# Impact of a well-defined indigenous starter on microbial communities and technological properties of raw milk during backslopping practice

Christel Couderc, Valérie Laroute, Nathalie Aubry, Charlotte Paës, Muriel

Cocaign-Bousquet, Marie-Line Daveran-Mingot, Michael Mourez

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Christel Couderc, Valérie Laroute, Nathalie Aubry, Charlotte Paës, Muriel Cocaign-Bousquet, et al. Impact of a well-defined indigenous starter on microbial communities and technological properties of raw milk during backslopping practice. Food Microbiology, 2026, 137 (105047), pp.105047. 10.1016/j.fm.2026.105047. hal-05522932

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# Impact of a well-defined indigenous starter on microbial communities and technological properties of raw milk during backslopping practice

Christel Couderc<sup>a, *, †</sup>, Valérie Laroute<sup>b</sup>, Nathalie Aubry<sup>b</sup>, Charlotte Paës<sup>c, d</sup>, Muriel Cocaign-Bousquet<sup>b, e</sup>, Marie-Line Daveran-Mingot<sup>b, e, 1</sup>, Michael Mourez<sup>a, 1</sup>

## ARTICLE INFO

Keywords:

Backslopping

Evolution

Indigenous starter

Raw milk

Bacterial community

Acidification

Aroma production

## ABSTRACT

Backslopping, which involves using the whey from a previous batch as a starter for a new production run, is one of the oldest traditional cheese-making methods and is still widely used today. However, this technique can sometimes lead to acidification failures, necessitating the use of industrial starters to quickly restart production and minimise economic loss.

This study investigated the effect of using a well-defined indigenous starter culture on raw milk during backslopping. The microbial composition and technological characteristics of successive subcultures were analyzed during one month of backslopping with and without the starter added.

The results showed that the indigenous starter remained highly stable in skim milk, predominantly drove homolactic fermentation. The raw milk alone displayed greater initial microbial diversity and metabolic variability but then stabilised and *Lactococcus lactis* became dominant from day four onwards of backslopping. The combination of the indigenous starter with the raw milk, rapidly established *L. lactis* dominance, directing the ecosystem towards homolactic fermentation from the start of backslopping.

The addition of the indigenous starter impacted particularly subdominant populations and technological properties, resulting in an acidification that met the requirements of the Rocamadour PDO from the start of the backslopping. From day fifteen onwards, comparison of bacterial communities with and without the starter added became differentiated, demonstrating that adding the indigenous starter reduced variability in bacterial composition.

Notably, coexistence between the indigenous starter and the raw milk flora was observed, suggesting that this could be a promising option for farmer producers to strengthen the link between the product and its microbial terroir.

## 1. Introduction

Traditional fermented products hold a significant place as cultural heritage, revered for their unique production techniques that have been carefully transmitted through generations. These products serve as a reflection of their terroir, expressing the geographical, climatic, and cultural characteristics of the regions where they are produced (Montel et al., 2014; Sepe and Argüello, 2019). In recognition of their

authenticity and regional significance, the European Union has awarded the Protected Designation of Origin (PDO) label to 190 cheeses, of which 22 are made exclusively from goat's milk. Notably, 15 of these goat milk cheeses originate from France, highlighting the country's rich tradition in artisanal cheese-making (EC, 2024).

Historically, the production of these cheeses, particularly on farms, has relied on the backslopping method, where whey from a previous lactic fermentation is used to inoculate a new batch of milk (Gastrow

<footnote><sup>a</sup> Oci'Food, Ecole d'Ingénieurs de Purpan, Université de Toulouse, Toulouse, France</footnote>

<footnote><sup>b</sup> TBI, Université de Toulouse, CNRS, INRAE, INSA, Toulouse, France</footnote>

<footnote><sup>c</sup> Département Sciences Animales et Système d'élevage, Ecole d'Ingénieurs de Purpan, Université de Toulouse, Toulouse, France</footnote>

<footnote><sup>d</sup> GenPhySE, INRAE, ENVT, Université De Toulouse, Toulouse, France</footnote>

<footnote><sup>e</sup> Université Toulouse 3 - Paul Sabatier, Université de Toulouse, Toulouse, France</footnote>

<footnote>* Corresponding author. Oci'Food, Ecole d'Ingénieurs de Purpan, 75 voie du Toec, F-31076, Toulouse, France.</footnote>

<footnote>E-mail addresses: christel.couderc@purpan.fr (C. Couderc), vlaroute@insa-toulouse.fr (V. Laroute), aubry@insa-toulouse.fr (N. Aubry), charlotte.paes@purpan.fr (C. Paës), cocaign@insa-toulouse.fr (M. Cocaign-Bousquet), daveran@insa-toulouse.fr (M.-L. Daveran-Mingot), michael.mourez@purpan.fr (M. Mourez).</footnote>

<footnote n="1">¹ These authors contributed equally to this paper.</footnote>
et al., 2020). This traditional method is still widely used in the pro-
duction of PDO-labelled goat cheeses in France, with 6 out of the 15 PDO
cheeses adopting this technique. The method is favoured for its practi-
cality, cost-effectiveness, and its ability to impart specific and desirable
characteristics to the cheese (Demarigny et al., 2011; Demarigny and
Gerber, 2014).

However, the backslopping method is not without its challenges. The repeated use of whey from previous batches can lead to significant variations in the acidification profiles, which in turn can cause inconsistencies in the texture, flavor, and safety of the cheese. These variations increase the risk of producing defective products, posing important economic challenges for producers who must then take corrective measures to restore the desired acidification process (Demarigny and Gerber, 2014; Gastrow et al., 2020). To address these issues, cheese producers are often faced with two options: either use a natural whey starter sourced from neighbouring farms or incorporate industrial starters into their production process.

The use of industrial starters, typically consisting of a limited number
of industrially produced strains such as *Lactococcus lactis*, is common
practice in the production of various soft cheeses (Fernández et al.,
2011). While this approach is effective in standardising the acidification
process and ensuring product consistency, it also significantly reduces
the local microbial diversity within the cheese. This can lead to more
uniform and standardised products and have an impact on organo-
leptic/aromatic characteristics, even for cheeses with PDO labels (Frau
et al., 2016; Nieto-Arribas et al., 2009; Rasovic et al., 2017).

In contrast, the development and use of indigenous starters, con-
sisting of strains isolated directly from raw milk within the PDO area,
represents a promising alternative for cheese producers. This approach
makes it possible to maintain the technological advantages offered by
the selected strains, while preserving the local microbial diversity that
contributes to maintaining the unique characteristics of PDO cheeses.
This approach also promotes the sustainability of traditional cheese-
making practices and strengthens the link between the product and its
terroir.

In this study, we investigated whether a selected indigenous starter
could influence backslopping practices by impacting the balance of
microbial communities in raw milk and their key technological prop-
erties. To investigate this, an indigenous starter culture specifically
designed for Rocamadour PDO cheese was used (Couderc et al., 2022).
This starter, consisting of three *Lactococcus lactis* strains isolated from
raw goat's milk in the Rocamadour PDO region, was evaluated for the
first time during one month of backslopping.

## 2. Materials and methods

2.1. Preparation of the indigenous starter

Cultures of *Lactococcus lactis* strains EIP07A, EIP13D and EIP20B were prepared by overnight incubation in M17 broth (BD Difco) supplemented with 10 g/L of glucose at 30 °C (Couderc et al., 2022). The optical density at 600 nm (OD<sub>600nm</sub>) of each overnight culture was adjusted to 0.5 to give 1.10<sup>9</sup> CFU/ml (using a correlation between CFU/ml and OD<sub>600nm</sub> based on previous experiments, data not shown). Cultures were then centrifuged at 4000 rpm for 10 min, supernatants removed, and the pellets washed twice with 0.9 % NaCl. Then 10 ml of sterile skim milk was added to the washed pellets. An equal volume of each strain culture was added to constitute the indigenous starter.

## 2.2. Raw goat milk used for the experiment

The raw goat's milk used was PDO raw milk collected from "Les
Fermiers du Rocamadour" and stored at 4 °C for 48 h before being
delivered to our laboratory. The pH value of the raw milk was 6.88. A
total of 10 L of raw milk was divided into 100 ml sterile vials for the
backslopping experimentation and 50 ml sterile vials for the

acidification study. All aliquots were frozen at -20 °C and then thawed
before the analyses.

2.3. *Experimental backslopping*

On day 0, the start of the backslopping experiment, three evolution
paths were monitored in parallel (Fig. 1); i) the evolution of the indig-
enous starter in skim milk (Sigma-Aldrich, Saint-Quentin-Fallavier,
France) (S), three tubes corresponded to three independent experi-
ments (S-A, S-B and S-C) containing 100 ml of sterile skim milk, each
inoculated with 1 ml of the indigenous starter (1.10<sup>7</sup> CFU/ml final), ii)
the evolution of the indigenous starter in raw milk (RMS), three tubes
(RMS-A, RMS-B and RMS-C) containing 100 ml of raw goat's milk
inoculated with 1 ml of the indigenous starter (1.10<sup>7</sup> CFU/ml final) and,
iii) the evolution of raw milk (RM), three tubes (RM-A, RM-B and RM-C)
containing 100 ml of raw goat's milk.

The experimental backslopping was carried out for 30 consecutive
days at 22 °C. Every 24 h, all fermented milks were vortexed for 30 s.
Then 1 % (v/v) of these fermented milks were inoculated into sterile
skim milk (S) or raw goat's milk (RM and RMS).

All analyses were carried out on the three evolution paths (S, RM and RMS) at nine time points: days 0 (D0 not fermented, 2, 4, 6, 8, 10, 15, 20 and 30). Two ml were taken for HPLC analysis and five ml for DNA extraction. These samples were stored at - 20 °C until analysis.

2.4. Acidifying properties of fermented milks

To study the acidification kinetics of the three evolutions (S, RM and
RMS), final pH (pH 24h), latency time (Ta, hours: minutes) and
maximum speed (Vm, UpH/h) were used as descriptors. At nine time
points (D0, D2, D4, D6, D8, D10, D15, D20 and D30), a defined pro-
portion (0.1 %, v/v) of each type of fermented milk (S, RM and RMS)
was taken to inoculate a separate 40 ml of skim milk for S evolution and
40 ml of raw goat's milk for RM and RMS evolutions. The analyses were
carried out at 22 °C using the iCinac system (AMS Alliance, Frepillon,
France) (Spinnler and Corrieu, 1989).

2.5. Determination of free amino acids by the OPA method

Free amino acids were determined by the method of Garbowska et al.
(2020). Briefly, five ml of each sample were added to one ml of sterile
distilled water and ten ml of 0.75 N trichloroacetic acid (TCA). This
mixture was vortexed and centrifuged at 4000×g, 4 °C for 30 min. The
supernatants were then filtered through a 0.45 µm syringe acetate cel-
lulose membrane filter (Grosseron, Coueron, France). One hundred and
fifty µL of the filtrate were added to three mL of o-phthalialdehyde
(OPA) reagent, mixed and incubated for 2 min at room temperature.
Absorbance was measured using a UV-VIS spectrophotometer (Thermo
Fisher Scientific, Illkirch, France) at 340 nm.

2.6. High performance liquid chromatography

The concentrations of substrates (lactose, galactose and citrate) and
fermentation end products (lactate, acetate, acetoin and ethanol) were
measured by high-performance liquid chromatography (HPLC, Agilent
Technologies, Waldbronn, Germany) as described in the work of Pass-
erini et al. (2013).

2.7. DNA metabarcoding

The DNA metabarcoding analyses were performed as described by
Abi Khalil et al. (2022). Briefly, five ml of fermented milk were added to
45 ml of sterile peptone water and mixed three times for 1 min. Five ml
of the prepared dilution were placed in a sterile tube and centrifuged for
3 min at 4,500×g at 4 °C. The supernatant was eliminated, and the total
microbial DNA was extracted from the pellet using the DNeasy
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Experimental design of the study. Three evolution paths were monitored in parallel for one month. S: evolution of the indigenous starter in sterile skim milk (replicates S-A, S-B and S-C), M: evolution of the indigenous starter in raw milk (RMS) (replicates RMS-A, RMS-B and RMS-C) and RM: evolution of the raw milk (replicates RM-A, RM-B and RM-C). Microbial and physicochemical analyses were performed on the three evolution paths at nine time points (D0, D2, D4, D6, D8, D10, D15, D20 and D30).</figcaption></figure>

PowerFood Microbial Kit (Qiagen Hilden, Germany) according to the supplier's instructions. The DNA purity was evaluated by the ratio of the absorbance at 260 nm and 280 nm and concentration adjusted to 10 ng/µL. Mock communities (a mix of equimolar DNA concentrations 10 ng/µL of several known species) were added for bacterial sequencing.

The V3 - V4 region of the 16S rRNA gene (amplicon size, 426 bp) was amplified. This first PCR was performed to add Illumina adapters and frameshifts to the target genes. To construct the library, a second PCR was then performed by the GeT-Biopuces platform (Toulouse, France) using Illumina MiSeq technology, generating reads of 2 × 250 bp.

The resulting sequences were analyzed using FROGS pipeline (the data of evolution S was treated separately and was used to check that the experiment was running smoothly by verifying that, as expected, only the *Lactococcus lactis* species were present throughout the evolution). Taxonomic affiliations were performed using DAIRYdb database version 2.0 (Meola et al., 2019). Multi-affiliations were resolved manually using the affiliation Explorer (Mariadassou, 2021).

Statistical analysis of microbial communities' diversity was performed using the phyloseq R package implemented in FROGS (FROGS-STAT) (McMurdie and Holmes, 2013). Before assessing diversity indices, Amplicon Sequencing Variants' (ASVs) tables were then normalized by rarefaction based on the lowest number of sequences in samples (79,690 sequences). Species diversity within each sample was estimated using the α-diversity indices (richness, Shannon and inverse Simpson).

Jaccard's dissimilarity index was used to estimate species diversity between samples (β-diversity) and were represented by hierarchical ascending classification (HAC) using the ward two linkage method (D0, D2, D4, D6, D8, D10, D15, D20 and D30).

Based on the HAC result, two multidimensional scaling (MDS) plots and clustering were realized. The first included samples from the early stages (D2, D4 and D6) and the second from the late stages (D15, D20 and D30). Subsequently, a multivariate analysis of variance (PERMANOVA) using the Adonis test was used to detect significant differences between the communities depending on whether or not the starter was added to the raw milk (RM, RMS) as an experimental variable.

## 2.8. Statistical analysis

Statistical analyses of the technological descriptors were performed using repeated measures ANOVA in R. Technological descriptors were then subjected to a Student's test to compare means per evolution (S, RM and RMS) and for nine time points (D0, D2, D4, D6, D8, D10, D15, D20 and D30). Mean values were considered significantly different at *p*-value <0.05.

Ta, start time of the acidification process corresponding to a ΔpH of 0.08 UpH (Ta; hours: minutes); Vmax, maximum acidification rate (UpH/h); pH 24 h, pH value at 24 h; FAAs, measure of free amino acids (OD340nm).

## 3. Results

To study the influence of the addition of a well-defined indigenous starter to raw milk on the microbial communities and technological properties, the practice of backslopping was carried out for 30 consecutive cycles on three types of matrices monitored in parallel: i) sterile skim milk supplemented at D0 with the indigenous starter at 1.10⁷ CFU/ml (evolution S), ii) raw goat's milk from the Rocamadour PDO area (evolution RM) and iii) raw goat's milk from the Rocamadour PDO area supplemented at D0 with the indigenous starter at 1.10⁷ CFU/ml indigenous starter (evolution RMS). Three replicates A, B and C were realized for each evolution (Fig. 1).

The analysis of the microbial ecosystem and the study of technological characteristics (lag time, maximum rate, final pH, total proteolytic activity, sugar consumption, organic acid production) were studied over nine time points of backslopping at D0, D2, D4, D6, D8, D10, D15, D20 and D30.

### 3.1. Adding the indigenous starter, rapidly stabilised technological properties and reduced proteolytic activity of raw milk

The technological property data for the three evolutions (S, RM and RMS) were presented in Fig. 2A for the pH descriptor (pH 24h), in Fig. 2B for the maximum acidification rate (Vm; UpH/h) and in Fig. 2C for the latency time (Ta; hours: minutes). All values associated with the
<figure><img src="image_4.png" /><figcaption>**Fig. 2.A.** Plots of the final pH descriptor (pH at 24h, UpH) obtained in the three evolution paths (S in blue, RM in pink and RMS in green) at nine stages of backslopping (D0, D2, D4, D6, D8, D10, D15, D20 and D30). Each point corresponds to a replicate (A, B and C). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 2.B.** Plots of the maximum speed descriptor (Vm, UpH/h) obtained in the three evolution paths (S in blue, RM in pink and RMS in green) at nine stages of backslopping (D0, D2, D4, D6, D8, D10, D15, D20 and D30). Each point corresponds to a replicate (A, B and C). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

figures were presented in the supplementary data, Table S1.

Regarding the evolution of the indigenous starter in skim milk (S), after 24 h, the action of the indigenous starter alone resulted in a pH level of 4.5. Throughout the evolution, the pH remained between 4.3 and 4.4, demonstrating the indigenous starter's acidifying capacity was compatible with the acidification required for producing Rocamadour PDO cheese.

From the first day of backslopping, the indigenous starter played its role as acidification starter in the raw milk (D0-RMS), reaching the values expected by the specifications for a Rocamadour PDO cheese, with an end pH of 4.38 in 24 h.

RM evolution reduced the pH by only 0.54 UpH in 24 h. After two days of backslopping, RM (D2-RM), produced an acidification comparable to RMS (D2-RMS), with an end pH of 4.33 in 24 h.

From D6, no significant difference was observed regarding acidification between the three evolutions studied, with a pH of 4.33 in 24 h for S, 4.32 for RMS and 4.31 for RM.

<figure><img src="image_6.png" /><figcaption>**Fig. 2.C.** Plots of the latency time (Ta, h) obtained in the three evolution paths (S in blue, RM in pink and RMS in green) at nine stages of backslopping (D0, D2, D4, D6, D8, D10, D15, D20 and D30). Each point corresponds to a replicate (A, B and C). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

For the acidification rate descriptor (Vm), at the start of the experiment (D0), the Vm value for the RM evolution was 0.08 UpH/h. This indicated that raw milk alone had not yet begun its acidification phase after 24 h. In contrast, the S and RMS evolutions had VM values of 0.2 and 0.3 UpH/h, respectively. Then between D2 and D10, the Vm values were significantly higher during backslopping of RM (Vm between 0.3 and 0.4 UpH/h) than those during backslopping of RMS and S (Vm between 0.2 and 0.3 UpH/h). Taken together, these results showed that there was little to no difference between the profiles of the evolution paths containing the indigenous starter, whether it was backslopped into raw milk (RMS) or skim milk (S), but the profile of the evolution path in raw milk alone (RM) was different.

Regarding the latency time descriptor (Ta), at D0, the acidification process of RM (D0-RM) started more than 19 h after inoculation, but Ta sharply decreased at the end of the second backslopping cycle (D2-RM) to reach a Ta of 2 h 35 min (supplementary data, Table S1). These results are consistent with the pH values obtained (pH = 6.34 on D0-RM and pH = 4.33 on D2-RM) and showed an early adaptation in the acidification process of raw milk. From day four onwards, the Ta for the evolution of S was always significantly higher than for the two observed evolution paths, RM and RMS.

Amino acids concentration, determined by the OPA method, showed that the evolution path of RM presented a higher free amino acids concentration from D4 onwards, compared to those of the evolution path of RMS and S, which were similar from D6 onwards (Fig. 2D).

Overall, the analysis of the technological properties showed significant differences in the technological parameters between RM and RMS up to D15 (supplementary data, Table S1). Thereafter, from D15 onwards, all technological parameters were similar except for amino acids concentration. This suggested that the addition of the indigenous starter to the raw milk (RMS evolution) i) allowed acidification at pH 4.4 to be imposed from the start of backslopping, ii) had the effect of reducing the overall proteolytic activity of the raw milk, iii) imposed its proteolytic activity.

## 3.2. Combining the indigenous starter with raw milk (RMS) resulted in immediate microbial dominance by *Lactococcus lactis*

The composition of the population was characterised by DNA metabarcoding in the three evolutions. As expected, the milk from the backslopping S contained only *Lactococcus lactis* species (data not shown). By contrast, *Pseudomonas* sp. was the predominant species in the initial raw milk (D0-RM), representing 82.5 % of total sequences,
<figure><img src="image_7.png" /><figcaption>**Fig. 2.D.** Plots of the proteolytic activity (OD340nm) obtained in the three evolution paths (S in blue, RM in pink and RMS in green) at nine stages of backslopping (D0, D2, D4, D6, D8, D10, D15, D20 and D30). Each point corresponds to a replicate (A, B and C). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

followed by *Staphylococcus* sp. (11.4 %), *Lactococcus lactis* (2.4 %), *Acinetobacter bereziniae* (1.6 %), *Hafnia alvei* (0.6 %) and *Pantoea coffeiphila* (0.5 %). Other bacterial species were also present but at relative abundances of less than 0.5 % of total sequences (Fig. 3A, supplementary data S2.A).

After the addition of the indigenous starter to the raw milk at 1.10⁷ CFU/ml (D0-RMS), *Lactococcus lactis* dominated the microbial ecosystem, accounting for 70.2 % of the total sequences, followed by the *Pseudomonas* sp. (26.5 %) and *Staphylococcus* sp. (2.4 %) (Fig. 3B, supplementary data S2.B).

At D2, the composition of RM changed, with a sharp increase in the species *Lactococcus lactis* (62.8 %), accompanied by a sharp decrease in

the species *Pseudomonas* sp. (0.6 %). *Hafnia alvei* also increased, representing 4.8 % of the total sequences. The species *Citrobacter freundii* and *Serratia nematodiphila*, which were not detected at D0, were present with 27.1 % and 1.3 % relative abundances respectively (Fig. 3A). RMS evolved differently: after two cycles of backslopping, *Lactococcus lactis* increased more than in RM backslopping, representing the majority of the population with a relative abundance of 93.4 % (D2-RMS). *Pseudomonas* sp. was present with a relative abundance of 5.1 %, followed by *Hafnia alvei* (0.7 %) and *Citrobacter freundii* (0.6 %) (Fig. 3B).

Between D4 and D6, *Lactococcus lactis* was still the most abundant species in the RM evolution with an increase in its relative abundance: 89.2 % in D4-RM and 94.3 % in D6-RM. It was followed by *Hafnia alvei* (5.5 % at D4 and 3.6 % at D6), then *Citrobacter freundii* (4 % at D4 and 0.8 % at D6). *Pseudomonas* sp. remained stable with 0.8 % of total sequences at D4 and 0.9 % at D6. At the same points in time, the RMS evolution was slightly different: less *Lactococcus lactis* (77.7 % at D4 and 79.9 % at D6), and more *Citrobacter freundii* (D4 and D6, 10.5%), and *Pseudomonas* sp. (especially at D4, 7 %). The species *Serratia nematodiphila* was significantly present (2.6 % at D4 and 3.8 % at D6) (Fig. 3B, supplementary data Table S2B).

The bacterial community of the RM evolution from D8-RM to D20-RM was still dominated by *Lactococcus lactis* (>90.0 % of total sequences). The presence and the growth of the species *Klebsiella* sp. was only observed in replicate A (3.8 % in D8-RM-A and 9.7 % in D20-RM-A), while *Serratia nematodiphila* was only detected in replicates B and C (0.2 % in D8-RM-B and 0.4 % in D8-RM-C and 1.9 % in D20-RM-A and 0.4 % in D20-RM-C). The *Pseudomonas* population remained stable (1.6 % on D8-RM and 3.3 % on D20-RM; average of all replicates). All others were detected at relative abundances below 0.5 % of the total sequences (Fig. 3A).

RMS evolution was still dominated by *Lactococcus lactis*, but with a lower abundance than the RM evolution (80.7 % at D8-RMS and 81.6 % at D20-RMS). The populations of *Serratia nematodiphila*, *Citrobacter freundii*, *Hafnia alvei* and *Pseudomonas* sp. were variable between the three replicates but appeared to be higher than in the RM evolution (Fig. 3B).

<figure><img src="image_8.png" /><figcaption>**Fig. 3.A.** Analysis of bacterial communities in the milk from RM backslopping by sequencing the V3-V4 region of 16S rRNA at eight evolution stages: days 0, 2, 4, 6, 8, 10, 15, 20 and 30. The letters A, B and C correspond to the replicates. As an example of the nomenclature used, D0-RM-A corresponds to the RM evolution at D0 and replicate A.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 3.B.** Analysis of bacterial communities in RMS evolution by sequencing the V3-V4 region of 16S rRNA at eight evolution stages: days 0, 2, 4, 6, 8, 10, 15, 20 and 30. The letters A, B and C correspond to the replicates. As an example of the nomenclature used, D0-RMS-A corresponds to the RMS evolution at D0 and replicate A.</figcaption></figure>

After 30 cycles of backslopping (D30), replicate D30-RM-A still differed from the two others replicates D30-RM-B and D30-RM-C. Replicate D30-RM-A contained 85.5 % of *Lactococcus lactis*, the replicates B and C were very close in their microbial populations with 98.9 % of *Lactococcus lactis* (Fig. 3A).

At the end of the experiment, RMS evolution appeared to be more stable between replicates: only three species were present with more than 0.5 % of the total sequences, *Lactococcus lactis* with 84.6 % of total

sequences followed by *Serratia nematodiphila* with 10.6 % and *Pseudomonas* sp. with 4.2 %.

Species richness was assessed using the α-diversity indices Richness, Chao1, Shannon, and inverse Simpson (supplementary data, Table S3). Comparison of Richness and Chao1 indices showed that over 70 % of bacterial diversity was identified in each sample (except for D10-RM-C and D30-RM-A).

In terms of bacterial diversity indices, D4-RMS samples showed the

<figure><img src="image_10.png" /><figcaption>**Fig. 4.A.** Hierarchical Ascending Classification (HAC) based on the Jacquard dissimilarity indices of the RM and RMS evolutions. Samples are colour coded according to the type of evolution paths: pink for RM and blue for RMS. Three replicates were analyzed per sample (A, B and C). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>
highest richness index with an average of 74 ASVs. Richness indices started to decrease from the twentieth day of backslopping for both types of evolution path (RMS and RM). Overall, the indices of samples of RM and RMS evolutions showed similar trends over the course of successive backslopping, with slightly higher indices in RMS evolution (supplementary data, Table S3).

The highest Shannon and inverse Simpson indices were observed on the initial samples J0-RM (Shannon index 1.6 and Simpson index 2.3, average of replicates) and J0-RMS (Shannon index 1.0 and Simpson index 1.9, average of replicates), which means that a higher bacterial diversity was observed at the beginning of each evolution (supplementary data, Table S3).

From the second day of backslopping until the end of evolution (D30), a general decrease in the values of the Shannon and inverse Simpson indices was observed. This means that the practice of backslopping led to a reduction in bacterial diversity over time, with the gradual dominance of one bacterial species, *Lactococcus lactis*. Overall, the Shannon and Simpson indices were higher in the RMS evolution.

Based on the Jacquard dissimilarity indices, hierarchical ascending classification (HAC) showed that the fermented milks were grouped according to evolutionary stages (Fig. 4A).

In fact, two distinct branches make it possible to distinguish two evolutionary periods: the first group included almost all the samples of days 2, 4 and 6 of evolution, regardless of the type of evolution followed (RM or RMS) and the second group contained two subgroups, the first subgroup (I) containing only the two initial samples (D0-RM and D0-RMS) and the second subgroup (II) containing all the samples belonging to days 15, 20 and 30 of the evolution (except D2-RMS-A).

Backslopping steps D8 to D10 appeared to be intermediate stages in the evolution of the composition of the bacterial ecosystem, since samples of these stages was found in both groups.

Based on Jacquard's dissimilarity indices, two MDSs

(multidimensional scaling) were performed, the first on samples belonging to the early stages (D2, D4, D6; Fig. 4B) and the second on samples in the late stages (D15, D20 and D30; Fig. 4C).

Although the first MDS analysis allowed the early stage samples (D2, D4 and D6) to be visually grouped according to the type of evolution (RM or RMS), no significant differences were found (PERMONAVA statistical analysis) (Fig. 4B).

The second MDS based on the late phases (D15, D20 and D30) also allowed us to visually distinguish the type of evolution (RM or RMS). The PERMANOVA statistical analysis applied to the Jacquard dissimilarity indices confirmed significant differences between the bacterial communities (RM or RMS) (*p*-value <0.001, R² = 23.5 %) (Fig. 4C). This means that the starter had an effect on the microbial populations (in terms of composition and variability) that emerged during the late phases of backslopping, depending on whether the starter was added or not (RM or RMS). It is also interesting to note that the addition of the starter to the milk reduced the variability of the microbial composition of the communities from D15 onwards.

These patterns for early and late stages were also observed using the Bray-Curtis index (data not shown).

### 3.3. The addition of the starter to raw milk redirected the fermentative metabolism of the ecosystem

During successive backslopping cycles (D0 to D30) in the three scenarios studied, lactose concentration decreased while lactate production increased (Fig. 5A, 5B, 5C; Supplementary Data Tables S4A, S4.B, S4.C). This trend indicated the presence of lactose-hydrolysing (*LacE*⁺) strains in all ecosystems.

In the S evolution scenario, the presence of *LacE*⁺ strains were consistent with the metabolic characteristics of the indigenous starter strains (EIP07A, EIP13D, EIP20B) (Fig. 5A). All three strains were *LacE*⁺,

<figure><img src="image_11.png" /><figcaption>**Fig. 4.B.** Multidimensional scale (MDS) based on the relative abundance of ASVs in the 16S rRNA-targeted bacterial microbiota of the two evolutions RM (orange) and RMS (blue) during three early stages of backslopping (D2, D4, D6). Three replicates were analyzed per sample (A, B and C). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>**Fig. 4.C.** Multidimensional scale (MDS) based on the relative abundance of ASVs in the 16S rRNA-targeted bacterial microbiota of both RM (orange) and RMS (blue) evolutions during the last three stages of backslopping, late stages: D15, D20 and D30. Three replicates were analyzed per sample (A, B and C). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>**Fig. 5.A.** Plots of lactose, galactose and citrate consumption and lactate, acetate, ethanol and acetoin production (mM) in the evolution of S at 8 stages of backslopping: days 0, 2, 4, 6, 8, 10, 15, 20 and 30. Three replicates were analyzed per sample (A, B and C).</figcaption></figure>

and two of them were classified as acidifying strains (EIP13D and EIP20B) (Couderc et al., 2022). In contrast, lactose consumption was more pronounced in the RM and RMS evolutions, leading to higher lactate production, suggesting the presence of a more important *LacE*⁺ population in these two evolutions (Fig. 5B and 5C).

Additionally, other lactose fermentation products such as acetate, acetoin, and ethanol were detected, but their profiles varied across the three scenarios.

As expected, lactate was the main fermentation product in S

evolution throughout the experiment (D0 to D30), a low concentration of acetate production was observed (<2 mM) (Fig. 5A).

In the RM evolution, ethanol production was observed transiently on the second day of backslopping (3.4 mM, average of the three replicates), with high acetate production (12.3 mM, average of the three replicates) (Supplementary Data Table S4B). This observation suggested the presence of microorganisms with a tendency to heterolactic fermentation, followed by a transition to mixed fermentation from D4 onwards, with the disappearance of ethanol and the production of
<figure><img src="image_14.png" /><figcaption>**Fig. 5.B.** Plots of lactose, galactose and citrate consumption and lactate, acetate, ethanol and acetoin production (mM) in the evolution of RM at 8 stages of backslopping: days 0, 2, 4, 6, 8, 10, 15, 20 and 30. Three replicates were analyzed per sample (A, B and C).</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>**Fig. 5.C.** Plots of lactose, galactose and citrate consumption and lactate, acetate, ethanol and acetoin production (mM) in the evolution of RMS at 8 stages of backslopping: days 0, 2, 4, 6, 8, 10, 15, 20 and 30. Three replicates were analyzed per sample (A, B and C).</figcaption></figure>

acetate in lower concentration (Fig. 5B). In the RMS evolution, the heterofermentative phase was not observed, indicating that the addition of the starter allowed a rapid shift towards mixed fermentation (Fig. 5C).

### 3.4. Addition of the starter did not prevent expression of endogenous milk microbiota

The evolution of citrate consumption and the production of acetate and/or acetoin are shown in Fig. 5A for the S evolution and Fig. 5B and C for the RM and RMS evolutions.

In evolution S, no citrate consumption was observed throughout the experiment (Fig. 5A and supplementary data, Table S4A). These results were consistent with the characteristics of the three starter strains that lacked the genetic predisposition to metabolise citrate (*citP- and citDEF-*) (Couderc et al., 2022).

In the RM evolution, during the hetero-fermentation phase (D2), citrate was completely consumed by the raw milk micro-organisms to form acetate, and no acetoin production was observed at this stage. This aroma appeared from D4 and reached a concentration of 1.0 mM (average of the three replicates) at a residual citrate value of 3.9 mM (average of the 3 replicates, Supplementary data, Table S4B). After 10 days, RM evolution showed heterogeneity between the responses of the three replicates (Fig. 5B, supplementary data, Table S4B). RM-B and RM-C consumed all the citrate, with an increase in acetoin production (3.2 mM, average of the 2 replicates). In RM-A, citrate consumption was

lower (3.7 mM), resulting in lower acetoin production (1.1 mM). This heterogeneity of response between replicates was observed until the end of the experiment, i.e. until the 30th cycle of RM evolution (Fig. 5B, supplementary data, Table S4B).

During the first days of backslopping (from D0 to D4), no citrate consumption and no acetoin production were observed in the RMS evolution (Fig. 5C, supplementary data, Table S4C). Then, gradually (from D6 to D20), in a heterogeneous manner according to the replicates, citrate was partially or totally consumed, and the presence of acetoin was observed (0–2.3 mM). Acetoin appeared in all three replicates from D8 onwards, meaning that from the eighth day of backslopping all ecosystems were able to produce acetoin. Finally, at D30, the micro-organisms present in the ecosystem had consumed all the citrate and were producing acetoin in the range of 0.9–2.2 mM (Fig. 5C, supplementary data, Table S4C).

In this study, acetoin production was attributed to the endogenous flora of the raw milk (RM), as the strains from the starter culture alone (S) were unable to produce it. The addition of the starter to the raw milk (RMS) did not prevent the emergence of ecosystems capable of producing acetoin from the eighth day of backslopping. This observation suggested that this indigenous starter did not inhibit the expression of the microbial flora native to raw milk.
## 4. Discussion

In this study, the influence of a defined indigenous starter on raw milk during 30 cycles of backslopping was studied. For that, the evolution of raw goat's milk (RM) and the evolution of the indigenous starter in raw milk (RMS) and in skim milk (S) were monitored for 30 days in successive cultures. The microbial ecosystem and technological characteristics of the three evolution paths were analyzed over nine time points (D0, D2, D4, D6, D8, D10, D15, D20 and D30).

The stability of the technological properties is particularly interesting and contributes to the robustness of the starter. The RMS evolution showed a rapid adaptation of the microbial communities, with acidification capacities stabilizing from the first day of backslopping (D0-RMS), demonstrating the effectiveness of the indigenous starter throughout the experiment. The analysis of the acidification parameters in RM evolution, such as the latency time and the final pH, highlighted a phase of adaptation and then stabilization of raw milk, which enabled it to acquire the technological capacities expected of a starter (pH = 4.4 in less than 24 h). It was interesting to note that in the first 15 days in RMS evolution, the maximum acidification rate was lower than in the RM evolution. This could mean that very efficiently acidifying strains were selected during the evolution in raw milk.

Indeed, in highly selective environments such as dairy processing conditions, the intrinsic properties of lactic acid fermentation and antagonistic interactions, reduce species diversity and enrich the microbiota in favour of a small number of lactic acid bacterial species. These adaptation phases have been well described in fermentations using backslopping (Gapp et al., 2024; Wahengbam et al., 2015). Natural fermentation by indigenous microorganisms leads to the selection of strains through competitive selection and adaptation to the substrate, resulting in dynamic changes in the structure and populations of the microbial community until stable ecosystems with characteristic sensory properties are obtained (Bertani et al., 2020).

In this study, the RM evolution showed dynamic changes in its microbial composition. This observation was made on the second day of backslopping, leading to a change in the fermentative pathway (heterofermentative to mixed) and then to stability in the acidification capacity of the community with the dominance of *Lactococcus lactis* species.

The dominance of *Lactococcus lactis* was observed throughout the association between indigenous starter and raw milk (RMS). These results were consistent with the work of Demarigny et al. (2011) on Tomme de Savoie, and Penland et al. (2021), on Pelardon (a soft cheese close to Rocamadour), who also highlighted the dominance of this species during backslopping.

Surprisingly, *Serratia nematodiphila* was the second most abundant species after *Lactococcus lactis* in the RMS evolution. The genus *Serratia* is well known to be a part of the flora commonly found in raw goat's milk (Tormo, 2010). To date, no studies have shown pathogenicity of *S. nematodiphila* species (Khoa et al., 2016; Mahlen, 2011). However, to our knowledge, it was the first observation of this species in goat's milk. This soil species has interesting properties, such as the ability to synthesize glycolipids with antibacterial properties, which have been shown to have antimicrobial potential against *Pseudomonas aeruginosa* and *Bacillus pumilus* (Panjiar et al., 2020). These two species are considered to be of concern in terms of product spoilage and also in terms of public health, and their control would be of real industrial interest (Oliveira et al., 2016). The review proposed by Rubio-Ribeaux et al. (2023) also highlights the interest of glycolipids produced by microorganisms as biosurfactants for the agro-food industry. Therefore, it would be interesting to try to isolate this species in order to characterize it and study its antimicrobial and biosurfactant potential.

The composition of the microbial communities (β-diversity) allowed to distinguish the samples according to the evolution paths (RM or RMS) from the fifteenth day of the backslopping practice. The introduction of a starter culture impacted the structure of the bacterial populations,

especially the subdominant populations, leading to different evolutionary dynamics. These results showed that the addition of the starter to raw milk affected the microbial dynamics of subdominant species and altered the microbial balance in the long term.

In a future study with the indigenous starter, it would be interesting to continue after the acidification stage and produce cheeses to see if the difference observed is confirmed during ripening. It should be noted that Silvetti et al. (2017) demonstrated that the microbial community composition of Silter PDO cheese did not differ statistically according to the type of starter used (indigenous or raw milk) or the stage of ripening.

Variations in microbial composition were observed in the three replicates of the evolution of raw milk (RM), similar observations were made by Zotta et al. (2022) and Bertani et al. (2020). Indeed, they found that the use of separate backslopping lines for the production of natural whey starter resulted in small but significant differences in the composition of the microbial communities. Others studies demonstrated that repeated incubation cycles (up to 13) with nine different natural milk cultures resulted in differential evolution of species composition (Parente et al., 2016). Another study on Mabisi, a fermented milk produced in Zambia, showed that 16 days of backslopping revealed fluctuations in microbial population dynamics between replicates (Groenenboom et al., 2022). These results are in line with those of the present work, since differences were also observed in the three replicates of the evolution of the raw milk (RM). In this study, the evolution of RM showed a variation in population dynamics in the three replicates with one of the three containing more than 5 % *Klebsiella oxytoca* after one month of backslopping practice. This species is known to be a spoilage organism in the cheese industry (Massa et al., 1992). Thus, it was important to note that in the RMS evolution, the addition of the indigenous starter did not allow the development of this species and suggested that the use of the indigenous starter was one of the keys to minimizing the variation observed and avoiding the development of undesirable bacteria such as spoilage organisms.

In addition, the presence of ecosystems capable of producing acetoin from the eighth day of backslopping practice was observed in the RMS evolution. This characteristic was associated with raw milk (RM) since the indigenous starter alone (S) was not able to produce this compound. The presence of this metabolite suggested a coexistence between the natural community of raw milk (acetoin producers) and the three *Lactococcus lactis* of the indigenous starter (no producers). It also meant that of using the indigenous starter did not prevent the expression of the endogenous raw milk flora.

## 5. Conclusion

This study showed that, under backslopping conditions, the well-designed indigenous starter culture played a structuring role within the dairy microbial ecosystem by shaping not only the dominant population but also the dynamics of sub-dominant bacterial groups.

It also demonstrated the effectiveness of using an indigenous starter to establish a microbial ecosystem that met the technological requirements from the start of backslopping practice. The indigenous starter not only performed the expected technological functions, but also coexisted with the natural raw milk flora, resulting in the formation of a community able to produce acetoin, a compound valued for its buttery and nutty flavour in fermented dairy products.

By maintaining technological stability, reducing variability in bacterial composition and not inhibiting the endogenous bacteria in raw milk, the indigenous starter was an interesting alternative to industrial options for farmer producers.

### CRediT authorship contribution statement

Christel Couderc: Writing – review & editing, Writing – original draft, Visualization, Validation, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation,
Conceptualization. **Valérie Laroute:** Writing – review & editing, Methodology, Formal analysis, Data curation. **Nathalie Aubry:** Methodology. **Charlotte Paës:** Data curation. **Muriel Cocaign-Bousquet:** Writing – review & editing, Validation, Conceptualization. **Marie-Line Daveran-Mingot:** Writing – review & editing, Validation, Supervision, Project administration, Conceptualization. **Michael Mourez:** Writing – review & editing, Validation, Supervision, Project administration, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

This research was supported by an "EMERGENCE, TRANSVERSALITÉS et INTERNATIONAL" project (ETI, 2023) and implemented by an exploratory DIA (INP Toulouse, France) entitled: "Indigenous LactoStart". The authors thank Benoît Bonizzoni from "Les Fermiers du Rocamadour" and Charlotte Faury from "the Syndicat AOP Rocamadour" for their guidance and for supplying the PDO Rocamadour raw goat milk used in this study.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.fm.2026.105047.

## Data availability

Data will be made available on request.

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