Tween 80™-induced changes in fatty acid profile of selected mesophilic lactobacilli

Fatty acid profiles are crucial for the functionality and viability of lactobacilli used in food applications. Tween 80™, a common culture media additive, is known to influence bacterial growth and composition. This study investigated how Tween 80™ supplementation impacts the fatty acid profiles of six mesophilic lactobacilli strains (Lacticaseibacillus spp., Limosilactobacillus spp., Lactiplantibacillus plantarum). Analysis of eleven strains revealed 29 distinct fatty acids. Tween 80™ supplementation significantly altered their fatty acid composition. Notably, there was a shift towards saturated fatty acids and changes within the unsaturated fatty acid profile. While some unsaturated fatty acids decreased, there was a concurrent rise in cyclic derivatives like lactobacillic acid (derived from vaccenic acid) and dihydrosterculic acid (derived from oleic acid). This suggests that despite the presence of Tween 80™ as an oleic acid source, lactobacilli prioritize the synthesis of these cyclic derivatives from precursor unsaturated fatty acids. Myristic acid and dihydrosterculic acid levels varied across strains. Interestingly, palmitic acid content increased, potentially reflecting enhanced incorporation of oleic acid from Tween 80™ into membranes. Conversely, cis-vaccenic acid levels consistently decreased across all strains. The observed fatty acid profiles differed from previous studies, likely due to a combination of factors including strain-specific variations and growth condition differences (media type, temperature, harvesting point). However, this study highlights the consistent impact of Tween 80™ on the fatty acid composition of lactobacilli, regardless of these variations. In conclusion, Tween 80™ significantly alters fatty acid profiles, influencing saturation levels and specific fatty acid proportions. This work reveals key factors, including stimulated synthesis of lactobacillic acid, competition for oleic acid incorporation, and strain-specific responses to myristic and dihydrosterculic acids. The consistent reduction in cis-vaccenic acid and the presence of cyclic derivatives warrant further investigation to elucidate their roles in response to Tween 80™ supplementation.


Introduction
The fatty acid composition of bacterial cells of the lactobacilli determines their survival and depends on many environmental factors (Johnsson et al., 1995;Partanen et al., 2001;Corcoran et al., 2007;Montanari et al., 2010;Li et al., 2011;Tan et al., 2012;Terraf et al., 2012;Al-Naseri et al., 2013;Hansen et al., 2015;Reitermayer et al., 2018).Corcoran et al. (2007) proved this by showing that oleic acid (C18:1,cis-9) and cis-vaccenic acid (C18:1,cis-11) have a protective effect on Lacticaseibacillus rhamnosus GG cells suspended in artificial gastric juice.These researchers have shown that only in the case of these two fatty acids, the number of bacterial cells was greater than the number of cells in the control sample.Other tested fatty acids, including stearic (C18:0), elaidic (C18:1,trans-9), linoleic (C18:2,cis-9,cis-12), cis-9,trans-11-octadecadienoic, and trans-10,cis-12-octadecadienoic, caused a decrease in the number of viable cells (to a level of 3.89 log CFU/mL in case of trans-10,cis-12-octadecadienoic acid and to 4.75 log CFU/mL for stearic and elaidic acids).This phenomenon can be explained by the fact that oleic (C18:1,cis-9) and cis-vaccenic (C18:1,cis-11) acids are the substrates for the synthesis of fatty acid in various ways required for cell survival and modification of cell membrane fluidity.Lactic acid bacteria (LAB), lactobacilli including, are equipped with the mechanisms for transforming these fatty acids in their cyclic, polyunsaturated, or conjugated forms (Henderson and McNeill, 1966;Weeks and Wakil, 1970;Partanen et al., 2001;Corcoran et al., 2007;Tan et al., 2012).Transformation of linoleic (C18:2,cis-9,cis-12) or conjugated acids to oleic acid (C18:1,cis-9) requires the action of specific enzymes if LAB cells lack access to external sources of oleic acid.In the absence of external oleic acid, some LAB strains may resort to this conversion pathway to maintain a certain level of oleic acid, potentially leading to a decrease in the overall protective action of these polyunsaturated fatty acids.
One of the external sources of fatty acids for bacterial cells is their living environment, including culture media.A key example is oleic acid, a fatty acid found in many natural sources.However, bacteria can also utilize oleic acid provided in the form of Tween 80 ™ .Tween 80 ™ is a nonionic surfactant, commonly used in the food industry as an emulsifier, dispersant, and stabilizer.It finds application in microbiology as a common additive to media for cultivating lactobacilli (Johnsson et al., 1995;Partanen et al., 2001;Montanari et al., 2010;Li et al., 2018).It could be also employed in food fermentation processes for its beneficial effects on microbial growth and product quality (Li et al., 2018).
Chemically, Tween 80 ™ is a synthetic molecule derived from sorbitan (a cyclic polyol) and esterified with oleic acid (C18:1,cis-9).This combination results in a molecule with a hydrophilic head (sorbitan) and a hydrophobic tail (oleic acid), allowing Tween 80 ™ to act as a surface-active agent.Tween 80 ™ improves the solubility and bioavailability of nutrients, promoting microbial growth and fermentation efficiency.By facilitating nutrient uptake and metabolism, Tween 80 ™ can lead to higher yields of fermented products (Li et al., 2018).The combination of oleic acid (C18:1,cis-9) with sorbitol permits the solubility of fatty acid in aqueous solution and a uniform dispersion of particles in growth medium, thereby making it more accessible to acid bacterial cells.Corcoran et al. (2007) compared the effect of adding Tween 80 ™ to the survival of four strains of lactobacilli suspended in artificial gastric juice: Lacticaseibacillus rhamnosus GG, Lacticaseibacillus rhamnosus E800, Lacticaseibacillus paracasei subsp.paracasei NFBC 338, and Ligilactobacillus salivarius UCC 500.In each case they confirmed the protective effect of Tween 80 ™ on cell viability.No Tween 80 ™ resulted in a decrease in the number of LAB viable cells in at least two logarithmic cycles after incubation in artificial gastric juice for 90 min.The cited studies are confirmed by other references (Johnsson et al., 1995;Broadbent et al., 2014).
The most widely used methods in study cellular fatty acid (as methyl esters) of Firmicutes (including lactobacilli) is matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) based on the principle of ionization and mass analysis of molecules, and it is particularly well-suited for identifying bacteria because it can analyze intact bacterial cells rather than extracting and purifying specific molecules (Anderson et al., 2014;Foschi et al., 2017;Nacef et al., 2017).As a result of these advantages, MALDI-TOF is becoming the preferred method for the identification of lactobacilli in many laboratories.MALDI-TOF (matrixassisted laser desorption ionization time-of-flight mass spectrometry) is another method that is increasingly being used for the study of bacterial cellular fatty acid.It is a rapid, sensitive, and easy-to-use technique that can provide accurate species identification.However, the Sherlock Microbial Identification System (by MIDI Inc.) is a reliable method for species identification, particularly for lactobacilli, and is often used in conjunction with other methods, such as sequencing, to provide a complete taxonomic profile (Brondz, 2002;Kunitsky et al., 2006;Alexander, 2012).The alternative method referred to in the statement is likely to be gas chromatography-mass spectrometry (GC-MS), which is a more sophisticated technique that can provide more detailed information about the composition of fatty acids.However, GC-MS is a more expensive and time-consuming method, so it is not always the most practical choice.MIDI is a faster and more cost-effective method, and it is often sufficient for routine identification of lactobacilli.In general, MIDI is a suitable choice for routine identification of lactobacilli when speed and cost are important, while MALDI-TOF is the more advanced method and is the best choice for routine identification and specialized applications.GC-MS is most appropriate for more detailed studies or when species identification is challenging.
This study investigates the influence of Tween 80 ™ (oleic acid source) supplementation in growth media on the FA profile of selected mesophilic lactobacilli strains.We aim to compare their abilities to modulate FA synthesis and transformation upon incorporation of this external oleic acid (C18:1,cis-9) source.

Materials
Eleven lactobacilli strains of the lactobacilli (Table 1) deposited in the laboratory collection in the form of monocultures freeze-dried or frozen were used in our research.Cultures of each bacterial strain were conducted in three independent experiments.First, the cultures were revived in the MRS (de Mann, Rogosa, and Sharpe) broth supplemented with Tween 80 ™ (Sigma-Aldrich) in portions of 50 mL (incubation at 37 °C for approx.12 h).Tween 80 ™ is known to influence membrane fluidity and potentially impact fatty acid synthesis in lactobacilli (Al-Naseri et al., 2013;Zotta et al., 2017b;Reitermayer et al., 2018).Subsequently, the cultures were inoculated onto the surface of three parallel MRS agar plates with Tween 80 ™ and three parallel MRS agar plates without Tween 80 ™ (in two independent replicates for each growth medium).Cultures were incubated at 37 °C for 24 h under anaerobic conditions.Anaerocult ™ A (Merck) was used to produce an anaerobic milieu in the anaerobic jar ™ (Merck).Anaerocult ™ A is recommended for the cultivation of obligatory and facultative anaerobes.To minimize growth phase variation, we carefully harvested cells for biomass collection from the defined center sector of each plate using the secondary inoculum.This ensured a representative sample of the cultured population for fatty acid analysis (Asadi et al., 2015).
The extraction of fatty acids: separating and detecting conditions of fatty acid methyl esters using GC-MS The fatty acids were extracted from the entire bacterial biomass according to recommendations (Haack et al., 1994;Buyer, 2002;Kankaanpaa et al., 2004).Each bacterial biomass sample was prepared for analysis and analyzed in two replicates.The chromatographic separation of fatty acid methyl esters was carried out by gas chromatography coupled with mass spectrometer (GC-MS QP 2010 Shimadzu, Shimadzu Corporation, Japan) using polar column 007-23-30-0.2F(30 m × 0.25 mm × 0.20 μm; Quadrex, Quadrex Corporation, United States).The sample was injected under a split ratio of 1: 25 at the dispenser temperature equal to 230 °C.The chromatographic separation was conducted in the following conditions: Initial temperature of column of 60 °C, 2 min isotherm, the temperature increase of 4 °C/min to 220 °C, 10 min isotherm.The carrier gas was helium with a flow rate of 0.37 mL/min.The following detector conditions were used: Temperature of the ion source of 200 °C, temperature of the line connecting GC with MS of 220 °C, detector voltage of 1.45 kV, and quadrupole filter sweep in a range 50-400 m/z.The following standards were used for identifying fatty acids: oleic acid (Sigma Aldrich, United States), anteiso12-methyltetradecanoic acid (Sigma Aldrich, United States), 2-hydroxytetradecanoic acid (Sigma Aldrich, United States), nonadecanoic acid (Sigma Aldrich, United States), the BAME (bacterial acid methyl esters; Sigma Aldrich, United States), GLC-674 and GLC-617 (Nu-Chek-Prep., United States), and isomers of methyl esters of linoleic acid 18:2 (cis-9,trans-11 and trans-10,cis-12; Nu-Chek-Prep., United States).The composition of bacterial acid methyl   1S.In other cases of fatty acids (including lactobacillic acid), the comparisons were made with the literature data (Brondz, 2002;Christie et al., 2007;Montanari et al., 2010).The data obtained from GC-MS was analyzed using GCMS Solution v.2.50 (Shimadzu Corporation).

Statistical analysis
The statistical analysis was carried out using the statistical program Statistica v.10.The analysis of variance (ANOVA) was employed.In this study, ANOVA was complemented by Tukey's test, conducted at a significance level of 0.05, to scrutinize the mutual influence of the factors and identify any statistically significant differences between them.Principal components analysis (PCA) was conducted to assess the relationship between fatty acid profiles and the presence or absence of Tween 80 ™ in the growth medium.In this case, PCA was used to identify the main components that influenced the variation in fatty acid profiles.

Lacticaseibacillus rhamnosus
The determined fatty acid profile of L. rhamnosus GG, L. rhamnosus MB, and L. rhamnosus ATCC 4769 cells demonstrated the presence of 29 fatty acids after the incubation on the growth medium with Tween 80 ™ (Table 2).
The strains of L. rhamnosus used in our study were characterized by an intense growth on the MRS agar without Tween 80 ™ .The character of the growth of this species' colonies was different from other lactobacilli species used for this study.The colonies on the line of loop movement were snow-white with characteristic papules and damming, and so obtaining the biomass required for the analysis was not difficult.Nineteen fatty acids were identified in the fatty acid profile of L. rhamnosus GG cells, while the presence of 21 fatty acids was noted in the case of L. rhamnosus MB and L. rhamnosus ATCC 7469 cells (Table 2).
rhamnosus ATCC 7469 cells cultured without Tween 80 ™ showed lactobacillic acid (cycC19:0,cis-10,11) dominance in their fatty acid profiles.This difference is likely due to the lack of separation of cyclic acids in the Corcoran et al. (2007) study.

Lacticaseibacillus casei and Lacticaseibacillus paracasei
The identified fatty acid profile of L. casei 393, L. casei 431, and L. paracasei subsp.paracasei MB grown on MRS agar with Tween 80 ™ contained 29 and 27 fatty acids for L. casei and L.
We observed significant differences in the proportions of saturated, unsaturated, and cyclic fatty acids compared to previous studies (Rizzo et al., 1987;Kankaanpaa et al., 2004;Machado et al., 2004;Liong and Shah, 2005;Corsetti et al., 2010;Rodrigues et al., 2012).Notably, our findings revealed substantial levels of dihydrosterculic and lactobacillic acids, cyclic derivatives of oleic and vaccenic acids, respectively.These results contrast with some prior studies that may have missed these cyclic acids due to separation and detection challenges (Machado et al., 2004;Liong and Shah, 2005) 2004) which we did not observe.These differences could be attributed to various factors, including strain variations and growth conditions.While comparisons for L. paracasei subsp.paracasei MB were limited due to a scarcity of data specifically for this subspecies, it's important to acknowledge that the L. casei group taxonomy has undergone revisions in recent years.Some strains previously classified as L. casei may now be identified as L. paracasei.Future studies that incorporate a wider range of Lactobacillus species, including recently reclassified members of the casei group, along with standardized protocols, would offer valuable insights into the influence of growth media components on bacterial fatty acid composition.

Lactiplantibacillus plantarum
Our study examined three strains of L. plantarum, all of which exhibited efficient growth on MRS agar with or without Tween 80 ™ .Interestingly, the fatty acid profiles of L. plantarum 299v and L. plantarum B.01149cultured on growth medium with Tween 80 ™ exhibiting profiles with 24 fatty acids, and B.01834 exhibiting a 24-fatty acid profile.Cultivation on growth medium with the addition of Tween 80 ™ resulted in the fatty acid profile of all three strains examined being dominated by palmitic acid (C16:0), dihydrosterculic acid, cisvaccinic acid and lactobacillic acid (cycC19:0,cis-10,11), with the involvement of the last two of these fatty acids differentiating the strains examined (Table 5).Removal of Tween 80 ™ from the growth medium resulted in a decrease in the share of 11 fatty acids (4 saturated and 7 unsaturated) in the fatty acid profile and an increase in the share of 9 fatty acids (6 saturated and 3 unsaturated) in the fatty acid profile.It is also worth mentioning that the overall contribution of CLA to the fatty acid profile of the tested strains was also statistically reduced due to the removal of Tween 80 ™ from the growth medium.
The first principal component separated the cases into two main groups based on the growth medium with or without Tween 80 ™ (Figure 1A).Notably, some L. casei strains did not cluster together, suggesting a potential taxonomic misclassification or high variability within this group.The high share of oleic acid (C18:1,cis-9) and CLA was important for the separation of the bacteria cultured on the growth medium with Tween 80 ™ , while linoelaidic (C18:2,trans-9,trans-12), palmitelaidic (C16:1,trans-9), and cis-vaccenic (C18:1,cis-11) acids were important in case of growth medium without Tween 80 ™ (Figure 1B).This allowed for the separation of strains cultured on growth medium with Tween 80 ™ into two main groups: those cultured with Tween 80 ™ and cultured without Tween 80 ™ .In contrast, no significant differentiation was observed for strains grown on the same growth medium without Tween 80 ™ .
Recent taxonomic revisions suggest that some L. casei strains might be reclassified as L. paracasei (Ghosh et al., 2019).Unfortunately, we could not perform phylogenetic analysis on all our strains due to the lack of a genome sequence for C-431.Despite this uncertainty, we observed distinct fatty acid profiles among the three L. casei strains (potentially L. paracasei).However, further studies with a larger and well-characterized strain collection are necessary to confirm the discriminatory power of fatty acid profiles for differentiation between these closely related species.
The exact effects of the presence of Tween 80 ™ in the growth medium vary depending on the type of lactobacilli, but in general, Tween 80 ™ can increase saturated fatty acids and reduce unsaturated fatty acids.The presence of Tween 80 ™ in the growth medium generally increased the proportion of lactobacillic acid (cycC19:0,cis-10,11) in the fatty acid profile of all lactobacilli strains.This was due to the stimulation of the  5 (Continued) The percentage fatty acids composition (mean ± SD, n = 6) in mesophilic Lactiplantibacillus plantarum strains profiles obtained from the cell cultured in a growth medium with Tween 80 TM (with T80) and without Tween 80 TM (no T80).synthesis of lactobacillic acid (cycC19:0,cis-10,11) by lactobacilli cells in response to the absence of oleic acid (C18:1,cis-9) from Tween 80 ™ .The presence of Tween 80 ™ in the growth medium had a mixed effect on the proportion of myristic acid (C14:0) and dihydrosterculic acid (cycC19:0,cis-9,10) in the fatty acid profile.In some lactobacilli strains, the proportion of myristic acid and dihydrosterculic acid (cycC19:0,cis-9,10) increased, while in other species, the proportion of myristic acid and dihydrosterculic acid (cycC19:0,cis-9,10) decreased.The presence of Tween 80 ™ in the growth medium significantly impacts the fatty acid profile of lactobacilli, generally increased the proportion of palmitic acid in the fatty acid profile of all lactobacilli strains.This was due to the incorporation of oleic acid (C18:1,cis-9) from Tween 80 ™ into the cell membranes of lactobacilli cells, which increased the demand for saturated fatty acids to maintain the fluidity of the cell membranes.While our data suggests a decrease in cisvaccenic acid (C18:1,cis-11) in the fatty acid profile of all lactobacilli strains, further investigation is needed to elucidate the underlying mechanisms.Existing literature suggests that Tween 80 ™ might suppress early fatty acids synthesis, potentially affecting cis-vaccenic acid (C18:1,cis-11) production later in the growth cycle (Jacques et al., 1980;Zotta et al., 2017b;Reitermayer et al., 2018).Future studies employing time-course experiments could provide valuable insights into the dynamics of fatty acid biosynthesis and incorporation in the presence of Tween 80 ™ , allowing for a more definitive understanding of potential competition or alternative pathways involved.

Conclusion
Our study investigated the influence of Tween 80 ™ , a widely used surfactant in culturing media, on the fatty acid profile of selected mesophilic lactobacilli strains.The fatty acid composition of mesophilic lactobacilli cells plays a crucial role in determining their membrane properties, which are essential for various cellular functions, including cell growth, motility, and stress resistance.Our study investigated the impact of Tween 80 ™ , a common surfactant used in bacterial culturing media, on the fatty acid profile of selected mesophilic lactobacilli strains.
Tween 80 ™ has a significant impact on the fatty acid profile of lactobacilli.The presence of Tween 80 ™ in the growth medium can alter the proportion of various fatty acids, including saturated and unsaturated.The specific changes in fatty acid composition vary depending on the lactobacilli strains and species.One important aspect to consider is the role of the culturing environment in shaping the fatty acid profile of lactobacilli.Culture media can introduce external fatty acids, such as the oleic acid (C18:1,cis-9) present in Tween 80 ™ , a common additive.Notably, oleic acid originating in Tween 80 ™ can be incorporated into lactobacilli membranes, contributing to the complex interplay between environmental factors and bacterial fatty acid biosynthesis in shaping membrane properties.
Our results reveal that the presence of Tween 80 ™ in the growth medium decreases the abundance of cis-vaccenic acid (C18:1,cis-11), a key contributor to membrane fluidity.This suggests that Tween 80 ™ may not only act as an external fatty acid source but also potentially compete with the bacteria's own synthesis of cis-vaccenic acid (C18:1,cis-11).While competition with oleic acid (C18:1,cis-9) from Tween 80 ™ for membrane incorporation is a possibility, further investigation is needed.Existing literature suggests that Tween 80 ™ may initially suppress fatty acid synthesis, potentially impacting the availability of precursors for later cis-vaccenic acid (C18:1,cis-11) production.This decrease in cis-vaccenic acid (C18: 1,cis-11) may lead to an increase in membrane rigidity, which could negatively impact cell growth, motility, and stress resistance.
Conversely, the addition of Tween 80 ™ to the growth medium had the opposite effect, increasing the proportion of cis-vaccenic acid (C18:1,cis-11), likely through precursor availability.This increase may help to maintain membrane fluidity, which is important for these cellular functions.The presence of Tween 80 ™ can increase the proportion of palmitic acid (C16:0) and lactobacillic acid (cycC19:0,cis-10,11). Palmitic acid is a saturated fatty acid that is involved in maintaining cell membrane structure and function.Lactobacillic acid (cycC19:0,cis-10,11) is a cyclopropane fatty acid that is unique to lactobacilli and is thought to play a role in their interactions with other organisms.
In conclusion, our study demonstrates that Tween 80 ™ significantly impact the fatty acid composition of lactobacilli.The presence of Tween 80 ™ can reduce the proportion of oleic acid and cis-vaccenic acid (C18:1,cis-11).Oleic acid is a major component of the cell membranes of lactobacilli, and Tween 80 ™ can compete with oleic acid for incorporation into the membranes.The results of our study are particularly relevant to the industrial application of mesophilic lactobacilli.These changes in fatty acid composition can in turn lead to changes in membrane properties, which could have implications for cell function.
Future studies could explore the specific mechanisms by which Tween 80 ™ influences fatty acid synthesis and the potential for strain-specific adaptations.Additionally, investigating the functional consequences of altered membrane fluidity on cell function would provide valuable insights for optimizing probiotic performance.

TABLE 1
The collection of lactobacilli strains used in this study.

TABLE 2
The percentage fatty acids composition (mean ± SD, n = 6) in mesophilic Lacticaseibacillus rhamnosus strains profiles obtained from the cell cultured in a growth medium with Tween 80 TM (with T80) and without Tween 80 TM (no T80).

TABLE 2 (
Continued) The percentage fatty acids composition (mean ± SD, n = 6) in mesophilic Lacticaseibacillus rhamnosus strains profiles obtained from the cell cultured in a growth medium with Tween 80 TM (with T80) and without Tween 80 TM (no T80).

TABLE 3
. For L. casei, our dominant fatty acids The percentage fatty acids composition (mean ± SD, n = 6) in mesophilic Lacticaseibacillus casei and Lacticaseibacillus paracasei strains profiles obtained from the cell cultured in a growth medium with Tween 80 TM (with T80) and without Tween 80 TM (no T80).

TABLE 3 (
Continued) The percentage fatty acids composition (mean ± SD, n = 6) in mesophilic Lacticaseibacillus casei and Lacticaseibacillus paracasei strains profiles obtained from the cell cultured in a growth medium with Tween 80 TM (with T80) and without Tween 80 TM (no T80). ,b,c,d,e,f -Means with different lowercase letters the same line indicate a significant difference at the significance level of 0.05 (Tukey HSD). a

TABLE 4
The percentage fatty acids composition (mean ± SD, n = 6) in mesophilic Limosilactobacillus fermentum and Limosilactobacillus reuteri strains profiles obtained from the cell cultured in a growth medium with Tween 80 TM (with T80) and without Tween 80 TM (no T80).

TABLE 4 (
Continued) The percentage fatty acids composition (mean ± SD, n = 6) in mesophilic Limosilactobacillus fermentum and Limosilactobacillus reuteri strains profiles obtained from the cell cultured in a growth medium with Tween 80 TM (with T80) and without Tween 80 TM (no T80).-Means with different lowercase letters the same line indicate a significant difference at the significance level of 0.05 (Tukey HSD).The percentage fatty acids composition (mean ± SD, n = 6) in mesophilic Lactiplantibacillus plantarum strains profiles obtained from the cell cultured in a growth medium with Tween 80 TM (with T80) and without Tween 80 TM (no T80).