# Joining environmental impacts and product quality in Life Cycle Assessment: The case of the production and storage of lactic acid bacteria concentrates

Maite Gagneten, Camille Quentier, Stephanie Passot, Stéphanie Cenard,
Fernanda Fonseca, Caroline Pénicaud

► To cite this version:

Maite Gagneten, Camille Quentier, Stephanie Passot, Stéphanie Cenard, Fernanda Fonseca, et al. "Joining environmental impacts and product quality in Life Cycle Assessment: The case of the production and storage of lactic acid bacteria concentrates." Cleaner Environmental Systems, 2024, 15, pp.100245. 10.1016/j.cesys.2024.100245. hal-05077075

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# Joining environmental impacts and product quality in Life Cycle Assessment: The case of the production and storage of lactic acid bacteria concentrates

Maite Gagneten<sup>a</sup>, Camille Quentier<sup>b</sup>, Stéphanie Passot<sup>b</sup>, Stéphanie Cenard<sup>b</sup>,
Fernanda Fonseca<sup>b</sup>, Caroline Pénicaud<sup>b, c</sup>

## ARTICLE INFO

**Keywords:**

Life cycle assessment

Biological activity

Freezing

Freeze-drying

Spray-drying

Storage

## ABSTRACT

Lactic acid bacteria are widely used in the food and pharmaceutical industries to produce fermented foods and probiotics. This study assesses the environmental impacts of fifteen production and storage strategies for lactic acid bacteria concentrates from two strains. A life cycle assessment was performed using pilot-scale data. Environmental impacts per functional unit were weighted based on biological activity using two approaches: one by final biological activity after storage and the other by activity at each production stage. Fermentation emerged as a key phase in both methods, with higher yields significantly reducing environmental impacts. Stabilization and storage also contributed substantially to both environmental impact and loss of biological activity. The choice of stabilization method and storage conditions depends on the strain and expected storage duration. The weighting method affected the results: weighting by final biological quality highlighted the preservation of activity while weighting by each production stage better reflected the material and energy flows responsible for environmental damage. This study identifies crucial stages for improving the sustainability of lactic acid bacteria production and suggests that integrating environmental and quality factors is essential when designing production processes.

## 1. Introduction

Lactic acid bacteria (LAB) are widely used in the food and pharma-
ceutical industries to produce fermented foods and probiotics. With the
increasing popularity of these products, the demand for starter cultures
is growing. Together with other microbe products, the global market is
projected to grow from $10.5 billion in 2022 to $12.1 billion by 2027
(BCC Research, 2023). The production of LAB concentrates is a
multi-faceted process with successive steps from fermentation and har-
vesting to storage. Moreover, to commercialize living microorganisms,
their liquid form needs to be concentrated to reduce transport costs,
while their sensitive structure has to be transformed into a more stable
form that preserves their functions after production and during storage
(Goderska, 2012). To this purpose, freezing and drying are used to sta-
bilize the cells and storage under controlled conditions, usually cold

temperatures, is needed to maintain their quality until use (Fonseca
et al., 2015; Pénicaud et al., 2018). The unit operations involved in these
processes, such as heating and refrigeration, are known to be highly
energy-consuming. Specifically, according to a report by the European
Commission Joint Research Centre, cooling and freezing operations
account for around 30% of the total electricity consumed by the food
industry (European Commission. Joint Research Centre, 2015) and are
associated with refrigerant liquids leak which is another important
source of air pollutants. Moreover, although freezing and drying enable
products to have higher physicochemical stability and longer shelf life,
the operations can damage cells, causing loss of viability and function-
ality. This deleterious consequence can be minimized by cells'
pre-adaptation during fermentation, adding protective molecules,
encapsulation, and applying favorable operating conditions during sta-
bilization. It is thus imperative to revisit LAB production and

<footnote><sup>a</sup> Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, ITAPROQ (UBA-CONICET), Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina</footnote>

<footnote><sup>b</sup> Université Paris-Saclay, INRAE, AgroParisTech, UMR SayFood, 91120, Palaiseau, France</footnote>

<footnote>* Corresponding author. Université Paris-Saclay, INRAE, AgroParisTech, 22 place de l'Agronomie, Palaiseau, 91120, France.

**E-mail address:** caroline.penicaud@inrae.fr (C. Pénicaud).

@cpenicaud, @MaiteGag (C. Pénicaud)</footnote>
preservation processes with an integrated perspective that considers product quality, process efficiency, and environmental impacts.

Life Cycle Assessment (LCA) is a standardized methodology (ISO 14040, 2006) that appears to be one of the most recognized scientific methods for systemic and multi-criteria assessment and quantification of the environmental impacts of processes through the whole food chain. The academic, public, and business communities widely use it for research and decision-making. However, in addition to covering the complete production systems in the LCA studies, the analysis of the environmental performance of LAB production systems must also consider the functionality preservation of the microorganisms during processing, making it essential to adopt a holistic approach. To take the product's quality into account, most studies either incorporate the final quality parameter in the functional unit (Priyadarshini et al., 2022; Svanes and Johnsen, 2019) or weigh the resulting impact scores by the desired quality property at the end of its production or use (Igos et al., 2013). However, the production of LAB is a multi-stage process, where the biological activity of the microorganisms can be affected to different extents at different steps. Moreover, the degree of damage may depend on the process parameters and the bacterial strains, among many other variables. In a previous work by our research group, the environmental impacts of the production of stabilized *Lb. bulgaricus* CFL1 by freezing or freeze-drying were assessed by LCA. The impact scores were weighted by the physiological state of the bacteria at the end of the process as a quality parameter. The results showed that no environmental benefit was observed if the storage temperature of freeze-dried products was increased from -20 °C to ambient temperature as a consequence of the high loss of bacteria quality in such storage conditions. The most eco-friendly scenario to preserve LAB showed to depend on the storage period: freezing was found to be better for short storage periods (less than two months) while freeze-drying for periods over seven months (Pénicaud et al., 2018). However, in that work, the most common approach of considering the quality parameter in the functional unit only at the end of the process limits the ability of the method to analyze the hotspots of the process since valuable information on bacteria quality loss along the process is lost.

Considering all the above, the present work aims to follow up and broaden the previous study. With this purpose, *Lactobacillus delbrueckii* subsp. *bulgaricus* CFL1 and *Lactiplantibacillus plantarum* WCFS1 were chosen for the study due to their widespread use in the food industry and their different sensitivity to stress (Arena et al., 2019; Golowczyc et al., 2010; Meneghel et al., 2017; Passot et al., 2012). Freezing and freeze-drying were assessed as stabilization methods since they are two of the most used strategies to stabilize LAB (Fonseca et al., 2019). However, freezing requires storage at very low temperatures (commonly under -50 °C), whereas freeze-drying is a high-cost and energy-intensive process (Marin and René, 2000). Therefore, spray-drying, widely used in the food industry, was also evaluated as a more economical and time-efficient alternative (Joshi et al., 2022; Martín et al., 2015). Also, protective solutions containing different fructo-oligosaccharides (FOS) were evaluated. The environmental impacts of the production of these FOS were assessed in previous work by Gerbino et al. (2022) and were incorporated into the study. Finally, storage at different temperatures was evaluated to study the effect of storage at freezing, refrigerated, and ambient temperatures. The specific objectives of this work were: 1) describe the environmental impact profile of five different production and storage systems of LAB concentrates by identifying the hotspots of each process, 2) compare the environmental impacts of the production and stabilization of two different LAB strains, three different protective solutions, and three different stabilization strategies, and 3) analyze the influence of applying two different methods to account the product's quality, defined as their acidifying activity, in the functional unit. The final objective was to contribute to the design of more eco-friendly processes to produce LAB starters.

## 2. Materials and methods

### 2.1. Production of LAB concentrates

Two lactic acid bacteria (LAB) strains, *Lactobacillus delbrueckii* subsp. *bulgaricus* CFL1 (CIRM-BIA; Rennes, France) and *Lactiplantibacillus plantarum* WCFS1 (NIZO Food Research B.V., The Netherlands) were studied in this work. The production of the LAB concentrates was performed at a pilot scale following the process presented in Fig. 1.

This process consisted of different steps: growth medium preparation, fermentation, harvest, concentration, protection, stabilization, storage, sterilization, and equipment cleaning. Three different protectants, consisting mainly of a mixture of maltodextrin (Glucidex IT6), sodium ascorbate, and one of the following three fructo-oligosaccharides, were tested in a cell-to-protective solution weight ratio of 1:2. The three protectants were: a commercial fructo-oligosaccharide (Orafti® P95, Beneo Orafti; Tienen, Belgium) (FOS 1); a fructo-oligosaccharide produced by enzymatic synthesis, without purification (FOS 2); and a purified fructo-oligosaccharide produced by enzymatic synthesis (FOS 6). Also, three stabilization processes were studied: freezing, freeze-drying, and spray-drying. Samples were stored for up to 28 days. Frozen samples were stored at -80 °C, while freeze- and spray-dried samples were held at 4 °C to consider refrigerated storage, or 23 °C as ambient temperature storage. Two independent replicates of the entire production and storage process were conducted. The detailed conditions for each step are described in the accompanying data paper (Gagneten et al.). The combination of the different strains, protection, and stabilization strategies are summarized in Table 1.

### 2.2. Bacteria quality measurements

The bacteria quality was measured after protection, after each stabilization procedure, and after storage for 7, 14, and 28 days at each tested condition (indicated with orange stars in Fig. 1). For analysis, frozen samples were thawed, and the dried samples were rehydrated in a 0.9% (w/w) NaCl sterilized saline solution. The amount of NaCl solution added was calculated to reach the same dry matter of the protected bacterial suspension before freeze-drying. The quality was assessed in terms of specific acidifying activity ($t_{spe}$ in min/log(CFU/mL)), defined as the ratio between the acidifying activity and the decimal logarithm of the cell cultivability (Gautier et al., 2013; Velly et al., 2015). The lower the $t_{spe}$, the better the physiological state of the bacteria.

To determine cell culturability, serial dilutions in sterilized saline solution were prepared and inoculated in triplicate in MRS agar (Biokar Diagnostics, France). Colonies were enumerated from those plates containing between 30 and 300 colonies, after incubation for 24h at 37 °C for *Lpb. plantarum* WCFS1 and 48h at 42 °C under anaerobic conditions (GENbox96124, BioMérieux, Marcy l'Etoile, France) for *Lb. bulgaricus* CFL1. The colony-forming units (CFU) were then multiplied by the dilution factor to determine the original concentration of cells, and results were expressed in log(CFU/mL). The acidifying activity was measured using the Cinac system (AMS, Guidonia, Italia) (Corrieu et al., 1988), according to Meneghel et al. (2017), with some modifications. A volume of 100 µl of *Lb. bulgaricus* CFL1 or 10 µl of *Lpb. plantarum* WCFS1 cell suspension or rehydrated samples were incubated in skim milk at 42 °C or in a glucose-yeast extract medium at 37 °C for each strain, respectively. The time necessary to reach a pH drop of 1.5 points (in minutes) was the parameter used to characterize the acidifying activity. For both biological activity measurements, three analytical replicates were conducted over each production replicate, and nonparametric Kruskal-Wallis tests were performed considering a significance level of 95% ($p < 0.05$). With these two parameters, the ratio between the acidifying activity to cell culturability was computed to obtain the specific acidifying activity ($t_{spe}$, in min/(log(CFU/mL)), an indicator of the LAB's biological activity that takes into account the physiological state and viability.
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Production process of the LAB concentrates. Black complete lines represent the production steps and associated material inputs and outputs; gray complete lines represent the cleaning and sterilization steps and their associated material inputs and outputs; the dashed black line represents the system boundaries; orange stars indicate the stages at which intermediate products were generated and acidification activity measured.</figcaption></figure>

<table><caption>**Table 1**
Variations in the scenarios under study.</caption><thead><tr><th>Variability factor</th><th>Alternative</th></tr></thead><tbody><tr><td>Strain</td><td>Lactobacillus delbrueckii subsp. bulgaricus CFL1<br>Lactiplantibacillus plantarum WCFS1</td></tr><tr><td>Protectant (Glucidex IT6 + FOS + sodium ascorbate)</td><td>FOS 1<br>FOS 2<br>FOS 6</td></tr><tr><td>Stabilization strategy</td><td>Freezing and storage at -80 °C<br>Freeze-drying and storage at 4 °C<br>Freeze-drying and storage at 23 °C<br>Spray-drying and storage at 4 °C<br>Spray-drying and storage at 23 °C</td></tr></tbody></table>

## 2.3. Environmental impact assessment

The environmental impacts of the different scenarios were calculated using the Life Cycle Assessment (LCA) methodology according to the standardized steps of the ISO 14040 standard (ISO 14040, 2006).

### 2.3.1. Goal and scope

The LCA study was performed with two primary goals: 1) to identify the process hotspots, i.e., the steps that contribute the most to environmental degradation, and 2) to compare the environmental performance between the different scenarios summarized in Table 1. All the variability factors were crossed, resulting in 30 scenarios to compare. The study included all the processes described in Fig. 1, from the inputs' production to the stabilized LAB's final storage. However, the transport and end-use were excluded from the analysis because the production systems do not change depending on the targeted end-use. An

attributional analysis was performed since the aim of this study was to describe the relevant flows associated with the production of a specific product and compare the environmental performance of different processing alternatives without co-products, and because the market supply chains were clearly defined in the experimental work (Ekvall, 2019).

#### 2.3.1.1. Functional unit.
The LAB chosen for this study are commonly used to produce yogurt, cheese, or other fermented products. Despite the different end-uses, the production stages of the LAB concentrates do not change. For all these applications, the value of the bacteria is determined by its technological performance, defined by its physiological state. The different LAB strains, protection solutions, and stabilization strategies result in bacteria with different biological activity. Therefore, first, the raw LCIA results were divided by the mass of the final product to obtain the LCIA related to the functional unit of 90 g of protected LAB. To consider the functional quality, the environmental impact scores were weighted using the t<sub>spe</sub> measured for each LAB according to Equation (1).

$$ \text{Weighted score} = \text{Characterized impact value} * t_{\text{spe}} \qquad \text{Eq. 1} $$

A high t<sub>spe</sub> value indicates low performance of the bacterial starter and, therefore, large quantities of starters to be used to reach the targeted acidifying activity. Consequently, at equivalent LCIA results (characterized impact values), a scenario producing low-performance LAB concentrates (high t<sub>spe</sub> value) will have a higher weighted score than a scenario that leads to starters of better quality.

In a first approach each scenario's total environmental impact per mass-based functional unit were multiplied by the final t<sub>spe</sub> value at the end of the storage. Alternatively, a second approach was studied, computing the total environmental impact as the sum of the impact of each production step (i.e., fermentation, stabilization, storage) per mass-based functional unit multiplied by the corresponding t<sub>spe</sub> measured
after that step. Since medium preparation and cleaning and sterilization are related to the obtention of the biomass rather to its preservation, and because no t<sub>spe</sub> loss was expected between fermentation and harvesting, the environmental impact of these steps was multiplied by the t<sub>spe</sub> value determined after protection.

### 2.3.2. Life cycle inventory (LCI)

The analysis considered all the known material and energy inputs and outputs. Primary data were directly measured onsite to provide high-quality data for LCA with regards to their reliability, completeness, and temporal, geographical, and technological correlations. The included mass flows were equipment materials, culture media, glucose, NaOH, protectants, cleaning products, wastes, cooling water, steam, compressed air used by the fermenter, compressed air used by the spray-drier, and refrigerant leakages. The energy flows were only electrical consumptions.

The central background database used was Ecoinvent 3.6, using the cut-off system model complemented with Agribalyse 3.0 for ingredients of the growth medium. The pilot plant process was located in Spain, so the electricity generation and imports/exports from this country were considered, while tap and wastewater data from Europe was used. A Life Cycle Inventory of the production of the three protectants was available from a previous study published in a data paper (Gerbino et al., 2022).

The data collection, mass and energy flows, equipment details, and background data information are available in the associated dataset (https://doi.org/10.57745/HTC3UB) and described in details in a data paper (Gagneten et al.).

### 2.3.3. Life cycle impact assessment

The Life Cycle Impact Assessment (LCIA) was performed using SimaPro 9.1.0.11 software and "EF 3.0 Method (adapted) V1.04/EF 3.0 normalization and weighting set" (Fazio et al., 2018). All the method's midpoint impact categories were calculated, and the raw LCA results are available in the associated dataset (https://doi.org/10.57745/HTC3UB) described in a data paper (Gagneten et al.). However, the following indicators are discussed: climate change, ozone depletion, ionizing radiation, photochemical ozone formation, particulate matter, acidification, eutrophication (marine), eutrophication (freshwater), eutrophication (terrestrial), land use, water use, resource use (fossil), and resource use (minerals and metals). The three remaining indicators (human toxicity (non-cancer), human toxicity (cancer), and ecotoxicity (freshwater)) were not included in the study since they are poorly modeled due to the lack of definition of the specific substances, their stability, the kind of water body, among others, and insufficient development of the characterization factors (Sala et al., 2018).

## 3. Results and discussion

### 3.1. Bacterial quality

The functionality of the LAB was assessed in terms of their specific acidifying activity (t<sub>spe</sub>) to weigh the environmental impact results by this quality parameter. The t<sub>spe</sub> of each bacteria protected and stabilized by each strategy after each production step and after 28 days of storage is available in the associated dataset (https://doi.org/10.57745/HTC3UB) described in a data paper (Gagneten et al.). Table 2 shows the t<sub>spe</sub> increase (Δt<sub>spe</sub>) after stabilization and after storage at 23 °C.

No significant loss of specific acidifying activity was observed after freezing (Δt<sub>spe</sub> < 3 min/logUFC/mL). This was expected as freezing is frequently used to preserve LAB since it leads to high cell viability and activity (Gagneten et al., 2024). On the other hand, although both drying methods caused important activity losses for *Lb. bulgaricus* CFL1 (between 28 and 45 min/logUFC/mL), spray-drying of *Lpb. plantarum* WCFS1 caused smaller reductions (between 19 and 32 min/logUFC/mL) and freeze-drying even less or none (between 5 and 10

<caption>Table 2</caption>

Specific acidifying activity variation (Δt<sub>spe</sub>, in min/(log(CFU/mL) after stabilization and after 28 days of storage at 23 °C. Data are presented as median and interquartile ranges (IQR).

<table><thead><tr><th rowspan="2">Strain and stabilization scenario</th><th colspan="2">Δt<sub>spe</sub> stabilization</th><th colspan="2">Δt<sub>spe</sub> storage at 23 °C</th></tr><tr><th>Median</th><th>IQR</th><th>Median</th><th>IQR</th></tr></thead><tbody><tr><td>CFL1 FOS1 Freezing</td><td>2.2</td><td>0.9</td><td>-</td><td>-</td></tr><tr><td>CFL1 FOS1 Freeze-drying</td><td>28</td><td>3</td><td>14</td><td>1</td></tr><tr><td>CFL1 FOS1 Spray-drying</td><td>45</td><td>9</td><td>24</td><td>3</td></tr><tr><td>CFL1 FOS2 Freezing</td><td>0.6</td><td>0.4</td><td>-</td><td>-</td></tr><tr><td>CFL1 FOS2 Freeze-drying</td><td>42</td><td>3</td><td>24</td><td>3</td></tr><tr><td>CFL1 FOS2 Spray-drying</td><td>39</td><td>5</td><td>17</td><td>3</td></tr><tr><td>CFL1 FOS6 Freezing</td><td>0.8</td><td>0.9</td><td>-</td><td>-</td></tr><tr><td>CFL1 FOS6 Freeze-drying</td><td>40</td><td>1</td><td>26.3</td><td>0.6</td></tr><tr><td>CFL1 FOS6 Spray-drying</td><td>42</td><td>4</td><td>10</td><td>4</td></tr><tr><td>WCFS1 FOS1 Freezing</td><td>-2</td><td>3</td><td>-</td><td>-</td></tr><tr><td>WCFS1 FOS1 Freeze-drying</td><td>4.8</td><td>0.3</td><td>7.3</td><td>0.3</td></tr><tr><td>WCFS1 FOS1 Spray-drying</td><td>32</td><td>1</td><td>19.0</td><td>4</td></tr><tr><td>WCFS1 FOS2 Freezing</td><td>-1</td><td>1</td><td>-</td><td>-</td></tr><tr><td>WCFS1 FOS2 Freeze-drying</td><td>7</td><td>3</td><td>1.5</td><td>0.7</td></tr><tr><td>WCFS1 FOS2 Spray-drying</td><td>19</td><td>5</td><td>5.3</td><td>0.2</td></tr><tr><td>WCFS1 FOS6 Freezing</td><td>1</td><td>1</td><td>-</td><td>-</td></tr><tr><td>WCFS1 FOS6Freeze-drying</td><td>10</td><td>1</td><td>-6</td><td>1</td></tr><tr><td>WCFS1 FOS6 Spray-drying</td><td>21.2</td><td>0.4</td><td>7.1</td><td>0.2</td></tr></tbody></table>

min/logUFC/mL). The differences observed between both LAB were also expected since *Lb. bulgaricus* CFL1 is known to be more sensitive to drying, while *Lpb. Plantarum* WCFS1 has shown more resistance (Gagneten et al., 2024). Freeze-drying is the most frequently used method for drying LAB due to its high survival rates in comparison to other drying techniques (Ermis, 2021). On the other hand, spray-drying is usually preferred in the food industry due to its higher productivity when compared to freeze-drying but it is still limited to preserve LAB since it presents more challenges to maintaining cell viability (Huang et al., 2017). FOS 1 seemed to show a slightly better protective effect than FOS 2 and FOS 6 for freeze-drying, especially of *Lb. bulgaricus* CFL1, but was the worst protectant for spray-drying of *Lpb. plantarum* WCFS1. No major differences were observed between the protective effect of FOS 2 and FOS 6. The FOS in the protective mixture provides small sugars that may substitute water during drying (Romano et al., 2016). Variations in the production and purification processes of the FOS result in differing sugar chain lengths, which in turn lead to varied protective effects. Regarding storage, at -80 and 4 °C, no significant activity losses were observed. On the contrary, storage at 23 °C caused significant acidifying activity losses for both dried samples of *Lb. bulgaricus* CFL1, but smaller ones for spray-dried and especially freeze-dried samples of *Lpb. plantarum* WCFS1 (Table 2). During storage, FOS 1 also showed better cell preservation for freeze-dried samples of *Lb. bulgaricus* CFL1 but worse for spray-dried samples, while FOS 2 and FOS 6 were better protectors for *Lpb. plantarum* WCFS1 for both drying methods. Pénicaud et al. (2018) also reported important losses of specific acidifying activity after freeze-drying of *Lb. bulgaricus* CFL1 and, similarly, found three months of frozen storage at -45 °C of *Lb. bulgaricus* CFL1 did not alter the bacteria, while storage at ambient temperatures caused important losses of specific acidifying activity on the freeze-dried cells.

### 3.2. Contribution analysis

A contribution analysis was performed to identify the production process stages that significantly influenced the environmental impacts. Fig. 2 shows the relative environmental impacts of each process step to produce *Lb. bulgaricus* CFL1 protected with FOS 1 and stabilized either by one of the five different stabilization strategies and stored for up to 28 days. The contribution of each stage of the process is reported as the percentage of the total impacts. Analogous analyses were performed for *Lpb. plantarum* WCFS1 and for all the other combined scenarios.

The fermentation stage represented a significant part of the impacts, primarily driven by liquid waste production, along with electricity
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Contribution analysis of the environmental impacts (calculated by EF3.0 method) for the production of *Lb. bulgaricus* CFL1 strain protected with FOS 1, stabilized and stored 28 days, either by one of the five different strategies: freezing and storage at -80 °C (A), freeze-drying and storage at 4 °C (B), freeze-drying and storage at 23 °C (C), spray-drying and storage at 4 °C (D), or spray-drying and storage at 23 °C (E). The relative contributions of the different production steps are shown, adding to a total of 100% for each impact category. The exact numbers (%) are provided only for climate change indicators to lighten the Figure.</figcaption></figure>

consumption and, to a lesser degree, tap water usage. It was the main contributor in the freezing scenario (~42% of the impacts on average) and the spray-drying scenarios (~36%–38% for storage at 4 and 23 °C respectively), and the second contributor in the freeze-drying ones (~34%). Pénicaud et al. (2018) reached similar results in their LCA of the production *Lb. bulgaricus* CFL1 concentrates. In their study, fermentation was also the most significant hotspot when freezing was used as a stabilization strategy and an equally important one for the freeze-drying alternative. The authors attributed the high relative impacts of this stage to the use of natural gas for sterilizing the fermenter and tap water employed to cool the fermenter during biomass production and after sterilization. This, together with the high amounts of electricity consumption and the generation of liquid waste containing organic residues, resulted in the increased contribution of this stage. Differing from the work by Pénicaud et al. (2018) in which the French electricity grid (dominated by nuclear power) was considered, in our study, the energy used for production corresponded to the Spanish electricity grid, which is still highly dependent on non-renewable resources like petroleum, natural gas and coal (International Energy Agency, 2023) (~62% of the total energy and ~31% of the electricity mix by 2021 according to DG ENER and Eurostat reports (DG ENER and Eurostat reports, 2022)). These electricity resources have average emission levels around 0.60 kg CO₂eq.kWh⁻¹, while renewable resources like wind power or solar photovoltaic have values of 0.03 and 0.09 kg CO₂eq.kWh⁻¹, respectively (Evans et al., 2009). Therefore, the impacts of this stage could be significantly reduced if electricity was generated entirely from renewable resources. Regarding water consumption, a recirculating loop with a cooling system was used to cool the

fermenter. Water reuse should be revised in detail to reduce consumption in this step.

The absolute impact of the fermentation stage was the same for all five scenarios (e.g., 15 kg CO₂ eq for climate change to produce *Lb. bulgaricus* CFL1) since precisely the same fermentation conditions were applied in all scenarios for a given strain. Yet, since different fermentation conditions were used between the two studied strains, the contribution analysis for *Lb. bulgaricus* CFL1 and *Lpb. plantarum* WCFS1 protected with FOS 1 and stabilized by freezing are contrasted in Fig. 3.

From this comparison, it was observed that the contribution of the fermentation was much smaller for *Lpb. plantarum* WCFS1 than for *Lb. bulgaricus* CFL1 (27% versus 42%, respectively). However, this significant difference was not due to the differences in the fermentation conditions themselves but to the fact that higher yields were reached for the cell growth of the first strain than for the second one. Hence, when adjusted to the functional unit of 90 g of protected cells, the contribution of the fermentation was lower for the strain that produced more biomass.

When returning to the contribution analysis for *Lb. bulgaricus* CFL1 (Fig. 2), the stabilization stage represented a significant part of the impacts, which depended strongly on the stabilization process used. For freezing, the contribution to the total impacts was ~9% due to the freezer itself and the packaging of the products. For spray-drying, it was up to ~37%, surprisingly due to compressed air consumption (about 40 m³) which significantly affected the Eutrophication of freshwater and Resource use, mineral, and metals categories. Spray-drying represented one of the main contributors together with fermentation. For freeze-drying, it was up to ~41%, resulting in the main contributor to this
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Contribution analysis of the environmental impacts (calculated by EF3.0 method) for the production of *Lb. bulgaricus* CFL1 (A) and *Lpb. plantarum* WCFS1 (B) protected with FOS 1 and stabilized by freezing and storage at –80 °C. The relative contributions of the different production steps are shown, adding to a total of 100% for each impact category. The exact numbers (%) are provided only for climate change indicators to lighten the Figure.</figcaption></figure>

scenario, largely driven by electricity consumption, with refrigerant leakages also playing a significant role in the Ozone depletion indicator. Pénicaud et al. (2018) reached similar results for producing frozen and freeze-dried bacteria. Indeed, freeze-drying is a highly energy-intensive process: to stabilize 90 g of protected bacteria, freeze-drying required 38 kWh of electricity, while spray-drying required on average 1.3 kWh, and 0.4 kWh were needed for freezing (48h at –80 °C). If freeze-drying is to be used, it is imperative to optimize the process to reduce its electricity consumption. In this sense, Deprá et al. (2020) revealed that a combination of pressurized filtration and freeze-drying would be the best alternative for energy consumption in the industrial drying processes of microalgae. Nevertheless, the effect of such a process on the LAB quality should be evaluated.

The contribution of the storage step to the environmental impacts was high for the freezing scenario, which was stored at –80 °C, also due to high electricity consumption (6.6 kWh) and the refrigerant leakage (0.14 kg). In this scenario, the contribution of the storage step (~24% of the impacts) was the second contributor to the total environmental impacts after fermentation. In contrast, for the freeze- and spray-drying scenarios, the storage step had a much smaller contribution (between ~5% and ~8% for storage at 4 °C and ~3% for storage at 23 °C), since less or even no energy and refrigerant liquid were used for storage at higher temperatures. The impact category Ozone depletion showed an exceptionally high contribution for this step for both refrigerated storage conditions of the frozen and spray-dried scenarios because of the refrigerant leakage related to cold storage. The contribution was lower for the refrigerated storage of the freeze-dried samples only because it was exceeded by the contribution of the dehydration step to this impact category. Nevertheless, the contribution of the refrigerant leakage related to cold storage should be qualified because the value was calculated and not measured due to unavailability of the refill experimental data, and may not necessarily match the refrigerant leakage needed to refill over time.

The cleaning and sterilization of equipment and the medium preparation steps did not contribute significantly to the environmental impacts (~4–~6%), except for the impact category Resource use, mineral, and metals, for which the culture medium caused a significant impact (~21% for freezing and freeze-drying scenarios) due to the raw materials used, such as glucose. In this regard, Smetana et al. (2017) showed that using food waste as a carbon source instead of glucose could reduce the environmental impacts associated with microalgae cultivation. Cerdeira et al. (2021) used apple bagasse, a by-product from the production of juice and cider, as a source of carbon supplemented with yeast extract, Tween, manganese, and magnesium as a substrate to obtain biomass of *Lpb. plantarum*. Moreover, the authors suggested that the supplementation could be replaced by other food waste rich in nutrients. This proposes a potential site of improvement that should be further analyzed.

The impacts generated during the harvesting and protection stage represented around 9–12% of the impacts for all the scenarios. Approximately 90% of these impacts were attributed to the protective solution and, more precisely, to the production of oligosaccharides. The environmental impacts of the production of these FOS were assessed in a previous work by Gerbino et al. (2022). In that study, it was observed that the production of FOS 2 had lower impacts than FOS 1 and FOS 6 since purification was not performed in this system, and it was shown that purification was a source of significant impacts (Quentier et al., 2020). The difference between FOS 1 and FOS 6 is in the purification step, where the conditions for FOS desorption were different (ethanol concentration during elution), leading to slightly lower impacts for FOS 6 than FOS 1. These environmental impacts were the ones used in this study. The use of FOS 2 instead of FOS 1 or FOS 6 induced only a reduction of 2–3% of the environmental impacts of the production process, which can be considered negligible. FOS 1 or FOS 6 did not show any difference in the environmental impacts.

Similar results were reached for the contribution analysis of the different production and storage stages of *Lpb. plantarum* WCFS1 stabilized by the different studied strategies.

### 3.3. Comparison of the different production and storage strategies

Since storage significantly contributed to the environmental impacts for the scenarios stored at –80 °C and had a relevant contribution for storage at 4 °C, the environmental impacts were estimated for up to 12 months. To do this, since $t_{spe}$ showed a linear relationship with storage time, the results of this parameter for one month were extrapolated for up to 12 months, together of those of the mass and energy flows of the storage step. Fig. 4 shows the results for the category Climate change for *Lb. bulgaricus* CFL1 (Fig. 4A) and *Lpb. plantarum* WCFS1 (Fig. 4B) samples protected with FOS 1. The complete results for all the midpoint impact categories for both strains protected with FOS 1 are shown in Figure SM1. Analogous analyses were conducted for both strains protected with FOS 2 and FOS 6 (LCIA results available in the associated dataset (https://doi.org/10.57745/HTC3UB) described in a data paper (Gagneten et al.)), and only negligible differences were observed between the different protectants on the overall environmental impacts.

The Life Cycle Impact Assessment (Fig. 4 and SM1) revealed that immediately after production, freezing was the stabilization method with the lowest impacts for all the midpoint indicators except Ozone depletion. This was due to the freezing process being brief and requiring minimal energy consumption but emitting refrigerant leakage. Freezing was followed by spray-drying (except for the indicators of Eutrophication of freshwater and Use of minerals and metals, related to compressed air consumption), while freeze-drying had the highest impact in most categories, due to very high electricity consumption and use of refrigerant liquid. However, as the storage time increased, the very low
<figure><img src="image_6.png" /><figcaption>Fig. 4. Comparison of the environmental impacts described by the indicator Climate change for *Lb. bulgaricus* CFL1 (A) and *Lpb. plantarum* WCFS1 (B) protected with FOS1 and stabilized by freezing and stored at –80 °C (black), freeze-dried and stored at 4 °C (light blue) and 23 °C (green), and spray-dried and stored at 4 °C (orange) and 23 °C (red) as a function of storage time.</figcaption></figure>

temperatures used to store the frozen samples led to a rapid increase in the impacts of this scenario due to the high contribution of this step, as explained in the previous section. On the contrary, the environmental impacts increased at a much smaller rate during storage at 4 °C and

remained unchanged for storage at 23 °C. As a result, even for short storage periods (even as fast as one month for some indicators), the impacts of the frozen scenario overpassed one of the spray-dried ones, and for storage times over 3–5 months, it grew higher than that of the

<figure><img src="image_7.png" /><figcaption>Fig. 5. Contribution analysis of the environmental impacts (calculated by EF3.0 method) for the production of *Lb. bulgaricus* CFL1 strain protected with FOS 1 and stabilized either by freezing and storage at –80 °C (A), freeze-drying and storage at 4 °C (B), freeze-drying and storage at 23 °C (C), spray-drying and storage at 4 °C (D), or spray-drying and storage at 23 °C (E). Impact scores were weighted by the specific acidifying activity corresponding to each production step. The relative contributions of the different production steps are shown, adding to a total of 100% for each impact category. The exact numbers (%) are provided only for climate change indicators to lighten the Figure.</figcaption></figure>
freeze-dried ones. Spray-drying was the stabilization strategy with the lowest environmental impacts for most of the categories when storage was considered. As a result, spray-drying and storage at 23 °C appeared to be the best strategy for producing LAB concentrates when the product's biological quality was not considered.

### 3.4. Considering the specific acidifying activity in the functional unit: sensitivity analysis

To consider the biological activity of the bacteria in the analysis, the environmental impacts per mass-based functional unit, computed to up to 12 months of storage, were weighted by the $t_{spe}$ measured for each scenario. In the first approach, the impact scores were multiplied by the final $t_{spe}$ according to the storage time considered, whereas in the second approach, weighting the impact scores of each production step by the $t_{spe}$ measured after that corresponding step was analyzed.

#### 3.4.1. Contribution analysis

When the first approach was studied, the contribution analysis was the same as the unweighted analysis since all the production steps were affected by the same quality score for each system (Figs. 2 and 3).

On the contrary, when the second approach was applied, the contribution analysis (Fig. 5) changed because this latter weighting method uncovered the different degrees of quality loss caused by the various production steps and strategies.

Since freezing did not affect the biological activity of the cells and neither did storage at -80 °C, the contribution of each production stage for these scenarios did not change when the impact scores were weighted by the biological activity of each step (Fig. 5A) compared to the unweighted analysis (Fig. 2A). On the contrary, because freeze- and spray-drying caused a significant loss of specific acidifying activity, the contribution of the stabilization step for the drying scenarios was more significant when this weighting method was used. For *Lb. bulgaricus* CFL1, the contribution of the stabilization step increased from ~40% to ~50% for freeze-drying and from ~37% to ~50% for spray-drying. The same happened with the contribution of the storage step. When this second weighting method was used (Fig. 5), the storage step's contribution also increased compared to the results obtained with the first weighting method due to the loss of activity. For *Lpb. plantarum* WCFS1, spray-drying also caused significant specific acidifying activity losses, that led to a significantly higher contribution of the spray-drying step (from ~44% to ~56%) when the second weighting method was used. On the contrary, when the different weighting methods were compared, for freeze-drying this increase was negligible (less than 3%) due to the

better resistance of this strain to this drying-method. Also, the contribution of storage at 4 °C and 23 °C was not significantly altered by the weighting analysis for the freeze-dried samples of *Lpb. plantarum* WCFS1, as only a slight specific acidifying activity loss was observed during storage. In contrast, for the spray-dried samples, the contribution of the storage stage increased from ~13% to ~16% for storage at 4 °C and from ~6% to ~10% for storage at 23 °C. Because of the increase in the contribution of the stabilization and storage steps, the contribution of all the steps previous to stabilization and storage was lower compared to the previous weighting method for those scenarios. Also, the differences observed in the biological activity preservation efficiency between the different protectants were not significant enough to introduce appreciable differences in the contribution analysis.

#### 3.4.2. Comparison of the different production strategies

Fig. 6 shows the scenario comparison for *Lb. bulgaricus* CFL1 (Fig. 6A) and *Lpb. plantarum* WCFS1 (Fig. 6B) protected with FOS 1 for the indicator Climate change when the first weighting approach was applied. The results for all the midpoint impact categories for both strains protected with FOS 1 are shown in Figure SM2.

Analogous analyses were conducted for both strains protected with FOS 2 and FOS 6. When this weighting was applied, freezing and storage at -80 °C appeared as the strategy with the lowest impacts for almost all the midpoint indicators for *Lb. bulgaricus* CFL1 except for Particle matter after six months of storage and Ozone depletion throughout the whole storage period, due to leakages of refrigerant liquid. These results agree with those reported by Pénicaud et al. (2018), as previously described in the introduction. Either freeze- or spray-drying showed lower environmental burdens depending on the impact category. These results may seem unexpected when considering the contribution analysis, in which high contributions of the cold storage and the freeze-drying operation were observed, due to electricity consumption and leakages of refrigerant liquid. However, they can be explained by the quality of the product. Despite the high contribution found for storage at -80 °C, the better biological activity preservation observed in this scenario led to lower weighted environmental impacts. On the contrary, although spray-drying showed lower environmental impacts, when the unweighted scores were analyzed, due to the extensive quality loss, larger amounts of bacteria would be needed to reach the desired activity. As a result, the environmental scores of this scenario are augmented to equal or even pass those of the freeze-drying scenario. Similar results were reached with FOS 2 and 6, except for the drying strategies in the Climate change category, where FOS 2 and FOS 6 showed lower impacts for the spray-dried scenarios than the freeze-dried ones as FOS 2 and FOS 6

<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Comparison of the environmental impacts for the category Climate change weighted by the final specific acidifying activity for *Lb. bulgaricus* CFL1 (A) and *L. plantarum* WCFS1 (B) protected with FOS1 and stabilized by freezing and stored at -80 °C (black), freeze-dried and stored at 4 °C (light blue) and 23 °C (green) and spray-dried and stored at 4 °C (orange) and 23 °C (red) as a function of storage time.</figcaption></figure>
protected less efficiently freeze-dried bacteria compared to FOS 1 (Table 2). For *Lpb. plantarum* WCFS1, the tendencies were similar. However, since this strain was more resistant to drying, the advantages observed for the freezing scenario were less significative. As a result, for extended storage periods (more than 7 or 8 months), the environmental impacts of freezing and storage at -80 °C were more significant than those of the dried samples stored at 4 °C for this strain. Similar results were reached with FOS 2 and 6, although the impact scores were much smaller than with FOS 1, and, therefore, the differences between the different scenarios were less pronounced.

This analysis shows that when the first weighting approach is applied, the storage temperature has a considerable and over-dimensionalized influence on the environmental results. Even though cold storage may imply more use of resources and environmentally damaging emissions due to energy consumption and refrigerant leakage, better biological preservation at -80 and 4 °C led to lower environmental impacts than at 23 °C. Moreover, as the storage time augmented, the impact scores of both dried samples stored at ambient temperatures reached values almost three times higher than those stored at 4 °C due to the specific acidifying activity loss.

Fig. 7 shows the environmental impact results reached by the second

approach, that is, weighting by the specific acidifying activity after each production step, for *Lb. bulgaricus* CFL1 (Fig. 7A) and *Lpb. plantarum* WCFS1 (Fig. 7B) protected with FOS 1.

After production and for short storage periods (less than 2–8 months, depending on the indicator), freezing and storage at -80 °C was the scenario with the lowest environmental impacts, except for the Ozone depletion category (strongly linked to the use of refrigerant liquid) as observed by the first weighting method. However, when longer storage times were considered, the scenario with the lowest environmental impacts varied depending on the indicator. For *Lb. bulgaricus* CFL1, spray-drying and storage at 23 °C was the strategy with the lowest impacts for most of the impact categories: Climate change, Ozone depletion, Ionizing radiation, Photochemical ozone formation, Water use, and Resource use of fossils. In these categories, the environmental impacts of the process outweighed the preservation efficiency, as they are closely tied to energy and water consumption, as well as refrigerant leakage. Instead, freeze-drying and storage at 23 °C was the strategy with the lowest environmental impacts for the categories of Particle matter, Acidification, Eutrophication of freshwater, and Land use. In these categories, preservation efficiency had a greater influence than the process's environmental impacts, as these impact categories are not heavily

<figure><img src="image_9.png" /><figcaption>**Fig. 7.** Comparison of the environmental impacts weighted by the specific acidifying activity of each production step for *Lb. bulgaricus* CFL1 (A) and *Lpb. plantarum* WCFS1 (B) protected with FOS1 and stabilized by freezing and stored at -80 °C (black), freeze-dried and stored at 4 °C (light blue) and 23 °C (green), and spray-dried and stored at 4 °C (orange) and 23 °C (red) as a function of storage time. Each environmental indicator is presented separately.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Fig. 7. (continued).</figcaption></figure>

impacted by the energy and water demands of stabilization processes. On the other hand, for *Lpb. plantarum* WCFS1 freeze-drying and storage at 23 °C showed the lowest environmental burdens for almost all the indicators except for Ozone depletion, Ionizing radiation, Water use, and Resource use of fossils. This better performance of the freeze-drying scenario for *Lpb. plantarum* WCFS1 is mainly due to this drying method's much better biological preservation than spray-drying for this strain. By comparing the performance of the storage at 4 and 23 °C for the same drying method, it could be concluded that for both strains and all the impact categories except Resource use of minerals and metals, storage at ambient temperature seemed to cause lower environmental impacts, despite the more considerable losses of biological activity that may take place at higher storage temperatures. This surprising result may seem contradictory to the results reached by the first weighting method. However, it may be explained by the fact that since storage at ambient temperatures implies almost no additional use of natural resources and emissions, its contribution to the total environmental impacts is meager even when the biological loss at this stage is considered: even though higher than when storage at refrigerated temperatures, the loss of quality is not significant enough to cause a more considerable increase of the weighted environmental impacts than for the refrigerated storage, which implies more considerable environmental burdens.

When the different protective solutions were compared using this

second weighting approach, slight differences in the environmental impact results depending on the stabilization strategy were observed. For both freeze-dried LAB and the frozen ones of *Lpb. plantarum* WCFS1, the same overall conclusions as with FOS 1 were reached after production for most impact indicators using FOS 2 or FOS 6, while a lower impact (~14%) was observed for *Lb. bulgaricus* CFL1 frozen samples protected with FOS 2 (Figure SM3) and FOS 6 (Figure SM4). Spray-dried LAB protected with these latter protectants showed significantly smaller weighted impacts than their equivalent ones protected with FOS 1 (between ~15% for *Lb. bulgaricus* CFL1 and 25–34% for *Lpb. plantarum* WCFS1). This was the result of the added effect of the slightly lower environmental impacts of the production of the protectant solution (in the case of FOS 2) and the better preservation of the biological activity of these protectants, observed especially for the spray-dried samples. As a result, the spray-dried samples protected with FOS 2 and FOS 6 stored at 23 °C exhibited better environmental performance. With these protective solutions, the weighted environmental scores of this stabilization scenario matched or even overperformed the one of the freeze-dried scenarios in some of the indicators such as Acidification, Marine eutrophication, Terrestrial eutrophication, and Land use.

### 3.4.3. Discussion on the functional unit sensitivity analysis

Many studies have highlighted the importance of the selection and
definition of the functional unit for comparative LCA studies (Gonzalez-García et al., 2016; Pérez-López et al., 2014). The mass-based functional unit remains a classical approach. When quality is included in the functional unit, as stated before, the most used weighting methods consider only the final quality of the product - i.e., weighting the impact scores by the last biological activity of the bacteria (e.g. Pénicaud et al., 2018; Igos et al., 2013). However, the choice of what weighting approach to apply is not trivial since it can largely influence the results, and, as our results show, even different conclusions might be reached depending on which method is applied. Weighting by the final t<sub>spe</sub> vastly increased the overall environmental impacts since the contribution of all steps was affected by the extensive loss of activity at the end of the process, overestimating the impact of stages that might have only minor or even no harmful effect over the biological activity of the LAB. In this case, the environmental impacts are mainly determined by the preservation of the biological quality, and some relevant information for decision-making about the environmental burden of different process steps and production alternatives may be lost. On the other hand, weighting by the t<sub>spe</sub> determined after each step considers the activity loss implied in each production stage and enables better visualization of the evolution of the environmental impacts across the process and of the different alternatives for each production step. With this method, the contribution of the material and energy flows responsible for environmental damage are better reflected while also considering the biological activity. In return, this biological activity must be measured throughout the process, which can limit the use of such an approach due to the experimental work it generates.

### 3.5. Limitations of the study

This work was performed with experimental data obtained at a pilot scale. The complete LCI can be found in the research dataset (https://doi.org/10.57745/HTC3UB) described in the associated data paper (Gagneten et al.). It highlighted the critical stages of relevance with potential for improvement and the areas of concern for the further development of these processes, which can help to eco-design the production processes of LAB concentrates. Nevertheless, it is known that the dimension of the production facilities and equipment may significantly influence the system's environmental impacts (Pénicaud et al., 2018; Munoz Ortiz et al., 2006). Moreover, in the case of the current study, it would be interesting to analyze how the scale-up may influence the LCIA results and the biological quality of the LAB differently and, consequently, how this would affect the weighted results. A study of these systems at an industrial scale could enable the evaluation of this variable's significance.

In addition, transport was left out of the system's boundaries because the analysis focused on the environmental impacts emerging from a LAB starter producer. Nevertheless, including the transportation stage in the analysis could be interesting since different requirements would be necessary for the frozen and the dried products.

## 4. Conclusions

This study examined the environmental impacts of different stabilization and storage strategies to produce lactic acid bacteria concentrates from two strains commonly used by the food industry.

The fermentation stage was one of the hotspots of the process, mainly due to electricity and liquid waste; therefore, achieving high yields during fermentation can lead to significant reductions in the system's environmental impacts. This was the main reason why the production of stabilized *Lpb. plantarum* WCFS1 caused much smaller impacts than the production of stabilized *Lb. bulgaricus* CFL1. Regarding stabilization and storage, drying represented a significant part of the impacts and caused important losses in biological activity. Freeze-drying had higher environmental impacts than spray-drying, primarily due to electricity consumption and refrigerant leakage in freeze-drying, while spray-drying's

impacts were mainly attributed to compressed air usage. In return, freeze-drying preserved better the biological activity of *Lpb. plantarum* WCFS1 cells than spray-drying. On the other hand, while freezing preserved the biological quality of the bacteria better, the very low temperatures needed to preserve the frozen bacteria largely increased the environmental impacts of this scenario as it implies huge amounts of electricity and refrigerant leakages. Therefore, the most adequate stabilization method and storage conditions depend on the bacteria strain and the estimated storage time. The comparison of two different approaches to consider the biological quality in the functional unit showed that the choice of the weighting method had a relevant influence on the results. Weighting the environmental impact scores by the final biological quality led to results mainly influenced by the preservation of the biological activity. Therefore, freezing and storage at very low temperatures resulted in the strategy with the lowest impacts for up to 6 (for *Lpb. plantarum* WCFS1) or even 12 (for *Lb. bulgaricus* CFL1) months of storage. On the other hand, weighting the environmental impact scores by the acidifying activity measured after each production stage shows more influence of the mass and energy flows related to each process step while also considering the biological quality of the product at the end of the production process. With this approach, freezing and storage at -80 °C only showed the lowest environmental impacts for very short storage periods. When longer storage times were analyzed, storage at 23 °C was the storage condition that had the lowest impacts for both strains and drying methods. Spray-drying and storage at 23 °C was the strategy with the lowest impact for most of the impact categories for *Lb. bulgaricus* CFL1, while freeze-drying and storage at 23 °C, showed the lowest environmental burdens for almost all the indicators for *Lpb. plantarum* WCFS1. The choice of the protective solution had a minor effect on the results, mainly related to its efficiency in preserving the biological quality rather than to the different environmental scores of their production, since small amounts of these solutions were used. Lastly, when the final acidifying activity weighted the environmental scores, the contribution analysis did not change. In contrast, when the second weighting approach was applied, the contribution of those process steps that had a more deleterious effect on the cells increased.

As final remarks, considering the biological quality of the bacteria in the functional unit is fundamental to obtain meaningful results regarding the environmental impacts of producing dehydrated LAB concentrates for the industry. Moreover, the approach applied for this purpose must be carefully evaluated since it may significantly affect the results.

This study's results contribute to identifying stages that require special attention when eco-designing lactic acid bacteria concentrates production processes and the process parameters that may hold key opportunities to reduce environmental impacts. However, further research should be made at the industrial scale to encourage the implementation of this type of environmental assessment and joint environment-quality analysis as a systematic approach to designing production processes.

### CRediT authorship contribution statement

**Maite Gagneten:** Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Camille Quentier:** Validation, Resources, Methodology, Investigation, Formal analysis, Conceptualization. **Stéphanie Passot:** Writing – review & editing, Resources, Investigation, Conceptualization. **Stéphanie Cenard:** Resources, Investigation. **Fernanda Fonseca:** Writing – review & editing, Resources, Investigation, Funding acquisition, Conceptualization. **Caroline Pénicaud:** Writing – review & editing, Supervision, Project administration, Methodology, Formal analysis, Conceptualization.
## Data availability

The data generated and analyzed in this study are available in an open dataset (https://doi.org/10.57745/HTC3UB) described in the associated data paper (Gagneten et al.).

## Funding

This work received funding from the European Union's Horizon 2020 research and innovation program under grant agreement N° 777657. The authors have applied a CC-BY public copyright license to the present document, which will be applied to all subsequent versions up to the Author Accepted Manuscript arising from this submission, in accordance with the grant's open access conditions.

## 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.

## Acknowledgments

The authors want to acknowledge Jérôme Delettre, Baptiste Gauducheau, Maria Guerrero, Sonia Campoy, and Yanis Rodríguez for their experimental and technical support.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.cesys.2024.100245.

## Data availability

I have shared the link to my data in the submission files.

Life Cycle Assessment of the production of stabilized lactic acid bacteria at pilot scale (Original data) (Dataverse)

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