euphrasia and, together with Buttiauxella, constituted prominent proportions in the early fermentation phase (Supplementary Figures S2 and S3).
Because of the outstanding significance of Lactobacillales for fermentation of the four MPs, we analysed the composition and temporal succession of this bacterial order in more detail (Figure 5). In the preparations of Achillea and Euphrasia, fermented according to GHP method 33c, Weissella dominated on day one. Thereafter, Pediococcus spp. constituted the highest proportions, until Lactobacillus dominated in the final phase by more than 90% of the total bacterial community. The preparations of Mercurialis and Taraxacum, both fermented according to GHP method 34c, including the addition of whey, exhibited different temporal patterns (Figure 5). Differences occurred mainly in the initial phase, but later, Pediococcus spp. and Lactobacillus spp. also became dominant with highest proportions of the latter in the final phase. Here, Weissella was not detected. In the preparation of Taraxacum, Lactococcus was initially dominant, succeeded by an early short dominance of Lactobacillus on day one. Thereafter, Pediococcus was the dominant genus, although Lactobacillus increased constantly—similar to the preparations of Achillea and Euphrasia (Figure 5). In the Mercurialis preparation, Enterobacterales dominated first, and LAB prevailed since day 39 (Figure 4). Here, Lactococcus initially prevailed, but on day 3, Pediococcus became dominant and continued to do so until day 39 (Figure 5). Afterwards, Lactobacillus reached the highest proportions, as in the other fermentations, which, however, varied by replicate and time. In the preparations of Mercurialis and Taraxacum, whey introduced members of the genera Lactococcus, Brachyspira, Lactobacillus, and Clostridium (Supplementary Figure S2). However, on day 3, most of these taxa were not detected any more, indicating that they did not affect the further fermentation process and bacterial communities (Figure 5, Supplementary Figures S2 and S3). Only in the Taraxacum preparation did Lactobacillus proliferate rapidly in the very early phase, reaching a temporarily high dominance of more than 80% of total bacteria at day 1, followed by high abundances of Pediococcus.
The temporal succession of the bacterial communities during the entire fermentation was substantiated by an NMDS analysis. This analysis highlights species-specific features, as well as the general temporal development towards a more LAB dominated community in all preparations (Figure 6A, Supplementary Figure S4).
The fungal communities of the freshly macerated MPs were also distinct. Ascomycota dominated initially in the Mercurialis and Euphrasia preparations, whereas Basidiomycota were dominant in that of Taraxacum (Figure 7). Interestingly, in the preparations of Achillea 2016 Ascomycota and Basidiomycota initially shared similar proportions, whereas in that of Achillea 2018 Basidiomycota greatly dominated and constituted about 80% of all fungal reads. The succession of the fungal communities during fermentation of the four MPs seemed to be plant- and year-specific and did not show such general patterns as the bacterial communities (Figure 7). In the preparations of Taraxacum and Achillea 2018, Ascomycota continuously increased with a final strong dominance of Saccharomycetales. In contrast, in those of Euphrasia and Achillea 2016, different basidiomycotal orders increasingly dominated over time. In the Euphrasia preparation, Tremellales was the single most abundant order. The “warm treatment” of Achillea 2016 showed similar patterns, except at the final sampling date, when Ascomycota greatly dominated. In the preparation of Mercurialis, a high proportion of “other” fungi, belonging to other than the 19 most dominant taxa, increased to 40% in the initial three days, at the expense of Ascomycota (Figure 7, Supplementary Figure S2). Thereafter, Basidiomycota constituted increasing proportions of 10 to 20%. At the final sampling date, the fungal community was rather diverse again.
The reduced fungal diversity of the Taraxacum preparation was a result of the prevalence of Saccharomyces and Candida in the later fermentation stages (Supplementary Figure S2). The Achillea 2018 preparation showed a similar reduction of diversity and dominance of Candida, but the richness remained higher than in the preparation of Taraxacum (Supplementary Figure S3). In the other preparations, the diversity of genera generally remained much higher and more evenly distributed over time, except in that of Euphrasia, in which Vishniacozyma finally predominated (Supplementary Figure S2).
The NMDS analysis of the fungal communities illustrates the different temporal development during fermentation. The communities remained more diverse and differed more during the fermentation, with a less uniform temporal trend than the bacterial communities (Figure 6B, Supplementary Figure S4).
Looking at the temporal succession of the microbial and, in particular, the bacterial communities in the fermentation of the four MPs altogether, three general fermentation phases can be distinguished: (i) an early phase lasting until day 2; (ii) an intermediate phase between day 2 and days 14 to 28, with some variations of the different MPs; (iii) a late phase until the end of fermentation.

3.3. Fermentation of Achillea in 2016 and 2018

To examine annual variations in fermentation, we compared the fermentation of Achillea harvested in 2016 and 2018. The initial bacterial and fungal communities of the leaves and flowers differed between both years (Supplementary Figure S5), which resulted in a different richness and composition of the microbial communities during fermentation (Figure 3, Figure 4, Figure 5 and Figure 7). In 2018, Pseudomonadales, Brachyspirales, and Ascomycota initially, and during the first days and weeks, constituted much higher proportions than in 2016 (Figure 4 and Figure 7). However, in contrast to 2016, Lactobacillus was not detected until day 28, but it dominated the bacterial community thereafter (Figure 5). In 2018, Ascomycota constituted much higher proportions than in 2016, with a strong dominance of Candida, which did not exceed 2% of the total reads in 2016 (Supplementary Figure S3). These differences were also reflected in a lower pH in 2018 than in 2016 (Figure 2), as well as in the different NMDS patterns of both years (Figure 6 and Supplementary Figure S4).

3.4. Co-Occurrence of Bacteria and Fungi during Fermentation

The bacterial and fungal communities exhibited pronounced and plant-specific temporal changes, thus reflecting their different involvement in the breakdown of the plant material and during fermentation. Members of both communities presumably interacted during the fermentation process in various ways. The interactions may be very complex, but can be assessed at a first glimpse by the co-occurrence of the different taxa. Therefore, we carried out a SparCC-inferred co-occurrence network analysis for those MPs, for which sufficient data and replicates were available, i.e., Achillea, Taraxacum, and Mercurialis (until day 192). The results show distinct plant-specific network topologies.
For Achillea, this analysis showed four major and five minor clusters of closely connected taxa that had no, or only weak, connections to other parts of the network (Figure 8A). Four clusters, including one of the major ones, consisted exclusively of fungi—three exclusively of bacteria and only two clusters encompassed both fungi and bacteria. One of the latter clusters (dominating in the late fermentation phase) contained almost all LAB and only three fungal genera, including Candida. This fungal genus displayed a central position by co-occurring with several LAB within this cluster and with members of Enterobacterales in the adjacent cluster, thus dominating in the early fermentation phase. One Lactococcus connected it to the other LAB containing cluster, which was linked via one Lactobacillus to the fungi-exclusive cluster. A chord diagram further showed that LAB were linked only to few other microbial taxa, mainly to Brachyspirales, a few Enterobacterales, and Saccharomycetales (Supplementary Figure S6A).
The microbial communities fermenting Mercurialis exhibited four linearly linked clusters (Figure 8B). The largest cluster encompassed all LAB except two, all enterobacterial taxa except one, and about half of all fungi. The second largest cluster included two LAB, other bacteria, and few fungal taxa. This cluster was linked to the largest cluster via two other smaller clusters, which consisted almost exclusively of fungi. The chord diagram of these communities showed interlinked taxa from various bacterial and fungal groups (Supplementary Figure S6B).
The microbial communities of Taraxacum also exhibited four linearly linked clusters (Figure 8C). The network analysis displayed one cluster encompassing all taxa of the intermediate and late fermentation phases, all LAB except one, and few ascomycotal fungi. It was linked to the largest cluster consisting of the majority of fungi. This was linked via an unidentified Heliotiales taxon to a small cluster consisting of only three ascomycotal fungi and further to another cluster consisting of few Enterobacterales, other bacteria, and a few ascomycotal fungi. The chord analysis of the Taraxacum networks showed only few links of LAB to other microbes, whereas most other microbes were intensely linked among each other (Supplementary Figure S6C).

3.5. Effect of Cooling in the Fermentation of Achillea

Processing according to the GHP standards requires a recurring cooling to 4 °C twice per day during the first week of fermentation. Hence, in 2016, we tested the effect of cooling by comparing fermentation of Achillea, according to the GHP standard, with permanently warm fermentation at 37 °C until day 2 and, thereafter, at 20 °C. We detected no difference in diversity or pH but observed decreased proportions of Lactococcus and Weissella and increasing proportions of Lactobacillus in the warm treatment (Figure 5, Supplementary Figure S3). Further, Brachyspira and Pantoea constituted reduced proportions in the warm treatment, relative to the cold treatment (Supplementary Figure S3). Analyses of the 16S rRNA (cDNA, active bacteria) on days 5 and 7 showed that the genus Lactobacillus also constituted enhanced proportions of the active community in the warm treatment, whereas Weissella and Lactococcus were relatively more abundant in the cold treatment (Figure 9). Fungal communities exhibited only minor variations between both treatments (Supplementary Figure S3). These results demonstrate that cooling twice per day in the first week does have an impact on the succession of the microbial communities.

4. Discussion

Our observations show that, in all four preparations of MPs, the different and plant-specific bacterial communities adapted and changed during the first week of fermentation, with a decrease of the pH in all preparations to below 4.3. In contrast, the fungal communities did not reflect these fermentation patterns. Temporal changes of richness, EN, and composition of the latter communities varied over longer periods of time and exhibited more plant-specific features. We applied a spontaneous fermentation approach by the plant-specific microbiome, in order to account for its metabolic potential. This standardized approach, according to GHP standards, has been applied for more than 80 years. It defines pharmaceutical processes to secure consistent quality, which is monitored by specific analytical methods, such as thin layer and high-performance liquid chromatography, and controlled by national authorities (German Federal Institute for Drugs and Medical Devices). Here, we specify the view on the microbial succession. Based on the bacterial communities, we identified three phases during the fermentation of the four MPs, even though the duration of these phases differed for each MP.

4.1. Early Fermentation Phase

The first fermentation phase lasted two to three days and was characterized by a strong decrease in pH and richness, as well as a rapid increase of LAB, which was least pronounced in the Mercurialis preparation. Similar observations are well-known from autochthonous food fermentation [56,57,58,59] and the few reports on the fermentation of MPs, e.g., Calendula officinalis L. and Matricaria chamomilla L. [60]. The fungal communities did not show this phase, and changes in their community composition, including the proliferation of fermenting yeasts occurred later, presumably reflecting the slower growth and tolerance to low oxygen concentrations and pH [60]. Thus, a diverse and MP-specific fungal community prevailed, which contained yeasts, as well as filamentous fungi, was also observed in traditional fermentations of various food [1,61,62,63,64].
In the initial shift towards the dominance of LAB, preparations applied with whey according to GHP method 34c differed to 33c without whey. Lactococcus sp. and Lactobacillus spp. dominated the early LAB communities in 34c (Taraxacum and Mercurialis), whereas Weissella spp. greatly dominated in 33c (Achillea and Euphrasia). We assume the initial high abundance of Lactococcus originated from the added whey. Lactococcus spp. are preferred starter cultures for fermented dairy products, have a preferential growth at neutral pH, and reveal high acidification rates [65,66,67]. As Lactococcus rapidly decreased in the early fermentation phase, Mercurialis and Taraxacum may provide inappropriate fermentation conditions to this foreign strain and, thus, were rapidly overgrown by other LAB. In the preparation of Taraxacum, a Lactobacillus ZOTU was detected after the decline of Lactococcus. It presumably also originated from the added whey, as it was identical to the ZOTU in the added whey. In contrast to Lactococcus, this Lactobacillus strain might be well-adapted to the fermentation condition in Taraxacum after a short latency phase and has a high acidification capacity, as reported for several Lactobacillus spp. [66,68]. In fact, Taraxacum crude extracts seem to favor the rapid proliferation of Lactobacilli [36,69]. The heterofermentative genus Weissella, exclusive to Achillea and Euphrasia and greatly dominating their initial fermentation, is known as a very suitable starter for fermented food and in biotechnological applications [66,70,71]. The majority of all Weissella-ZOTUs were assigned to W. ciberia, a species known to reduce toxicity and increase anti-inflammatory properties in the MP Inula britannica [15]. The production of a broad range of bacteriocins, probiotic, and anti-inflammatory metabolites by different Weissella strains has been reported [15,70,71,72].
Besides LAB, other lineages were detected in the different preparations in the early fermentation phase. Brachyspira sp. of the Spirochaetaceae occurred in the preparations with added whey and rapidly disappeared. As this taxon was also detected in the whey itself, we assume that it was introduced along with the whey. However, Brachyspira sp. was also detected in the preparations of Achillea, where it decreased until day 84. We do not know how it was introduced into the Achillea preparations, possibly by a contamination. This obligate anaerobic genus is known from the intestines of mammals, in particular, pigs and cattle, and includes pathogenic strains [73]. We do not know whether it had any effect on the fermentation process of these MPs, but we did not observe anything that would specifically be attributable to Brachyspira. The enterobacterial fermentative Pantoea initially constituted high proportions in the preparations of Achillea and Euphrasia but rapidly declined in abundance. This genus is known from plant microbiomes, including that of Achillea [31,66,74,75], thus it is presumably part of the autochthonous microbiome of these MPs.

4.2. Intermediate Fermentation Phase

This phase included the temperature shift from 37 to 20 °C at day 3 and, finally, to 18 °C on day 20, according to the GHP method, as well as the removal of insoluble plant material on day 4 and bottling on day 7, and it featured a pH consistently below 4.5. The progressing fermentation was reflected in the dominance of Lactobacillales to >90% in three of the four MP preparations over this entire phase. In the preparation of Mercurialis, the enterobacterial genera Klebsiella, Kosakonia, and Pantoea constituted more than 65% until the first week, and only thereafter did Lactobacillales become dominant. The prevalence of Pediococcus spp. characterized the LAB community in all preparations until Lactobacillus spp. increased and marked the end of this phase. They typically occur or are applied in different fermentations in food processing, biotechnological applications, and the production of probiotics [65]. In a study on the spontaneous fermentations of C. officinalis L. and M. chamomilla L. over six weeks, which were not performed according to the GHP standard, Enterobacterales and Pseudomonadales greatly dominated, and LAB never exceeded 7% [60]. This dominance was irrespective of the pH, which was 2.8 in the preparation of M. chamomilla and 6.8 in that of C. officinalis. Enterobacterial species, as found in the fermentation of C. officinalis and M. chamomilla, are also used for processing of different types of food and tea [75,76,77,78,79]. The comparison to our MP preparations indicates that the application of the GHP methods leads to always low pH value and appears to favor fermentation by LAB, irrespective of their origin from whey or the specific MP-associated microbiome. However, our analyses suggest that the latter is the more important source.
The still high fungal diversity at the beginning of the intermediate phase decreased gradually, except in the preparation of Mercurialis, with plant-specific patterns. In the preparations of Taraxacum and Achillea 2018 members of the fermenting yeast genera Saccharomyces and Candida dominated to >95% at the end of this phase and beyond, whereas in the other preparations, they constituted only minor proportions. The other preparations, including those of Achillea 2016, ascomycotal, basidiomycotal, and other fungal taxa, constituted varying proportions but with a more balanced partitioning of each lineage. We have no clear-cut explanation for the differences in the fungal communities in the preparations of Achillea 2016 and 2018, despite similar bacterial communities in both years. The plant-associated bacterial and fungal communities differed between both years (Supplementary Figure S5), which may have caused the differences in the composition of the fungal communities but did not affect that of the bacterial communities during fermentation in both years. These differences may be critical for the comparability of the mature tincture of both years. However, a first test of the phenolic compounds, presumably processed by the fungal community, indicated no difference (S. Sauer et al., unpubl. data). Our data on the fungal communities support the assumption that their role during the early and intermediate fermentation phases was less related to the drop in pH and specific lactic acid fermentation. As shown in studies on the fermentation of food and beverages, fungi have unique capabilities to degrade complex lignocellulosic material and phenolic compounds, interact with bacteria, and produce novel compounds [80,81,82,83]. Hence, we assume that the distinct communities of filamentous fungi and yeasts actively contributed to the breakdown of the plant material and promoted the release of plant specific primary and secondary metabolites. Their interactions with bacteria are presumably of particular significance, as they are known to be of key importance in autochthonous fermentation of food [80]. Even though we are unable to specify these interactions, we showed that fungi and bacteria have MP-specific co-occurrence patterns, a first indication that systematic interactions exist (see below).

4.3. Late Fermentation Phase

This phase was typically characterized by a low number of remaining LAB genera. It started between day 42 for the preparation of Achillea 2018 and day 192 for that of Mercurialis. Due to the longer sampling intervals, it was impossible to determine the exact beginning of this phase. The pH remained below 4.3, but slightly increased at the final sampling of the preparations of Mercurialis and Achillea 2016, presumably via actively secreted alkaline compounds, such as ammonium, by persisting active fungi, as has been reported previously [84]. In all preparations, except in those of Mercurialis and Achillea 2018, at the final sampling, only few LAB genera (Lactobacillus and Pediococcus) remained. This is a common final fermentation feature in many applications [71,79,85,86,87,88]. It is presumably a result of the high acidification rate and ability of these taxa for slow and persisting growth at low substrate availability [65]. The non-LAB detected at the final sampling in Achillea 2018 may have been caused by contamination during sampling or if they were introduced otherwise, as they were not detected previously. The Mercurialis preparation, deviating in its bacterial succession during fermentation from the others by maintaining a higher diversity until the end; additionally, Enterobacteriaceae, presumably contained different organic compounds as a source of energy and carbon, thus leading to a differently composed microbiome, which would be interesting to study.
The fungal communities basically maintained their plant-specific characteristics of the intermediate phase in the late phase, even though relative proportions of single taxa shifted at the late and final samplings. Since only one replicate was available for the preparation of Euphrasia, the results need to be assessed cautiously. It is interesting that an elevated diversity remained in all preparations, except in that of Taraxacum, with the clear dominance of Saccharomyces. We do not know whether these fungi were still active or persistent in the resting stages, and we did not assess their abundances relative to that of bacteria. Hence, we cannot clearly evaluate the significance of the fungi, with regard to their metabolic contribution in the late phase.

4.4. Co-Occurrence of Bacteria and Fungi

It is well-established that different bacteria, as well as filamentous fungi and yeasts, are actively involved in processing different types of food during fermentation, including plant-derived, as well as dairy and health-promoting, products [80,89,90,91]. Filamentous fungi may breakdown complex polymeric compounds and provide bacteria with substrates for fermentation, but they may also produce specific compounds derived from bacterial fermentation products. Bacteria may also contribute to breaking down polymers, actively ferment monosaccharides, and produce fermentation and/or antibiotic products, which are different from those produced by filamentous fungi and yeasts. Spontaneous fermentation by autochthonous microbial communities takes advantage of these mutually beneficial features, but it is difficult and challenging to identify such interactions in the preparations we studied. A first attempt is to look for the co-occurrence of different taxa during fermentation, implying that they interact and possibly cooperate in the maturation of the fermenting products. For this reason, we applied co-occurrence networks of the bacterial and fungal taxa, in order to test our MP preparations for potential interactions. This analysis yielded plant-specific patterns, thus showing how differently LAB co-occurred with enterobacterial, yeast, and filamentous fungal genera. LAB are usually grouped closely together in one or two clusters, indicating their specific role in all preparations, as discussed above. These clusters were complemented differently by other bacterial species and fungal genera, and this was most pronounced in the Mercurialis preparation. Some fungal genera clustered together with no or few links to LAB or Enterobacterales, thus suggesting that only few fungal genera had the potential to interact with bacteria. These analyses suggest that only a limited number of fungi, LAB, and enterobacterial genera were the active players during fermentation. Interactions during fermentation are usually rather specific, e.g., by producing growth promoting compounds of mutual benefit or the joint breakdown of complex compounds [80,89,90,91]. Therefore, co-occurrence networks may help to identify and specifically investigate such interactions in follow-up studies. So far, only very few studies have applied co-occurrence networks in fermentation studies [92,93], with, to the best of our knowledge, none in the fermentation of MPs.

4.5. Recurring Cooling and Its Significance for the Fermenting Microbial Community

The comparison of the fermentation of Achillea, according to the GHP standard, with recurring cooling twice per day in the first week to the permanently warm fermentation revealed an earlier increase of Lactobacillus in the latter and prolonged higher proportions of Lactococcus, Weissella, Brachyspira, and Pantoea in the former treatment. Additionally, Candida and a few other fungal taxa maintained higher abundances in the cold treatment. Hence, cooling twice per day does have an impact on the fermentation during the first week and supports the significance of cooling, according to this GHP method for fermenting Achillea, as well as, presumably, other MPs by LAB. Possible effects of these successional differences on the chemical profile during this phase were not examined, but we speculate that they may occur as a result of cooling, due to the prolonged enhanced diversity of LAB. It has been shown that cooling to below 10 °C triggers a stress response in the Lactococcus strain, increasing specific protein expression, which was not detected at 30 °C [94]. Hence, it is also conceivable that the recurring cooling leads to the formation of chemical compounds, which is not produced at constantly warm conditions, thus maintaining a higher chemical diversity when applying the GHP standard.

5. Summary and Outlook

We investigated, for the first time, the composition and succession of microbial communities by high resolution amplicon sequencing of marker genes during the fermentation of MPs, according to GHP standards. Our study revealed that the fermentation of Achillea, Euphrasia, Mercurialis, and Taraxacum, according to GHP methods 33c and 34c, with autochthonous microbial communities of the MPs leads to a rather unified LAB dominated community in all MPs, despite a different and diverse plant-specific initial microbial community. During the course of the fermentation process, we identified three phases of variable length in each MP, mainly reflected in the bacterial communities.
The fungal communities developed differently and maintained a rather MP-specific composition. Co-occurrence analysis revealed plant-specific clusters of bacteria and fungi, which suggests that distinct interactions of a limited number of taxa occur and are presumably important for the formation of plant-specific chemical profiles in the tinctures. The comparison of the process performed, according to the GHP method, including recurring cooling to the permanently warm fermentation of Achillea in the first week, revealed that this treatment is important for the succession of the bacterial community in this GHP method for this MP, and it is presumably of general significance of all MPs processed, according to this method. In order to obtain refined insights into the significance of bacteria and fungi for fermentation and pharmaceutical applications of these and other MPs future studies need to quantify the abundance of bacteria and fungi during fermentation and link the analysis of microbial community with that of the plant-specific chemical profiles during fermentation and resulting tinctures.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation8080383/s1. Table S1: Timetable of the cooling phases of the four MPs during the first week of fermentation. Figure S1: Rarefaction curves of the bacterial and fungal communities assessed during fermentation of the preparations of Achillea, Mercurialis, Taraxacum and Euphrasia according to the GHP. Figure S2: Heat map of the 40 most abundant bacterial genera (upper panel) and fungal genera (lower panel) during fermentation of the preparations of Mercurialis, Taraxacum and Euphrasia according to GHP-methods. Sampling points (day) and the number of replicates at each sampling (1, 2, 3, in parentheses) are given at the bottom. The colour code indicates percentages of the abundance of the total bacterial community. Figure S3: Heat map of the 40 most abundant bacterial genera (upper panel) and fungal genera (lower panel) during the fermentation of the preparations of Achillea according to GHP method 33c and the permanently warm treatment in 2016. Sampling points (day) and the number of replicates at each sampling (1, 2, 3, in parentheses) are given at the bottom. FP = fresh plant material. The colour code indicates percentages of the abundance of the total bacterial community. Figure S4: Non-metric multidimensional scaling (NMDS) analysis of bacterial (A–D) and fungal (E–H) communities during the fermentation of Achillea (A,E), Mercurialis (B,F), Taraxacum (C,G) and Euphrasia (D,H) according to GHP standards. Achillea 2016 was also fermented at permanently warm conditions (treatment) during the initial 7 days (the warm treatment is marked with asterisks). Data of the bacterial communities of the whey added to the preparations of Mercurialis and Taraxacum are also displayed by a diamond. Isolines indicate age of the preparation in days. Figure S5: Composition of the bacterial and fungal communities of fresh leaf and flower material of Achillea growing at the same location and sampled in 2016 (July 22nd) and 2018 (June 26th). Shown are an NMDS of the epi- and endophytic bacterial (A) and fungal communities (B) of flower and leaf material. Heat map of the percentages of the 40 most abundant bacterial (C) and fungal genera (D) of both years. Cluster dendrograms of the relatedness of the leaf- and flower-associated bacterial (E) and fungal (F) communities of both years. Figure S6: Chord Network diagram on the order level of the bacterial and fungal communities during fermentation of the preparations of Achillea 2016 and 2018 (A), Mercurialis (B) and Taraxacum (C).

Author Contributions

S.S., D.R.K., T.B., F.C.S. and M.S. designed the study. S.S. carried out the experimental work, sample processing, and data analyses. L.D. carried out the bioinformatics and statistical analyses. F.M. conducted the co-occurrence analyses. S.S. wrote the manuscript, complemented by substantial contributions by M.S. and critical feedback by F.C.S. All authors reviewed the manuscript and approved it for publication. All authors have read and agreed to the published version of the manuscript.

Funding

This study received funding from WALA Heilmittel GmbH (funding no. 0101). The funder was involved in the study design, collection, analysis, interpretation of data, writing of this article, and decision to submit it for publication.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The bacterial 16S rRNA gene and fungal ITS amplicon sequencing data have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI, under accession number PRJEB51209 (https://www.ebi.ac.uk/ena/browser/view/PRJEB51209?show=reads).

Acknowledgments

We thank all colleagues of the plant laboratory of WALA Heilmittel GmbH for experimental support and practical assistance.

Conflicts of Interest

S.S., D.R.K. and F.C.S. are employed at WALA Heilmittel GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Fermentation 08 00383 g001 550
Figure 1. Flowchart of the fermentation, according to GHP methods 33c and 34c, during the first three weeks. Method 33c includes an initial supplementation with honey and lactose, and method 34c includes an initial supplementation with cow whey. Incubation was performed at 37 °C until day 3 and then at 20 °C. The preparations were cooled to 4 °C twice per day. On day 4, the plant material was removed and after 7 days, and ash of the incinerated plant material was added.
Figure 1. Flowchart of the fermentation, according to GHP methods 33c and 34c, during the first three weeks. Method 33c includes an initial supplementation with honey and lactose, and method 34c includes an initial supplementation with cow whey. Incubation was performed at 37 °C until day 3 and then at 20 °C. The preparations were cooled to 4 °C twice per day. On day 4, the plant material was removed and after 7 days, and ash of the incinerated plant material was added.
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Figure 2. pH values during fermentation of all MPs, according to GHP methods 33c (Achillea and Euphrasia) and 34c (Mercurialis and Taraxacum). Achillea 2016 was also fermented without cooling during the initial 7 days (“Achillea 2016—warm”). Note the log-scale of the time axis.
Figure 2. pH values during fermentation of all MPs, according to GHP methods 33c (Achillea and Euphrasia) and 34c (Mercurialis and Taraxacum). Achillea 2016 was also fermented without cooling during the initial 7 days (“Achillea 2016—warm”). Note the log-scale of the time axis.
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Figure 3. Richness and effective number of bacterial and fungal genera during fermentation, according to the GHP of Achillea, (A,B,E,F) and Mercurialis, Taraxacum, and Euphrasia (C,D,G,H). Achillea 2016 was also fermented at permanently warm conditions (Achillea 2016—warm) during the initial 7 days. Richness and effective number of the whey-associated bacterial community is shown as black dots on panels (C,G). Note the log-scale of the time axis. Each dataset was subsampled to 1000 reads per sample.
Figure 3. Richness and effective number of bacterial and fungal genera during fermentation, according to the GHP of Achillea, (A,B,E,F) and Mercurialis, Taraxacum, and Euphrasia (C,D,G,H). Achillea 2016 was also fermented at permanently warm conditions (Achillea 2016—warm) during the initial 7 days. Richness and effective number of the whey-associated bacterial community is shown as black dots on panels (C,G). Note the log-scale of the time axis. Each dataset was subsampled to 1000 reads per sample.
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Figure 4. Percentages of the 19 most abundant bacterial orders during the fermentation of Achillea (A–C), Mercurialis (D), Taraxacum (E), and Euphrasia (F), according to the GHP. Achillea 2016 was also fermented at permanently warm conditions during the initial 7 days (B).
Figure 4. Percentages of the 19 most abundant bacterial orders during the fermentation of Achillea (A–C), Mercurialis (D), Taraxacum (E), and Euphrasia (F), according to the GHP. Achillea 2016 was also fermented at permanently warm conditions during the initial 7 days (B).
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Figure 5. Percentages of Lactobacillales during the fermentation of Achillea (A–C), Mercurialis (D), Taraxacum (E), and Euphrasia (F). Achillea 2016 was also fermented at permanently warm conditions during the initial 7 days (B).
Figure 5. Percentages of Lactobacillales during the fermentation of Achillea (A–C), Mercurialis (D), Taraxacum (E), and Euphrasia (F). Achillea 2016 was also fermented at permanently warm conditions during the initial 7 days (B).
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Figure 6. Non-metric multidimensional scaling (NMDS) analysis of bacterial (A) and fungal (B) communities during the fermentation of Achillea, Mercurialis, Taraxacum, and Euphrasia, according to the GHP. Achillea 2016 was also fermented at permanently warm conditions during the initial 7 days. Isolines indicate age of the preparation in days.
Figure 6. Non-metric multidimensional scaling (NMDS) analysis of bacterial (A) and fungal (B) communities during the fermentation of Achillea, Mercurialis, Taraxacum, and Euphrasia, according to the GHP. Achillea 2016 was also fermented at permanently warm conditions during the initial 7 days. Isolines indicate age of the preparation in days.
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Figure 7. Percentages of the 19 most abundant fungal orders during the fermentation of Achillea (A–C), Mercurialis (D), Taraxacum (E), and Euphrasia (F), according to the GHP. Achillea 2016 was also fermented at permanently warm conditions during the initial 7 days (B).
Figure 7. Percentages of the 19 most abundant fungal orders during the fermentation of Achillea (A–C), Mercurialis (D), Taraxacum (E), and Euphrasia (F), according to the GHP. Achillea 2016 was also fermented at permanently warm conditions during the initial 7 days (B).
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Figure 8. SparCC network analysis of the co-occurring bacterial species and fungal genera in fermented preparations of Achillea (A), Taraxacum (B), and Mercurialis (C). Shown are positive correlations between bacterial and fungal taxa. Clusters were calculated by using edge-betweenness between vertices of undirected and unweighted networks. Different colors denote different clusters.
Figure 8. SparCC network analysis of the co-occurring bacterial species and fungal genera in fermented preparations of Achillea (A), Taraxacum (B), and Mercurialis (C). Shown are positive correlations between bacterial and fungal taxa. Clusters were calculated by using edge-betweenness between vertices of undirected and unweighted networks. Different colors denote different clusters.
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Figure 9. Heat map of the 16S cDNA (rRNA) analysis of the 20 most abundant bacterial genera at days 5 and 7 during the fermentation of the preparations of Achillea 2016, according to GHP method 33c and without cooling (warm). Shown are data of each triplicate. The color code indicates percentages of the abundance of the total bacterial community.
Figure 9. Heat map of the 16S cDNA (rRNA) analysis of the 20 most abundant bacterial genera at days 5 and 7 during the fermentation of the preparations of Achillea 2016, according to GHP method 33c and without cooling (warm). Shown are data of each triplicate. The color code indicates percentages of the abundance of the total bacterial community.
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Table
Table 1. Sampling schedule for the preparations of Achillea, Taraxacum, Mercurialis, and Euphrasia during the fermentation period. Sampling times of the four MPs are provided as days after start (0). Fermentation of Achillea 2016 was conducted with recurring cooling (according GHP) and “warm” treatment (for details, see text).
Table 1. Sampling schedule for the preparations of Achillea, Taraxacum, Mercurialis, and Euphrasia during the fermentation period. Sampling times of the four MPs are provided as days after start (0). Fermentation of Achillea 2016 was conducted with recurring cooling (according GHP) and “warm” treatment (for details, see text).
Month12–45–78–11≥12
Week12468121420212428334243464950535861100
Achillea 20160 123456713 56 140 291 424
Achillea 20180 12345671428425684 168 294 366
Taraxacum0 1 3 5 7 27 166 322
Mercurialis00.51 3 7 39 192226 301 352
Euphrasia0 1 3 7 97 226 342 406
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Sauer, S.; Dlugosch, L.; Milke, F.; Brinkhoff, T.; Kammerer, D.R.; Stintzing, F.C.; Simon, M. Succession of Bacterial and Fungal Communities during Fermentation of Medicinal Plants. Fermentation 2022, 8, 383. https://doi.org/10.3390/fermentation8080383

AMA Style

Sauer S, Dlugosch L, Milke F, Brinkhoff T, Kammerer DR, Stintzing FC, Simon M. Succession of Bacterial and Fungal Communities during Fermentation of Medicinal Plants. Fermentation. 2022; 8(8):383. https://doi.org/10.3390/fermentation8080383

Chicago/Turabian Style

Sauer, Simon, Leon Dlugosch, Felix Milke, Thorsten Brinkhoff, Dietmar R. Kammerer, Florian C. Stintzing, and Meinhard Simon. 2022. "Succession of Bacterial and Fungal Communities during Fermentation of Medicinal Plants" Fermentation 8, no. 8: 383. https://doi.org/10.3390/fermentation8080383

APA Style

Sauer, S., Dlugosch, L., Milke, F., Brinkhoff, T., Kammerer, D. R., Stintzing, F. C., & Simon, M. (2022). Succession of Bacterial and Fungal Communities during Fermentation of Medicinal Plants. Fermentation, 8(8), 383. https://doi.org/10.3390/fermentation8080383

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