*Article* **Lipid Remodeling in the Mitochondria upon Ageing during the Long-Lasting Cultivation of** *Endomyces magnusii*

**Elena P. Isakova 1,\*, Natalya N. Gessler 1, Daria I. Dergacheva 1, Vera M. Tereshina 2, Yulia I. Deryabina <sup>1</sup> and Marek Kieliszek <sup>3</sup>**


**Abstract:** In this study, we used *Endomyces magnusii* yeast with a complete respiratory chain and well-developed mitochondria system. This system is similar to the animal one which makes the yeast species an excellent model for studying ageing mechanisms. Mitochondria membranes play a vital role in the metabolic processes in a yeast cell. Mitochondria participate in the metabolism of several pivotal compounds including fatty acids (FAs) metabolism. The mitochondria respiratory activity, the membrane and storage lipids composition, and morphological changes in the culture during the long-lasting cultivation (for 168 h) were under investigation. High metabolic activity of *E. magnusii* might be related to the active function of mitochondria increasing in the 96- and 168-h growth phases. Cardiolipin (CL), phosphatidylethanolamine (PE), phosphatidylcholine (PC), and sterols (St) were dominant in the membrane lipids. The St and sphingolipids (SL) shares increased by a lot, whereas the CL and phosphatidylinositol (PI) + PE ones decreased in the membrane lipids. This was the main change in the membrane lipid composition during the cultivation. In contrast, the amount of PE and phosphatidylserine (PS) did not change. Index of Hydrogen Deficiency (IHD) of phospholipids (PL) FAs significantly declined due to a decrease in the linoleic acid share and an increase in the amount of palmitic and oleic acid. There were some storage lipids in the mitochondria where free fatty acids (FFAs) (73–99% of the total) dominated, reaching the highest level in the 96-h phase. Thus, we can conclude that upon long-lasting cultivation, for the yeast assimilating an "oxidative" substrate, the following factors are of great importance in keeping longevity: (1) a decrease in the IHD reduces double bonds and the peroxidation indices of various lipid classes; (2) the amount of long-chain FFAs declines. Moreover, the factor list providing a long lifespan should include some other physiological features in the yeast cell. The alternative oxidase activity induced in the early stationary growth phase and high mitochondria activity maintains intensive oxygen consumption. It determines the ATP production and physiological doses of reactive oxygen species (ROS), which could be regarded as a trend favoring the increased longevity.

**Keywords:** *Endomyces magnusii*; mitochondria; yeast; lipids; fatty acids; unsaturation degree

#### **1. Introduction**

Mitochondria participate in numerous cellular processes being critical for cell survival and death. Besides their best-known function of ATP generation, the mitochondria are entirely involved in cellular metabolism, partly maintaining calcium homeostasis and regulating cell adaption to various stresses. They are implicated in ROS signaling modulation, maintenance of oxidative homeostasis, and apoptosis regulation under stress conditions [1,2]. Indeed, mitochondria are involved in free radicals generation, with the

**Citation:** Isakova, E.P.; Gessler, N.N.; Dergacheva, D.I.; Tereshina, V.M.; Deryabina, Y.I.; Kieliszek, M. Lipid Remodeling in the Mitochondria upon Ageing during the Long-Lasting Cultivation of *Endomyces magnusii*. *Appl. Sci.* **2021**, *11*, 4069. https:// doi.org/10.3390/app11094069

Academic Editors: Francesca Silvagno and Burkhard Poeggeler

Received: 25 February 2021 Accepted: 28 April 2021 Published: 29 April 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

respiratory chain as the leading ROS producer. Excessive ROS production is supposed to result from mitochondria dysfunction [3]. Mitochondria are indispensable organelles of any eukaryotic cell, determining the organism's fitness and physiology [4]. The damage of mitochondrial function is a universal sign of ageing in a eukaryotic cell [4,5]. Some mechanisms causing damage to mitochondria bioenergetics include the accumulation of mutations and deletions in mtDNA, oxidation of mitochondria proteins, destabilization of macromolecular super-complexes in the respiratory chain, some changes in the lipid composition of the membranes, and modulation in mitochondria dynamics due to imbalance in organelle fusion, fission, and mitophagy [5]. The major ROS sources are mitochondrial respiratory chain, non-enzymatic reactions involving oxygen, phagocytosis, prostaglandin synthesis, the P450 cytochrome system, and ionizing radiation (in mammals). Thus, an increase in ROS generation due to an increased metabolic rate is considered the lifespan's principal determiner.

Not only the abundant ROS generated in pathological mitochondria but also ROS produced in the normal ones can lead to oxidative damage of the cells causing the ageing process. The hypothesis known as the "free radical theory" of ageing (FRTA) [6] declares the major statements of ageing in any aerobes, which later result in the mitochondrial theory of ageing. The theory supposes that mitochondria are the primary ROS sources in a cell and consequently participate in ageing and lifespan regulation [7]. Nevertheless, some new data have led to a more thorough overview of mitochondria ROS function, which is now considered to be signaling molecules in numerous pivotal biological processes, including ageing and lifespan [8].

The molecular mechanisms of the response to oxidative stress and the role of ROS in ageing have been widely and keenly studied for a long time. Hence, using simple eukaryotic models, namely yeast organisms, can be helpful. The *Saccharomyces cerevisiae* yeast is widely used in research on ageing mechanisms, and it contributes to understanding basic cellular and molecular processes [9]. Budding yeast research largely contributed to comprehending the ageing process and age-related diseases [10]. Yeasts, being the simplest unicellular eukaryotes, have many features in common with mammalian and human cells that permit studying both chronological and replicative ageing [11]. Moreover, the response of the yeast cell to oxidative stress is similar to that of mammals, including the sites of ROS generation in the electron transport chain and the performance of the major antioxidant enzyme complex [12].

Until recently, it was unknown if the composition of the mitochondrial membrane lipids during ageing in yeast can result in mitochondrial dysfunction. However, recent studies showed that in the *S. cerevisiae* yeast, a metabolic pathway of ceramide and sphingolipid synthesis is an essential node of the signaling network to determine the replicative and chronological lifespan [13–15]. High content of triacylglycerols (TAGs) is another aspect of lipid metabolism defining the lifespan of chronologically senescent yeast cells [15,16]. The neutral lipids are synthesized in the endoplasmic reticulum (ER) and then accumulate into lipid bodies (Lb), which determines the longevity of senescent yeast cells regardless of any other signaling pathways [15]. Their lipid composition can also distinguish mitochondrial inner and outer membranes. The outer mitochondrial membrane is a smooth lipid-rich envelope with pore-forming proteins. In contrast, the highly folded inner mitochondrial membrane is protein-rich harboring mainly enzymes of the respiratory chain [15]. PI is present in the outer mitochondrial membrane at a considerable amount. By contrast, CL and PE are enriched in the inner mitochondrial membrane. The presence of high CL levels in the mitochondrial membranes suggests that this PL is essential for efficient oxidative phosphorylation. The unsaturation degree of membrane lipids was found to influence respiratory properties and cytochrome content of mitochondria from *S. cerevisiae*. It was shown that a decrease in the amount of unsaturated FAs also declined the activity of several mitochondrial enzymes.

Vladimir Titorenko's team has methodically studied the relationship between the mitochondria lipidome and the life span of the yeast. The authors declared that ageing changes of the mitochondria lipid composition in the yeast include. (1) The levels of PE, CL and

monolysocardiolipin (MLCL) decrease in the mitochondrial membranes. (2) the levels of phosphatidic acid (PA), PS, PC and phosphatidylglycerol (PG) increase in the mitochondrial membranes [17,18]. The applied lithoholic bile acid was reported to penetrate into the mitochondria and accumulate in the inner mitochondria membrane. This leads to changes in the phospholipid composition of the mitochondria membranes [17–19]. The remodeling of mitochondrial phospholipids intensifies while a yeast culture ages and finally causes substantial changes in mitochondria membrane lipidome.

The composition of the mitochondria's respiratory chain in traditional yeast species, namely *S. cerevisiae* is quite invariable. In particular, their mitochondria have no complex I [20]. By contrast, the *Endomyces magnusii* yeast being an obligate aerobe possesses a complete respiratory system similar in its mitochondrial system to the animal one. The *E. magnusii* cannot grow in the presence of the inhibitors of the mitochondria transcription (ethidium bromide) and mitochondria translation (erythromycin). Under these conditions, the growth should undergo only at the expense of substrate and glycolytic phosphorylation. In our experiments, the glycerol-containing medium-low concentrations of ethidium bromide (5–15 μg/mL) led to the complete inhibition of the growth. We assayed the number of generations (Figure S3B). We could see similar results in the experiments with erythromycin, which in low concentration (5 mg/mL) blocked the growth of the *E. magnusii* (Figure S3A).

In the yeast with fermentative metabolism types such as *S. cerevisiae*, the membrane and mitochondrial apparatus are ill-developed. There are only scanty large mitochondria with small cristae. Their mitochondria have much fewer cristae and trend to irregularity in their shape, structure, and packing. However, the yeast of strongly-pronounced aerobic metabolism, namely *E. magnusii,* possesses well-developed membrane apparatus with abundant complex mitochondria with numerous cristae [21]. According to the above-mentioned statement, we consider that the *E. magnusii* species is a unique model close to the animal in the mitochondrial system that makes it a convenient model for studying some mechanisms, including ageing.

Previously, we demonstrated high viability capacity in the *E. magnusii* yeast upon chronological ageing [22]. For the 20-day cultivation of the strain in glycerol-containing (1%) medium, there were no signs of growth culture inhibition. The analysis of cell survival during culture ageing showed that after 168 h of growth (7 days), it decreased by not more than 10–15% compared to the logarithmic growth stage [22]. The yeast population in the stationary growth stage consists of long-lived quiescent cells, which can enter the cycle of cell fission cycle, and non-quiescent cells, which are short-lived and incapable of dividing, similar to the phenotype of ageing mammalian cells [13,23].

Considering the hypothesis of yeast population heterogeneity upon its shift into the stationary growth phase, our data, and the key role of the mitochondria in the ageing regulation, we wanted to systematically study the alterations in the lipid amount and composition, assayed the mitochondria metabolic activity, and the changes in cell morphology of *E. magnusii* mitochondria during long-term cultivation.

#### **2. Results**

#### *2.1. Long-Lasting Growth of the E. magnusii Yeast*

Figure 1 shows the growth curve of *E. magnusii* yeast upon assimilating 1% glycerol. The dynamics of cell growth displayed classical logarithmic dependence with a wellpronounced lag phase (from 0 to 6 h of cultivation), a phase of logarithmic (exponential) growth (from 6 to 20 h of cultivation), a diauxic shift phase defining the transition of the population into the stationary growth phase (from 22 to 30 h of cultivation), early stationary growth phase (from 30 to 48 h of cultivation), and late (48 h) and stationary phase (from 48 to 200 h of cultivation) (Figure 1).

**Figure 1.** The growth curve of *E. magnusii* yeast in glycerol-containing medium grown for 260 h. Absorbance was assayed every two hours in cell suspension at the wavelength of 590 nm. The curve is representative of four biological experiments. (The figure was partially used in the article [Microorganisms 2020, 8, 91; doi:10.3390/microorganisms8010091]).

Microscopic analysis of the morphology of *E. magnusii* cells in various growth stages showed that it varies greatly. In the logarithmic growth stage, the cells are round and oval, sometimes fusiform or pear-shaped, either with well-visible buds (in the process of division) or budded daughter cells (Figure 2A), whereas in the stationary stage (after 30 h of cultivation) the number of daughter cells decreased to 5% of the total number (Figure 2B). The population of the exponential phase had about 4% actively dividing cells with buds at the poles, about 10% cells with detached immature buds, and about 20% young daughter cells, which are smaller and regularly spherical (Figure 2A). In the stationary growth phase, the number of actively budding and young cells nearly halved, there was some heterogeneity in the cell population, some cells changed their shape, and some single pseudomycelial and mycelial forms appeared (Figure 2B). It should be noted that during long-lasting cultivation, the nuclei of *E. magnusii* cells changed their shape (increased by 1.5 times). In the stationary phase, the nuclei increased in their volume and became more round (Figure 2C,H). The morphometric features of this growth stage indicated the intensive growth and fission of *E. magnusii* cells (Table S1 in the Supplementary Section). At the same time, the population was highly heterogeneous without any signs of pathological changes or inclusions (Figure 2D). Moreover, the cells at this stage got a distinct thickened cell wall, large vacuoles, and small dense cytoplasmic inclusions, which are likely to be autophagic vacuoles or bodies (Figure 2H). It should be emphasised that the energy status of the cells in the logarithmic and stationary growth phases, confirmed by potentiometric staining, was maintained at a high level (Figure 2E,F).

Summarising all the obtained results including the changes in morphology, along with the functional heterogeneity of the *E. magnusii* culture, shown before [21], we studied the lipid amount and composition in the mitochondria at different growth stages upon ageing.

**Figure 2.** Micro images of the *E. magnusii* cells in the logarithmic (**A**,**C**,**E**,**G**) and late stationary (**B**,**D**,**F**,**H**) growth stages. (**A**,**B**)—The cells were stained with neutral red; (**C**,**D**). Fluorescent microimages of the cells, labelled with 0.3 μM DAPI for DNA. (**E**,**F**) the potential-dependent stain of the mitochondria in *E. magnusii* cells by 5 mM JC-1. The regions of high mitochondrial polarization are bright yellow due to the concentrated dye. The cells were examined after 30 min. The incubation medium contained 0.01 M phosphate buffer saline (PBS) and 1% glycerol, pH 7.4. To examine the Rh123-stained preparations, filters 02, 15 (Zeiss) were used (magnification 100×). Photos were taken using an AxioCam MRc camera. (**G**,**H**)-ultrastructure of *E. magnusii* cells. Nu—nucleus; V—vacuole; Mt–mitochondria; CW–cell wall.

#### *2.2. Respiratory Activity of E. magnusii Yeast Mitochondria*

We designed a unique method for mitochondria isolation modified especially for the yeast. The test for fraction contamination included electron microscopy of the organelles and the polarographic assay of the mitochondria function: (1) the electron microscopy images of the *E. magnusii* yeast (Figure S1 in the Supplementary Section) showed that the mitochondria fraction was pure and free from any inclusions and other organelles. Moreover, the mitochondria in the photos had a regular structure with well-developed cristae and intact outer and inner membranes; (2) the mitochondria could oxidize the respiratory substrates (pyruvate+malate) (Figure S2A in the Supplementary Section) and generate a high membrane potential without any co-factors application (NAD(P)H, cytochrome c) (Figure S2B in the Supplementary Section); (3) the assay of the mitochondria activity showed no endogenous respiration (without any substrate application), indicating neither other membranes nor enzymes (Figure S2A (a) in the Supplementary Section); (4) the mitochondria demonstrated high respiratory rates and phosphorylation, ADP/O ratios close to the theoretically expected maxima (Table S1 in the Supplementary Section) upon oxidation of NAD-dependent substrates. It says about good mitochondria integrity after the isolation. We could detect the activity of adenilate kinase, which is in the inter-membrane mitochondria space and synthesizes ADP from AMP and ATP (Figure S2C in the Supplementary Section). The enzyme cannot work if the membrane is damaged. However, its activity was observed even in the hypotonic incubation medium. We are also sure that the mitochondria fraction was pure because we modified the method of isolation of the mitochondria, especially, to bind and remove the lipid fraction from the suspension. In the mitochondrial isolation and washing medium, we use 1% bovine serum albumin (FAs free fraction V), which binds FAs in the medium. Moreover, the high degree of mitochondria coupling indicates no lipid contamination, as any lipid compounds lead to powerful uncoupling of respiration and phosphorylation [24]. We get live mitochondria with respiratory controls close to the maximum that is impossible if the fraction is contaminated (Table 1, Table S2 in the Supplementary Section).

**Table 1.** Respiratory activity and the inhibitory analysis of the *E. magnusii* yeasts mitochondria in different growth phases upon ageing.


\* V41, V42-the respiration rate in the second state (initial respiration) before and after the phosphorylation cycle. \*\* V3-the respiration rate in state 3 (the phosphorylation after the ADP addition). \*\*\* KCN was added in the concentration of 4 mM. \*\*\*\* SHAM was added in the concentration of 2 mM. The incubation medium for the experiments contained 0.6 M mannitol, 1 mM Tris-phosphate (pH 7.4); 1 mM EDTA, 20 mM pyruvate, 5 mM malate, and mitochondria corresponding to 0.4 mg protein/mL. a–f Means with the same letter did not differ significantly. Values are mean ± S.E.M from five independent experiments and three analytical replicates.

> Changes in respiratory activity is the main indicator of the cellular energy status. The mitochondria of most plants, fungi, and yeasts possess an alternative pathway of electron transport that is induced if the main (cytochrome) pathway is inhibited by KCN, azide, or antimycin A [25,26]. The electron flux is switched off at the reduced ubiquinone site and is specifically inhibited by hydroxamic acid derivatives. The dynamics of the oxygen uptake rate by *E. magnusii* mitochondria showed that it varied significantly during the growth and ageing of the culture (Table 1).

The respiration rate in the state 4 (initial respiration) was minimal at the logarithmic growth stage, slightly increased at the 24-h stage, increased threefold in the 48-h phase, and reached its peak by 96 h of growth. The respiration rate of mitochondria in the third state (phosphorylation after the ADP addition) was also altered, similar to that in the second one. However, the coupling degree of the mitochondria insignificantly decreased at the 24-h growth stage compared to that in the log-stage.

The proportion of the alternative electron transfer pathway showed that the mitochondria respiration at the mid-logarithmic stage was extremely sensitive to the inhibitor KCN. However, in the 24- and 48-h phases, we observed the induction of AO upon inhibiting the main (cytochrome) pathway. The level of the KCN-resistant respiration was 12–13% being blocked by the specific AO inhibitor salicylhydroxamic acid (SHAM). At the 96-h and 168-h phases of cultivation the share of cyanide resistance increased up to about 30 and 50%, respectively.

According to the model proposed by *Bahr and Bonner* [27], the alternative pathway becomes active only if the main pathway of electron transport cannot work (in case of inhibition or electron saturation), i.e., the activity of the KCN-resistant pathway is regulated by the main cytochrome chain. It can be assayed by its participation in the total cell respiration. The AO level indicates the respiration inhibited by the KCN + SHAM system, and its contribution into the total cell respiration is defined as the respiration share inhibited by SHAM only without KCN [27]. The attempts to assess the proportion of the alternative pathway in vivo corroborated no hypothesis of constitutive induction of AO. SHAM did not induce the respiratory rate inhibition in any experiments significantly (Table 1). Based on the data, we could suppose that during the growth and ageing of *E. magnusii*, the alternative pathway induction triggers; however, it is not likely to be related to the decrease in the cytochrome pathway activity.

To comprehend the mechanism of high energy activity of yeast mitochondria for 20 days, we tried to determine the alterations in the main lipid components of the *E. magnusii* mitochondria during long-lasting cultivation.

#### *2.3. Storage Lipids Profile of E. magnusii Yeast in the Different Growth Phases*

Figure 3 shows the total content and composition of neutral lipids in *E. magnusii* mitochondria. The major lipid fractions were presented by FFAs and diacylglycerols (DAGs). The FFAs level was high in all the mitochondria samples and reached 73–99% of the total lipids (Figure 3A). The maximum DAGs amount was found in the logarithmic growth phase. Of note that a certain share of them (10–15% of the total lipids) was also revealed at the later growth stages. The minority comprised TAGs and sterol esters (ESt). TAGs were detected only in the early growth stages (17 h of growth) while ESt appeared in the late stationary stages and their amount nearly doubled by 168 h of cultivation compared to that in the 96-h phase.

The amount of total storage lipids remained relatively stable during the growth culture up to 96 h when it increased nearly twofold with a concurrent decrease by 168 h of cultivation (Figure 3B).

In the next step of the study, we assayed the membrane lipids profile in the *E. magnusii* mitochondria.

#### *2.4. Membrane Lipids Profile of E. magnusii Mitochondria during Long-Lasting Cultivation*

The amount and composition of the mitochondrial membrane lipids also changed significantly during growth and ageing of the culture. The membrane lipid amount reached its peak of 118.120 mg/g mitochondrial protein in the early growth stationary stage (48 h of cultivation) (Figure 4B), whereas the minimum content of 59.086 mg/g mitochondrial protein was in the deep stationary phase (96 h). The dynamics of the mitochondria membrane lipids showed a gradual increase by 48 h of cultivation with a nearly twofold drop in the deep stationary phase. The main fractions were sterols St (10–40%), PE (10–15%), PC (13–22%), CL (47%), and lysophosphatidylethanolamines + PI (up to 13%) (Figure 4A).

**Figure 3.** The storage lipid composition in *E. magnusii* yeast in the different growth phases during long-lasting cultivation. (**A**)—the share of each storage lipid fraction,%; TAGs—triacylglycerols; DAGs—diacylglycerols; FFAs—free fatty acids; ESt sterol esters; Y1—unknown fraction (**B**)—the share of each storage lipid fraction (mg/g). <sup>a</sup>−<sup>h</sup> Means with the same letter did not differ significantly. Values are mean ± S.E.M from three independent experiments and three analytical replicates.

**Figure 4.** Membrane lipid composition of the mitochondria from *E. magnusii* cells raised in the different growth phases upon ageing during the seven-day cultivation. (**A**)—the share of each membrane lipid fraction; PE—phosphatidylethanolamines; PC—phosphatidylcholines; CL—cardiolipins; PS—phosphatidylserine; LPE + PI—lysophosphatidylethanolamine + phosphatidylinositols; LPC—lysophosphatidylcholines; SL—sphingolipids; St—sterols; X1, X2—unknown; (**B**)—the total membrane lipid content. a–m Means with the same letter did not differ significantly.

The PE fraction remained constant at the level of about 10% throughout the experiment with a concurrent increase up to 15% in the late stationary phase (168 h of cultivation). The PC fraction gradually increased up to 22% in the 48-h growth stage and nearly halved in the 168-h growth stage while the CL content reached its peak in the logarithmic growth stage (47%), decreased by more than two times in the 24-h stage (up to 18%) with a slight increase in the 48-h growth phase (up to 29.4%), and stabilized in the late stationary phase at 18%.

The LPE + PI fraction decreased during the cultivation from 13% to 4%. The St level changed in waves in different growth phases, reaching its maximum of 36–39% in the 24-h, 96-h, and 168-h growth phases (Figure 4A). The minor components of the lipid spectrum with the share of less than 10%, comprised lysophosphatidylcholines (LPC) and sphingolipids (SL). It is noteworthy that two unidentified lipid components were revealed: X1 was in the 24- and 48-h growth phases, and X2 was in 96- and 168-h growth stages.

The overall degree of acyl residue unsaturation, Index of Hydrogen Deficiency (IHD) in phospholipids determines the fluidity of the membrane lipid bilayer, which in turn may affect the adaptation and survival of the yeast under stress conditions, including ageing. Thus, we next assayed the FFAs composition and amount of the membrane lipids.

#### *2.5. FAs of the Main PLs in the Different Growth Phases*

To determine the degree of unsaturation, four main PLs fractions (PE, PC, CL, and PA), the share of which in the membrane lipids comprised more than 6%, were chromatographically isolated, and their FAs composition was analyzed. Figures 5 and 6 show the changes in the FAs composition and IHD of the mitochondrial membrane lipids in *E. magnusii*. The overall IHD in the membrane lipids was below 1.0 during the whole experiment. At the same time, the degree of unsaturation reached its maximum in the 48-h growth stage and its minimum at the 24-h growth stage (Figure 5B). Moreover, in the late stationary phase, the IHD was a bit higher than 0.6.

During long-lasting cultivation, the IHD changed due to significant alterations in the unsaturation of some fractions of the membrane lipids. Thus, IHD of PE remained at the same level in all the growth stages except for the 168-h stage, where it decreased sevenfold (Figure 5A). However, it was extremely stable in the PC and CL fractions, which dominate in the PLs of the mitochondrial membranes.

Dominating FAs in the mitochondrial PLs were palmitic (C16:0), oleic (C18:1), and linoleic (C18:2) acids (Figure 6A). In general, during the seven-day cultivation, we could observe the following consistent pattern: (1) palmitic acid (16:0) increased while the level of palmitoleic acid (C16:1) decreased; (2) oleic acid (C18:1) gradually increased with a sharp drop in the 168-h growth stage; and (3) the level of linoleic acid significantly decreased during cultivation being the highest in the early logarithmic stage (Figure 6A).

**Figure 6.** *Cont.*

**Figure 6.** *Cont.*

**Figure 6.** Fatty acid composition of the main membrane PLs in *E. magnusii* mitochondria in the different growth phases (**A**); PE—phosphatidylethanolamines (**B**); PC—phosphatidylcholines (**C**); CL—cardiolipins (**D**); PA—phosphatidic acids fraction (**E**). Values are mean ± S.E.M from three independent experiments and three analytical replicates.

The linoleic acid level in the PE fraction compared to that in the logarithmic stage decreased by 30%, 20%, and 63% in the 24-, 48-, and 96-h growth stages, respectively. Furthermore, by 168 h of cultivation, its level decreased by 26 times. On the contrary, the oleic acid level increased by 1.5, 1.96, and 2.3 times in the 24-, 48-, and 96-h growth stages, respectively, followed by more than a threefold decline in the 168-h growth stage (Figure 6B–E). The palmitic acid level in the PE fraction compared to that in the logarithmic stage doubled in the 168-h stage, but the palmitic acid level in the CL fraction remained constant of about 14–16% throughout the experiment. As for the oleic acid in the CL fraction, its level increased nearly twofold as the culture was ageing and reached 24–25% of the total fatty acid amount in the late stationary phase (Figure 6B–E).

At the same time, the linoleic acid level in the CL fraction decreased during the growth and ageing by about 10% (Figure 6B–E). As for PC, the palmitic acid level decreased by about two times in the 48-h stage and remained the same until the 96-h growth stage compared to that in the logarithmic stage. The amount of the oleic acid in the PC fraction increased by 2.3, 3.2, and 2.9 times in the 24-, 48-, and 96-h growth stages, respectively. Meanwhile, the linoleic acid level in the PC nearly halved during the growth and ageing of the culture. It is noteworthy that these FAs levels in the phosphatidic acids (PA) fraction also varied significantly. For example, the linoleic acid level decreased by more than 20% while the amount of the oleic acid doubled during the cultivation.

In the stage of the shift to the stationary growth phase, rather significant changes in the heptadecenoic acid (C17:1) level in the PE fraction were observed. Its amount increased by 6–8 times in the 96-h and 168-h growth stages compared to that in the logarithmic phase. Moreover, the main membrane PLs comprised negligibly small amounts of short-chain FAs, namely lauric acid (C12:0), myristoleic acid (C14:1), and pentadecenoic acid (C15:0).

#### **3. Discussion**

*E. magnusii* is a classical lower eukaryote of aerobic metabolism. It has a well-developed mitochondria system with a complete respiratory chain containing the invariable first coupling point [28]. It is capable of assimilating lots of substrates, including those substrates that determine the oxidative type of metabolism. The physiological features make significant advantages for the strain compared to the traditional *S. cerevisiae* yeast, used as a convenient tool to simulate processes in the unicellular eukaryotes model.

Earlier, we performed a systematic assay of the redox status of the *E. magnusii* yeast cultivated using two different types of substrates: a so-called "respiratory" substrate of glycerol and "fermentation" type substrate of glucose [22]. We revealed a significant difference in the survival of *E. magnusii* culture grown in glucose-containing medium, which showed a gradual decrease from 85% in the early stationary growth phase to 55% in the 168-h phase. The culture grown using glycerol showed a consistently high level of viability [22].

The data obtained suggest that the survival of the yeast culture during long-term cultivation and their high metabolic activity may be related to the active function of mitochondria. Their biogenesis is observed upon aerobic growth on non-fermentable substrates [22]. The yeast is capable of aerobic growth while using glycerol as a carbon source. The transition from "fermentative" to "respiratory" growth requires significant transcriptional rearrangements [29,30], which triggers the genes encoding the respiratory chain components, the elements of oxidative, osmotic, and general anti-stress responses, the enzymes of protein synthesis, glycerol assimilation and so on [20,31]. The mechanisms of yeast adaptation to various types of metabolism have been thoroughly studied, and it has been shown that 176 proteins of the *S. cerevisiae* mitochondria change while shifting from the fermentation type of metabolism (glucose, galactose) to the respiratory one (lactate) [32].

Analysis of the dynamics of the respiratory activity in *E. magnusii* mitochondria using the "oxidative" glycerol showed that it was maintained at a high level during the experiment (Table 1). It should be noted that the coupling parameters a bit decreased, however, the overall mitochondria functions kept up at a high level. The mitochondria AO is activated in the 96- and 168-h growth phases. The results agreed well with our published data on the dynamics of the ROS generation with the maximum in the 96-h growth stage [22]. Thus, the high ROS level in our studies could provoke the induction of the alternative cyanide-resistant pathway, which, in turn, is an element of the antioxidant defense system. This was shown in some papers [33–35]. The cyanide-resistant pathway of electron transport is not known to be related to energy storage and leads to a decrease in the ROS generation in the respiratory system, reducing the impact of the stress on the cell. Thus, this pathway induction is one of the ways for the cell to avoid excessive ROS generation under oxidative stress [36,37]. A large amount of storage lipids in the mitochondria, namely FFAs and DAGs, are considered as the second aspect of the high adaptation of *E. magnusii* upon long-lasting cultivation (Figure 3A,B). Abundant Lb in the yeast cells in the logarithmic (5–6 per a cell) and early stationary growth phases (6–7 per a cell), forming a structural complex with the mitochondria and nucleus (Figure 7A,B) testify to this.

The protective role of storage lipids, particularly TAGs and unsaturated FAs, is well known. TAGs can inhibit the yeast chronological ageing due to their accumulation in the Lb, which allows most of the unsaturated FAs to be deposited in TAGs by esterification [16,38]. In addition, the esterification of unsaturated FAs into TAGs slows down yeast chronological ageing by eliciting the liponecrotic cells. Some neutral lipids serve as a source of energy and precursors for membrane lipids synthesis [39]. The "model of secretory vesicles" based on the development of Lb from the secretory vesicles filled with TAGs is one of the possible models for forming Lb in a yeast cell. Lbs are usually about 300–400 nm in diameter and covered with a PL monolayer containing few proteins [39], mainly the enzymes of lipid metabolism [40]. Of note, the ratio of TAGs to ESt in Lbs from *S. cerevisiae* is usually 1:1 [41], and their TAGs core is surrounded by several ESt envelopes [42].

**Figure 7.** Image of *E. magnusii* yeast cells in the logarithmic (**A**) and early stationary growth phases (**B**). Nu—nucleus; Mt—mitochondria; Lb—lipid bodies.

In our studies, we found TAGs as a minority (about 4% of the total lipids) only in the mitochondria of the logarithmic growth stage (Figure 2A). During cultivation, they were replaced by FFAs with the highest level in the 24-h stage. DAGs kept up at about the same level (about 10–15%) throughout the experiment. It is probable that such an alteration in the storage lipids composition in the mitochondria is due to the depletion of the storage lipid resource during growth. In a yeast cell, there are three main pathways for synthesis of DAGs: (1) PA removal by phosphatidate phosphatase (*PAH1*); (2) degradation of phospholipids by phospholipases (*LRO1*, *DGA1*, *ARE1/2*); and (3) deacetylation of TAGs (*TGL2/3/4/5*, *AYR1*) [39]. We suppose that upon intensive growth, the third pathway of DAGs formation seems preferable, taking into account the disappearance of TAGs in the stationary growth phase.

At the same time, the high FFAs level in the *E. magnusii* mitochondria could be related to the presence of a minority of ESt (Figure 2A). The FFAs synthesis in yeast occurs via three main pathways: (1) de novo synthesis, (2) the complex and storage lipid degradation, and (3) external uptake [39]. Taking our data altogether, including the growing conditions of *E. magnusii*, we posit that in our experiments, the FFAs synthesis occurs de novo in the cytosol and mitochondria with a concurrent elongation and desaturation in the ER. The initial stage of the FFA synthesis is triggered by the acetyl-CoA carboxylase (the cytosolic enzyme is encoded by *ACC1*, the mitochondrial one is by *HFA1*). In this reaction, acetyl-CoA is carboxylated and forms malonyl-CoA serving as a two-carbon building block for the next FFAs synthesis reactions [39].

CL performs a lot of cell functions, being associated with all the major proteins of the mitochondrial respiratory system, and thereby increases the efficacy of the electron flux and the ADP/ATP exchange [40]. This PL corrects the catalytic activity and stability of the interactive proteins [41], is crucial for the biogenesis of mitochondria proteins [42], promotes mitochondrial fission/fusion [38], and participates in the cristae structure and morphology formation [43]. According to some researchers' data [43,44], the CL amount in the mitochondrial fraction of *S. cerevisiae* reaches 7.2% of the total membrane PL one, while for the mitochondria from *E. magnusii*, its level was much higher. In the logarithmic growth stage, the CL fraction made up about 50%, and in the stationary ones, it was not less than 18–20% (Figure 3A). It is probable that such a permanently high level of the CL fraction provides the high mitochondrial activity. In 2012, *Rostovtseva and Bezrukov* [45] showed that CL-rich areas of the outer mitochondrial membrane exhibited a higher activity of the mitochondrial VDAC porin involved in eliminating the ROS from the mitochondria. The CL fatty acids composition impacts VDAC activity greatly. This fact can be considered as the third most important point of the lipid composition adaptation in the yeast mitochondrial upon long-term cultivation.

Nevertheless, it is noteworthy that the PLs composition of the mitochondria in *E. magnusii* differs significantly from that in *Saccharomycetes*, where the dominating PLs fractions were PC, PE, and PI. Their shares made up 40.4% (PC), 26.7% (PE), and 14.6% (PI) [46,47]. In the *E. magnusii* mitochondria, these fractions are not dominating and reach no more than 10–20% of the total PLs amount (Figure 3A). However, among the major components, besides the CL fraction, the St share made up 40% in some samples. St are essential for supporting membrane integrity and eukaryotic cell viability. Taking into account our results on the "LB + MITO + Nucleus" complex in *E. magnusii* cells (Figure 7), we could speculate that the St fraction plays a crucial role in the functional interaction of the organelles upon ageing (Figure 2F–D).

Finally, the composition of FFAs in PLs is another important point of the lipid profile adaptation in the yeast mitochondria to the ageing processes. The main findings while assaying the FFAs composition of the mitochondria membrane lipids upon ageing were: (1) a smooth increase in the level of the palmitic acid (C16:0) against a fall in the palmitoleic acid (C16:1); (2) a gradual increase in the oleic acid amount (C18:1) with a sharp drop in the 168-h growth phase; and (3) a significant increase in the stearic acid level (C18:0) with a simultaneous decrease in the linoleic acid level (C18:2) throughout the whole experiment (Figure 5A).

Earlier, *Kieliszek* et al. [48] and his team showed a significant increase in the margarine (C17:0) and heptadecenoic (C17:1) acids level in the *Candida utilis* yeast grown either using 5% glycerol as a carbon source or enriched with selenium (20 mg/L) by two times and by 25%, respectively. Additionally, abundant margarine (C17:0; 12.19%) and heptadecenoic (C17:1; 9.31%) FAs were found in the *Yarrowia lipolytica* yeast when it was cultivated in batches using glycerol as a carbon source. Our findings showed that in the extremophilic *Y. lipolytica* cells under alkaline stress, the CL fraction had similar changes, namely the amount of saturated acyl residues in it increased [49]. An increase in the saturated FAs amount in the mitochondria of the culture under stress is likely to provide the integrity and rigidity of the membranes according to the homeoviscous adaptation hypothesis, which suggests these changes in the membrane lipid profile should facilitate its necessary fluidity [50].

#### **4. Materials and Methods**

#### *4.1. Yeast Strain and Culture Conditions*

*E. magnusii* yeast VKM Y261 strain was grown in batches of 100 mL in glycerol- (1%) containing media of the following composition (g/l): MgSO4-0.5, (NH4)2SO4-0.3, KH2PO4-8.6, NaCl-0.1, CaCl2-0.05, yeast extract–2.0, *L*-histidine–2.75 mg, *L*-methionine–2.75 mg, and *L*-tryptophan–2.75 mg at 28 ◦C as described previously [51]. Absorbance was assessed in cell suspension at the wavelength of 590 nm (A590) using a Specol-11 spectrophotometer (Carl Zeiss, Oberkochen, Germany). Cells were harvested at different stages of growth: logarithmic (A590 = 2.6–2.7), early stationary (24 h of growth, A590 = 4.0–4.1), late stationary (48 h of growth, A590 = 4.5–4.6), deep stationary 1 (96 h of growth, A590 = 4.4–4.7), deep stationary 2 (168 h of growth, A590 = 4.4–4.7).

#### *4.2. Potential-Dependent Staining*

Potential-dependent staining of mitochondria in the *E. magnusii* cells raised in the different growth phase by JC-1. Cells were incubated with 0.5 μM JC-1 and examined in 0, 15, 20, and 30 min. Incubation medium contained 0.01 M PBS, pH 7.4; 1% glycerol or glucose, respectively. Regions of high mitochondrial polarization are indicated by red fluorescence due to the concentrated dye. To examine the JC-1-stained preparations, filters 02, 15 (Zeiss, Oberkochen, Germany) were used (magnification ×100). The photos were taken using an AxioCam MRC camera (Microvisioneer, Esslingen am Neckar, Germany)

#### *4.3. Staining with Neutral Red*

Yeast cells were suspended in PBS, and a 200 μL sample of the cell suspension was mixed with 100 μL neutral red (0.1 mg/mL stock solution, dissolved in a 2% dihydrate sodium citrate solution) and incubated for 5 min at room temperature. Viability was examined under a light microscope using Gorjaev's chamber (×400) from at least 1.000 cells in one biological replicate. Viable cells were colorless, and dead ones were red.

#### *4.4. Transmission Electron Microscopy (TEM)*

TEM analysis of untreated *E. magnusii* yeast cells was performed as described previously [15]. Briefly, the yeast cells were raised in the logarithmic or stationary (24 h) growth phase, precipitated, fixed with 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) for 2 h, and then post-fixed in 1% OsO4 for an hour at room temperature. After dehydration, the samples were embedded in Epon 812. Ultrathin sections were prepared with an LKB-8800 ultratome using diamond knives. Thereafter, the sections were stained with uranyl acetate for 60 min and post-stained as described previously, and examined with a Jeol (JEM-100B) and Hitachi U-12 electron microscopes (Hitachi, Tokyo, Japan).

#### *4.5. Isolation of Mitochondria*

Mitochondria were isolated using the method described in [52] with minor modifications. The mitochondria thus obtained met all known criteria of physiological intactness, as inferred from high respiratory rates and ADP/O ratios close to their theoretically expected maxima. Mitochondria were fully active for at least 4 h after being isolated when kept on ice. Briefly, cells were harvested at different growth phases, washed twice with ice-cold water, resuspended (0.1 g wet cells/mL) in pre-spheroplast buffer (50 mM Tris-HCl buffer; pH 8.6, 4 mM dithiothreitol), incubated at room temperature for 10–15 min, then diluted with ice-cold water, pelleted at 3000× *g* for 10 min, washed twice to remove excess dithiothreitol, and incubated at 28 ◦C using gentle stirring for 15–20 min in spheroplast buffer (10 mM HEPES-buffer, pH 7.2, 1.1 M sorbitol) with Novozym 20 T from *Trichoderma harzianum* (Sigma-Aldrich, St. Louis, MO, USA) (2.5 mg/g cells) and lytic enzymes from snail gut juice (50 mg/g cells). Spheroplast formation was monitored by measuring the osmotic fragility in distilled water. The spheroplasts were rapidly cooled, pelleted by centrifugation at 3.000× *g* for 10 min, washed gently twice in post-spheroplast buffer (1.2 M sorbitol, 5 mM MgSO4, pH adjusted to 7.2), resuspended (0.1 g wet cells/mL) in grinding buffer (10 mM Tris-HCl, pH 7.2, containing 0.4 M mannitol, 1 mM EDTA, 0.05 mM EGTA, and 4 mg/mL BSA), and disrupted in an all-glass *Dounce* homogenizer (Kontes, Vineland, NJ, USA) with a low clearance pestle. The suspension was diluted with isolation buffer (10 mM Tris-HCl, pH 7.2, 0.6 M mannitol, 0.05 mM EDTA, 0.05 mM EGTA, and 4 mg/mL BSA) and centrifuged at 2000× *g* for 10 min. The supernatant was centrifuged once more at 7000× *g* for 20 min. The resulting pellet was washed in 10 mM Tris-HCl; pH 7.2, containing 0.6 M mannitol and 4 mg/mL BSA, resuspended in a smaller volume of the same buffer, and used immediately.

Mitochondrial protein was assayed by the method of Bradford with bovine serum albumin as the standard.

#### *4.6. Respiration Assessment*

Oxygen consumption by the yeast cells was assessed in vitro at 25 ◦C using electrodes covered by fluoroplastic film at a constant potential of 660 mV. Analysis of respiratory activity was performed using a multichannel microelectrode polarograph with data-analysis software Record-4 (Institute of Cell Biophysics of the Russian Academy of Sciences, Puschino, Russia). Oxygen consumption in mitochondrial suspensions was monitored polarographically with a Clark-type electrode in a medium containing 0.6 M mannitol, 1 mM Tris phosphate (pH 7.4), 1 mM EDTA, 20 mM pyruvate, 5 mM malate, and mitochondria corresponding to 0.4 mg protein/mL. All shown data traces are representative of four to six replicates.

#### *4.7. Preparation and Analysis of Lipids*

The lipids were extracted from the mitochondria by the Nichols method described in [53], which involved extraction with isopropanol and the isopropanol–chloroform mixture (1:1 and 1:2) at 70 ◦C, evaporation in a rotary evaporator, and extraction of the residue with chloroform-methanol (1:1) supplemented with 5% sodium chloride solution and water to remove water-soluble substances. After separating the mixture with a vortex, we dried the chloroform layer by passing it through water-free sodium sulphate, evaporated, and desiccated with a vacuum pump. The resulting pellet dissolved in a small amount of chloroform-methanol (2:1) was stored at −21 ◦C. The composition of storage lipids was assayed using an ascending thin layer chromatography on glass plates with silica gel 60 (Merck KGaA, Darmstadt, Germany). To separate storage lipids, the hexane: sulphuric ether: acetic acid (85:15:1) system [54] was used. To separate PLs and SLs SI60 Silica thin layer chromatography plates were activated and developed in two dimensions, first with chloroform/methanol/water (65/25/4, by volume) and second with chloroform/acetone/methanol/acetic acid/water 50/20/10/10/5, by volume) [55]. The lipids (100–200 μg) were applied to a plate. Lipid quantities were determined using the following standards: PC (Sigma, Saint-Luis, MO, USA) for PLs, a glycoceramide mixture (Larodan, Solna, Sweden) for SLs, and ergosterol (Sigma, Saint-Luis, MO, USA) for St. Samples of glycoceramides (5 and 10 μg) and PC (10 and 20 μg) were applied on the plates in the second direction. To develop the stains, the chromatograms were sprayed with 5% sulfuric acid in ethanol, followed by heating up to 180 ◦C. Quantitative densitometric analysis of the lipids was performed using the Dens software package (Lenkhrom, Sankt Peterburg, Russia) in the linear approximation regime using the calibration curves constructed with the standard solutions. Lipid data are presented in μg/g mitochondrial protein. PLs were identified using individual markers and qualitative tests for amino groups (with ninhydrin), choline-containing phospholipids (with the Dragendorff reagent), and glycolipids (with α-naphthol). Neutral lipids were identified with individual markers for MAGs, DAGs, and TAGs, St (ergosterol), FFAs, and hydrocarbons (Sigma, Saint-Luis, MO, USA). SLs were detected in the glycolipid fraction by the saponification method [56].

To assess the FAs composition of PLs, separate PLs were isolated using TLC with two plates, eluted with chloroform/methanol (1/1, *v/v*) for a night. Then, the supernatant was decanted, evaporated, 1 mL toluene and 2 mL of 2.5% H2SO4 dissolved in methanol and kept for two hours at +70 ◦C. FAs methyl ethers were extracted with hexane, dried, and analyzed by a Kristall 5000.1 gas chromatograph (Chromatek, Moscow, Russia) using an Optima-240 (60 m × 0.25 mm) capillary column (Macherey-Nagel GmbH and Co., Dueren, Germany). The temperature program was set from +130 to +240 ◦C. Eluting FAs were identified using the Supelco 37 Component FAME Mix (a mixture of FA methyl esters) (Supelco, Bellefonte, PA, USA). The degree of unsaturation was calculated by the equation: Δ/mole = 1.0 × (% monoene)/100 + 2.0 × (% dienes)/100 + 3.0 × (% trienes)/100 [57].

#### *4.8. Statistical analysis*

The experiments were performed in biological triplicates with a standard error of less than 5%. The influence of pH and temperatures on soluble carbohydrates and lipids was estimated using one-way ANOVA with R (R Core Team 2016). The significance of differences between the mean values in each group was tested by Tukey's test. Values were considered significant at *p* < 0.05.

#### **5. Conclusions**

The recent review by *Medkour* et al. [15], using the correlation profiling of lipidomes in various tissues of long- and short-lived mammalian species, stated the following tendencies of so-called "lipidomic signature" for increasing longevity and delayed ageing in mammals and humans: (1) A decrease in the degree of FAs unsaturation, which reduces both double bonds and the peroxidation indices of various lipid classes. (2) Declined concentrations of long-chain FFAs. (3) An increased ratio of monounsaturated (MUFA) to polyunsaturated (PUFA) FAs. (4) Decreased levels of some SLs, some LPCs, and PCs, as well as highly polyunsaturated TAGs and DAGs (5). An increased amount of some sphingomyelins and cholesterol esters, as well as TAGs and DAGs with low IHD. The authors concluded that identifying the key trends in the lipidomic signature is an essential first step towards the detection of lipid biomarkers for healthy ageing and extended life span. However, whether any of the trends mentioned above have a cause-and-effect relationship in slowing the ageing process and increase in life expectancy should be determined. Summarizing the obtained results, we can conclude that, with respect to yeast mitochondria, the first and second statements, and partially the fifth one, are doubtlessly true for the long-lasting cultivation and the initial stages of ageing in the lower eukaryotes using an "oxidative" type glycerol. Moreover, the list of the factors favoring a long lifespan should include some other physiological parameters of yeast cells. The mitochondrial AO activity induced in the early stationary growth phase and high mitochondrial activity maintaining intensive mitochondrial respiration, which in turn determines the ATP production and physiological doses of ROS that should be added to the list of the trends providing increased longevity.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/app11094069/s1. Table S1: The dinamics of different cell types, Figure S1: Micro images of transmission electron microscopy of the *E. magnusii* mitochondria fraction, Figure S2: A - Amperometric recording of oxygen consumption by the E. magnusii mitochondria respiring on pyruvate + malate. Numbers adjacent to traces are respiration rates in ng-atoms of O/min/mg of mitochondrial protein. The incubation medium contained 0.6 M mannitol, 0.2 mM Tris-phosphate, pH 7.2; 20 mM pyruvate + 5 mM malate as respiratory substrates, and mitochondria corresponding to 0.5 mg mitochondrial protein, added at MITO. The frame in the Figure (a) shows the curve of oxygen consumption without substrate application (endogenous respiration). B - Recording of ΔΨ generated by the *E. magnusii* mitochondria respiring on a 20 mM pyruvate + 5 mM malate. The incubation medium contained 0.4 M mannitol, 0.1 M KCl, 20 mM Tris-acetate, 0.4 mg of mitochondria protein, pH 7.4. C – the demonstration of the adenilate kinase activity in the intact mitochondria (the incubation medium composition is as in A), Table S2: The phosphorylating activities of the mitochondria using different substrates, Figure S3: Effect of erythromycin (A) and ethidium bromide (B) on the *E. magnusii* cell growth.

**Author Contributions:** Conceptualization: E.P.I.; methodology: E.P.I., N.N.G., and D.I.D.; validation: V.M.T.; formal analysis: M.K.; resources: E.P.I. and V.M.T.; writing—original draft preparation: E.P.I.; writing—review and editing: M.K.; supervision: Y.I.D.; project administration: E.P.I. and Y.I.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Review* **Nutritional and Health Potential of Probiotics: A Review**

**Muhammad Modassar Ali Nawaz Ranjha 1,\*, Bakhtawar Shafique 1, Maria Batool 2, Przemysław Łukasz Kowalczewski 3,\*, Qayyum Shehzad 4,5, Muhammad Usman 5, Muhammad Faisal Manzoor 6, Syeda Mahvish Zahra 1,7, Shazia Yaqub <sup>1</sup> and Rana Muhammad Aadil 8,\***


**Abstract:** Several products consist of probiotics that are available in markets, and their potential uses are growing day by day, mainly because some strains of probiotics promote the health of gut microbiota, especially *Furmicutes* and *Bacteroidetes*, and may prevent certain gastrointestinal tract (GIT) problems. Some common diseases are inversely linked with the consumption of probiotics, i.e., obesity, type 2 diabetes, autism, osteoporosis, and some immunological disorders, for which the disease progression gets delayed. In addition to disease mitigating properties, these microbes also improve oral, nutritional, and intestinal health, followed by a robust defensive mechanism against particular gut pathogens, specifically by antimicrobial substances and peptides producing probiotics (AMPs). All these positive attributes of probiotics depend upon the type of microbial strains dispensed. Lactic acid bacteria (LAB) and *Bifidobacteria* are the most common microbes used, but many other microbes are available, and their use depends upon origin and health-promoting properties. This review article focuses on the most common probiotics, their health benefits, and the alleviating mechanisms against chronic kidney diseases (CKD), type 1 diabetes (T1D), type 2 diabetes (T2D), gestational diabetes mellitus (GDM), and obesity.

**Keywords:** probiotics; health benefits; alleviating mechanism; CKD; T1D; T2D; obesity

#### **1. Introduction**

Good health is a fundamental need of human beings [1]. Humans' priority was to rely on natural resources for good health outcomes [2–5]. In the 20th century, it was observed that healthy children fed mothers' milk had *Bifidobacteria* in their gut microbiota [6]. It established a positive association between health and gut microbiota, and numerous studies have been conducted to investigate the proper mechanism of this association, and some found that some bacteria have a positive correlation with health [7]. The term probiotics were first used in 1965 by Stillwell and Lilley to describe these beneficial bacteria [8]. At the beginning of the 20th century, a Nobel laureate in Paris named Élie Metchnikoff, a professor

**Citation:** Ranjha, M.M.A.N.; Shafique, B.; Batool, M.; Kowalczewski, P.Ł.; Shehzad, Q.; Usman, M.; Manzoor, M.F.; Zahra, S.M.; Yaqub, S.; Aadil, R.M. Nutritional and Health Potential of Probiotics: A Review. *Appl. Sci.* **2021**, *11*, 11204. https://doi.org/10.3390/ app112311204

Academic Editors: Patrizia Messi and Anabela Raymundo

Received: 26 September 2021 Accepted: 23 November 2021 Published: 25 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

by profession noticed the health-enhancing properties of fermented dairy products. He then reported that the presence of lactic acid bacteria in fermented dairy products helps keep the defensive system activated, resulting in higher longevity of its consumers [9].

Some crucial characteristics of probiotics were introduced in the 1980s, and these include: (a) strains to have a beneficial impact, (b) non-toxic, non-allergic, and nonpathogenic, (c) available in large quantity as viable cells, (d) suitable for the environment of the gut, and (e) storable as well as stable [10]. Although the most commonly used probiotics are bacteria, especially lactic acid, molds and yeasts can also be probiotics [11]. In 2001, the Food and Agriculture Organization and the World Health Organization council defined probiotics, which was later refined in 2014 by Hill et al. as "live micro-organisms that, when administered in adequate amounts, confer a health benefit on the host," which can be understood to mean that probiotic strains must be (i) sufficiently characterized; (ii) safe for the intended use; (iii) supported by at least one positive human clinical trial conducted according to generally accepted scientific standards or as per recommendations and provisions of local/national authorities when applicable, and (iv) alive in the product at an efficacious dose throughout shelf life [12]. Examples include living bacteria containing *Lactobacillus*, *Bifidobacterium*, and some others [13].

From the start, probiotics have been considered beneficial to the health of gut microbiota, studies have also confirmed their positive associations with many other chronic diseases [14]. Although *lactobacillus* is the most commonly used probiotic, many other beneficial microbes are present, and their usage depends upon their origin and healthfriendly properties [15]. All bacterial strains have specific properties; some are useful in treating obesity, some treat diabetes mellitus (DM), some help with CKD, and some deal with osteoporosis. In addition to these highly prevalent diseases, several studies have confirmed the beneficial role of probiotics in the case of autism, irritable bowel syndrome (IBS), and wound healing [16,17].

Probiotics have also been investigated to promote oral health and strengthen the immunological system [18–20]. Additionally, they are an essential contributor in the field of agriculture as well as in food processing [21].

In this article, we review and discuss some essential probiotics that can be useful for human health, how they affect an individual's nutritional status, and how probiotics perform their action to prevent disorders. In addition, the health-promoting role of probiotics and action mechanisms in the case of several of the most prevalent diseases are reviewed.

#### *1.1. Nutritional Impacts of Probiotics*

Lactic acid bacteria (LAB) most commonly ferment fruit, vegetables, and cereals. It impacts taste, and, when fermenting rice bran, sprouts of bean sprouts, and buckwheat, it also produces bioactive components that benefit inflammatory, compromised immunity, glycemic imbalance, and fatigue conditions [22].

#### *1.2. Nutritional Impacts of Probiotics in Inflammation*

Currently, people are highly concerned about their health. There is an increasing focus on disease prevention compared to cures. The consideration of probiotics and their health benefits began about a century ago. It was discovered that the people of Bulgaria and Russia lived longer than other populations because they use sour milk that contains beneficial bacteria [23]. Probiotics play an important role in preventing and improving diseases like allergies, liver disorders, intestinal ailments, and metabolic syndromes leading to diabetes, cardiovascular diseases, and obesity [24]. *Escherichia coli* and LAB are extensively used to treat inflammatory bowel disease, colon cancer, and constipation because lactic acid bacteria directly deliver cytokines to the target sites within the host [21,25,26]. Investigators indicated a significant inhibiting role of non-pathogen apoptosis induction within carcinoma cells, which helps protect against colon cancer (HGC-27) and human colonic cancer cells (Caco-2, DLD-1, HT-29), especially with the action of *Escherichia coli* K-12 strains, *Lactobacillus rhamnosus*, and *Bifidobacterium latis* [27].

#### *1.3. Nutritional Impacts of Probiotics in Dental Carries*

LAB probiotics in cheese seem to reduce the number of mutant streptococci in saliva and therefore have a beneficial impact on dental carries; the LAB in cheese also prevents the demineralization of enamel and dental plaque [28]. However, the decrease in streptococci number is independent of the strain used, and the effect is not the same in all studies. Therefore, no exclusive report has been made on dental carries [29].

The health of an individual depends upon the status of a healthy microbiota. Pathogenic bacteria cause inflammation during acute infection, whereas symbiotic bacteria regulate the immune responses to such inflammations and protect the host from different diseases [30].

#### *1.4. Nutritional Impacts of Probiotics in Obesity, Diabetes and Associated Issues*

Gut bacteria play a vital role in the pathogenesis of bile duct diseases. Probiotics are also used for the treatment of liver diseases [31,32]. The result of a study on a boy suffering from primary sclerosing cholangitis treated with the combination of drugs and probiotics showed improvement in symptoms and laboratory tests [33]. According to the result of a study on a group suffering from type 2 diabetes mellitus, eating yogurt that contains probiotics (*Lactobacillus acidophilus* La5 and *Bifidobacterium lactis* Bb12) improved fasting glucose level and antioxidant ability, and it was therefore concluded that probiotic yogurt has a positive impact on patients with type 2 diabetes [34]. *Bifidobacteria* play a role in the development of obesity. Mothers who are overweight give birth to neonates with low levels of *Bifidobacterium*. A low number of *Bifidobacterium* at birth is associated with overweight issues later in childhood. The *Bifidobacterium* level in obese adults is lower as compared to that in lean people [35]. The gut microbiota provides opportunities for the fermentation of non-digestible compounds like fibers that support the microbes that produce short-chain fatty acids and gases [36]. The short-chain fructo-oligosaccharides increase intestinal magnesium absorption in postmenopausal women in whom magnesium deficiency leads to osteoporosis [37].

Probiotics also prevent and treat gastrointestinal inflammatory conditions like inflammatory bowel diseases, irritable bowel syndrome [38], allergies, and respiratory disorders [39]. When probiotics were administered in pregnant women, no increase or decrease in the risk of preterm birth and other pregnancy-related adverse effects was seen [40]. *E. coli*, *Staphylococcus aureus*, *Bifidobacterium*, and *Firmicutes* have been reported by researchers to have an impact on obesity, thus can be used as obesity inhibitors [41]. Type 2 diabetes (T2D) is one of the most prevalent diseases globally that can be cured by using probiotics as an adjuvant. It was proved that probiotics exhibit anti-diabetic and anti-inflammatory properties and therefore play a vital role in diabetes prevention and treatment. Probiotics have also been proven beneficial in autistic children, especially for GIT disturbance caused by this disorder. Bone mineral density has also been proven to be affected by these gut microbes, as these microbes lead to enhanced absorption of various nutrients and minerals. These microbes improve human health and improve the health of plants and crops, increasing the overall yield of the land [42].

Traditional probiotics have marginal ameliorative effects on diseases, so the next generation probiotics (NGP) now serve as new preventive and therapeutic tools. These NGP include *Prevotella copri* for controlling insulin resistance, *Akkermansia muciniphila*, and *Bacteroides thetaiotaomicron* that are used to reverse obesity and insulin resistance, and *Bacteroides fragilis* is used to reduce inflammation and facilitates anticancer effects [43].

#### **2. Probiotics and Their Benefits**

The use of probiotics in the form of live bacteria for health promotion in animals and human beings is emerging daily. Today, a vast array of fermented food items and beverages is available, accounting for approximately one-third of worldwide human diets [44]. The level of probiotics in foods range from 2 to 20 g/day depending on the component and desired effect and can be added to different food products, including cereals, biscuits, bread, sauces, yogurts, and drinks [45]. Curd is considered the most preferred source of

probiotics, as it is globally consumed [46]. Interest and development of functional foods consisting of both probiotics and prebiotics have increased due to increased awareness of their health-promoting properties. They positively affect gut health and decrease the risk of diseases, which is why they are used as therapy [47].

All probiotic products have different nutritional and therapeutic characteristics, due to various conditions, such as the genetic make-up of the strain, amount of the probiotics used in the product, the purpose it is used for, and its shelf life. The selection of probiotic strain depends on its production, impact, and health benefits in the host [48]. A probiotic food must have 106 CFU/g of probiotic micro-organism to achieve a health benefit. The dosage recommended for human consumption is 107–109 CFU/mg/day. It is known that the effect of probiotic food consumption depends on the specific strain used in that product [49]. To get the beneficial strains of probiotics, the following genera are of great importance: *Lactobacillus*, *Escherichia coli*, *Bifidobacterium*, *Enterococcus*, *Saccharomyces*, *Pediococcus*, *Streptococcus*, and *Leuconostoc*. The most common micro-organisms used as probiotics are lactic acid bacteria (LAB) and *Bifidobacteria* [50].

Strains of lactobacillus species that are commonly found in saliva samples include *L. paracasei*, *L. plantarum*, and *L. rhamnosus*. The *bifidobacterial* species are the anaerobes that are also found in the oral cavity, and both species are found in breast milk and are generally regarded as safe [51]. Several elements, such as nutrition, age, environmental difficulties, incompatibilities, illnesses, and treatment routes, strongly impact gut microbiota growth, maintenance, and functionality [52]. When selecting the strain it must be kept in mind that it should be originated from target and natural microflora as it is vital for its survival in an acidic environment during its travel to the intestine [53,54].

Probiotics have a positive effect on the immune system of the host and it was proven that they influence the healthy bacteria present in the gut or intestine [55]. Probiotics improve the immunity system by modifying the humoral and cellular immune response [56]. Figure 1 summarizes the benefits of different probiotics on health.

**Figure 1.** Benefits of probiotics on health [57].

The strains that are being selected also depend on the biosafety level, which means they should not be toxic or pathogenic [58]. They should be tested for safety parameters, including antibiotic susceptibility, antibiotic resistance gene, and hemolytic activity. Antimicrobial production is an important feature of probiotics against pathogens, but non-optimal antimicrobial activity will disrupt the healthy microbiota in the intestines and pose harmful effects [59]. Bile present in higher concentrations resulted in the lower growth of strains [60]. Probiotics can alter the pH of the surrounding environment, and hence they can compete with the pathogens present there.

In the same way, probiotics adhere to the adhesion sites on the mucosa, which decreases the chances of adhesion of pathogens and decreases the cases of probiotics being washed out [61]. Different tests are performed to identify the strain of bacteria, such as biochemical and molecular tests, then further techniques are used to differentiate between the strains of the same species, such as polymerase chain reaction (PCR) and gene sequencing tests [62,63]. Other tests are performed, such as hemolytic tests to determine whether the organisms are destroying red blood cells or not and platelet aggregation tests, which are crucial factors for pathogen activity [64]. Recommended storage temperature for probiotic foods is 4–5 ◦C, and the product must be used according to the information noted on the label, which should be clear [65].

#### *2.1. Significant Effects of Probiotics on Oral Health Status*

Probiotics primarily contribute to gastrointestinal health, but their diversity has been increasing and now they greatly contribute to oral health care [66]. Oral disease treatment is one of the most expensive health care treatments; that is why new approaches are being used to treat these infections, such as the use of probiotics [67]; in other words, oral lactic acid bacteria have been characterized after isolation and purification to use for oral health, especially to cure periodontal diseases, caries, and halitosis (bad breath) [19].

These ailments are some of the most widespread diseases throughout the world, but the treatment of these infections require potent antibiotics that cause severe side effects in the gastrointestinal tract. The use of probiotics as a relatively less harmful treatment has been advised to avoid such side effects [68].

Additionally, the buccal cavity is an excellent bacteriological medium, where microorganisms can grow very easily if certain factors are out of balance due to poor oral health, poor diet, or immunodeficiency. These parameters alter the pH of the mouth and create a favorable environment for micro-biota growth. Micro-organisms are deposited on the oral cavity like biofilm and create a bacterial hold and aggressively colonize the buccal cavity [69].

Probiotic strains of *Bifidobacterium*, *Lactobacillus*, and *Streptococcus* are primarily used to prevent or cure oral infections. Plaque or dental biofilms in the buccal cavity leads to poor oral health, but lactic acid bacteria are probiotics that interact with that biofilm/plaque and destroy the causative agents with LAB's antimicrobial activity [70].

Indirectly, probiotics enhance immunity and strengthen the immune system by the interaction of lactic acid bacteria with immuno-competent cells, which leads to modification in the production of cytokines and ensuing effects on the overall immune system [71]. Lactobacilli used as a probiotic in mild gingival inflammation causes a great decline of Interleukin-8 discharge in the gingival crevicular fluid [72]. Probiotics have been used in the treatment of periodontitis and gingivitis: they modify the buccal cavity homeostasis, with possible numerous health benefits such as combatting dysbiosis and moderating inflammatory pathways to minimize the inflammation of periodontitis [73,74].

Probiotics are also being used in the treatment of halitosis; for this purpose, bacteriocins producing *Streptococcus salivarius* K12 strain were developed. This strain treats the mouth odor by actively helping de-colonize bacteria like *Solobacterium moorei*, *Parvimonas micra*, and *Eubacterium sulci*, which produce volatile sulfur compounds(VSCs) on the tongue [75,76].

A new study demonstrated the effects of widely commercially used chewing gum containing probiotic bacteria (xylitol), which greatly impacted plaque and gingival scores by reducing *Streptococcus mutants* counts in plaque and saliva [77]. Probiotics played an essential role in the clinical manifestation of dental disorders and periodontal diseases and perhaps impacted halitosis as well. Probiotics provide long and short-term therapeutic effects. Advance studies about particular strains and oral micro-biome transplants could also increase probiotics' role or efficacy in oral health, and probiotics consumed as part of the diet may also enhance oral health and strong oral immunity or hygiene [78,79].

#### *2.2. Improvement of Intestinal Health through Probiotics*

Probiotics are known as important micro-organisms that are used to improve intestinal health, including beneficial micro-organisms known to retard the production of bacterial enzymes that cause colon cancers [80]. Colonic microflora is very important for human health [81]. These bacteria promote the normal functioning in the intestine and maintain the host's health [82]. Consumption of pre and probiotics alters gut microbiota by facilitating or inhibiting microbe growth [83].

These bacteria first colonize the intestinal track and then help establish the immune system with balanced cell responses [84]. During pregnancy, prebiotics and probiotics are supplemented in pregnant females to protect the fetus from many autoimmune diseases and syndromes (AIDS). These supplements immunize the child and protect against AIDS [85]. Recent investigations have shown that human intestinal microbiota comprises over 1000 microbes [86]. In one study performed on obese and diabetic patients, scientists concluded that their gut microbiota differs from non-diabetic patients and this modification occurred as a result of dietary changes of diabetics versus non-diabetics [87]. Several studies have shown that the microbiota influences energy homeostasis and controls body weight [88].

Drug and probiotic interference are well known; studies have revealed the probiotics and warfarin interaction. Intestinal bacteria are well known for the production of vitamin K, but absorption of antibiotics disturb the gut flora and result in the deficiency of vitamin K [89]. According to the study, many external factors like medication, radiotherapy, stress, and infections disturb the gut microflora growth and their ratios [90]. Other essential functions played by probiotics include improvement in flu/influenza; protection from dental caries; prevention of tonsillitis, respiratory infections, and urogenital health problems; and help in wound healing and throat infection through immunomodulatory action. Currently, probiotics are being sold in markets as dietary supplements proven to be so-called standard drug therapy [91].

#### *2.3. Role of Probiotics in Development of Immunity*

Probiotics and prebiotics exert beneficial effects on humans' health, including immune modulating capacity, modulation of cellular metabolism, epithelial barrier functions, or proliferation [92]. The growth of the gut microbiome is a dynamic process, and early colonization of *Bacteroides* and *Bifidobacterium* species might play a crucial role in immune regulation. Factors that can affect the early colonization of gut microbes in neo-nates include the mother's diet, antibiotic treatments, method of delivery, and surrounding environment [93]. On the surface of immune cells, Toll-like receptors (TLR) behave differently depending on the immune system's response, which allows distinguishing between pathogenic and native gut microbiota. Probiotics and their effector molecules affect the gut barrier by different methods such as modulation of mucus production, reduction in bacterial adhesion, and induction of IgA [94].

#### **3. Association of Probiotics in Prevention of Diseases**

Probiotics are very helpful in preventing chronic diseases by mediating their effects. They show positive effects on gut health and help in skin-related problems such as burns, scars, infections, wounds. They increase the skin's innate immunity and help to regenerate healthy skin [95]. The action of Saccharomyces cerevisiae dressing improved burn skin healing significantly as demonstrated by [96], whereas a hypothetical model of intestinal microbiota influencing wound healing through gut-brain-skin axes explains damaged tissue repair [97]. The distinction in gut microbiota constitution and decreased levels of *Bifidobacteria* and *lactobacillus* in infants' guts leads to the onset of allergic symptoms. Both the reports of specific probiotic strains and their results indicate that they can be used in the prevention of eczema. Dysbiosis, also known as dysbacteriosis, is a term used for imbalance in microbes inside the body, such as impaired microbiota, which is often related to inflammatory bowel disease, colonic cancer, metabolic syndrome, and allergic reactions. Improving the gut microbiota balance by different nutritional concepts or by ingesting specific micro-organisms led to significant improvement in health and decreased the risk of diseases or changing treatment mode [98].

The development of irritable bowel syndrome is associated with deviation in intestinal homeostasis, whose outcome is the uncontrolled immune response to gut microbiota by intestinal immune cells and epithelial cells, which results in complications including ulcers and fibrosis. A prebiotic is a valuable food substance that can be used to facilitate the growth of beneficial bacteria that modifies intestinal microbiota. Both probiotics and prebiotics are helpful for irritable bowel syndrome [97].

Probiotics have been involved in the healing process of intestinal ulcers and infected cutaneous wounds. Skin microbiota acts as a defensive barrier and can regulate the skin's inflammatory response to minor epidermal injury by decreasing and promoting cytokine production to maintain healthy skin [99]. The process through which probiotics show positive effects includes directly killing the pathogen, increasing the epithelial barrier, competitive displacement of pathogenic bacteria, and induction of fibroblasts [100].

Probiotics are also very beneficial for burn patients; they can reduce the bacterial load on the ulcer area [101]. Skin injuries cause disturbance in microbiota levels and increase the prevalence of bacteria that exert adverse effects on wounds. Additionally, having a wound causes stress, which results in alterations of neuro-endocrinal responses and impairs wound healing [102]. Chronic wounds are those that are difficult to heal and can exert a burden not only on the patient but also on the health care system, for example, diabetic foot ulcers (DFU), venous leg ulcers (VLU), and decubitus ulcers (DU). In chronic wounds, polymicrobial biofilms that promote pathogenic microbial growth and interfere in the process of wound healing are abundant and play an important role in the development of impaired wounds [103].

Probiotics play an important role in the treatment of autism by affecting the microbiome, which is present in the gut and responsible for imbalanced neuro-developmental conditions, like autism. If gene *Shank*3 is disturbed by gut microbes, it affects a person's behavior and can lead to autism [104]. There are numerous medicines are available for treatment, but they have side effects; to avoid these side effects, probiotics are used as an alternative therapy. Probiotics alter the gene that is responsible for neurodevelopment and maintains the gut environment to treat this disease [105]. This bacterial disorder promotes different pathophysiological gastrointestinal syndromes like bowel syndrome, obesity, diarrhea, and food allergies [106].

Dietary biotic aid is used to maintain the GIT flora and relieve pain, vomiting, nausea, and bowel syndrome. The most potent probiotic strains used to treat GIT disorder are *L. rhamnosus GG*, *L. reuteri 17938, VSL #3*, and *Bifidobacteria* species [107]. With probiotic intake, stool PCR tests of autistic children indicated increased colony count of Bifidobacteria and lactobacilli with a major bodyweight loss and great progress in the severity of autism and gastrointestinal disorders [108]. In pregnancy, Interleukin-6, 17a cytokines prompt autism spectrum disorder. Probiotics play an important role in inhibiting the production of cytokines and preventing the autism spectrum disorder induced by maternal immune activation [109].

Osteoporosis is a disease that affects the skeletal system, manifesting as low bone mass density, deterioration of the skeletal system, and greater bone brittleness and sensitivity to crack. Most cracks are in the distal forearm, femur, and back. These cracks especially occur in postmenopausal women [110]. The cause of bone loss is due to the low level of the estrogen hormone because estrogen performs a significant role in developing and sustaining bones [111].

In America, many people face the problem of osteoporosis, and most people are at high risk of low bone mass. Osteoporosis can occur in males and females at any period of life, but it predominantly occurs in older females [112]. Probiotics act as a therapeutic agent that helps treat many bone diseases such as osteoporosis and rheumatoid arthritis [113]. There are many mechanisms of probiotics that affect bones; the most latent influence of probiotics on bone happens through the integration of vitamins. In the metabolism of calcium, vitamin D, C, K, and folate are linked and are necessary for bone development [114].

*L. reuteri* is a probiotic that plays an essential role in alleviating osteoporosis and improving bone density [115]. Osteocalcin serum (OC), osteoblasts, i.e., bone composition parameters, low serum C-terminal telopeptide (CTX) and osteoclasts, i.e., bone resorption parameters are increased by *B. longum* supplementation. *B. longum* changed the structure of the femur [116]. When bone loss in postmenopausal osteoporosis occurs due to the estrogen level being inadequate, intestinal and bacterial antigens traverse the arbitrated intestinal epithelium wall and inaugurate the immune response. Probiotics boost the intestinal epithelial wall, balance the deviant host immune responses, support intestinal calcium absorption, restore intestinal microbial diversity to prevent bone resorption, and help in the latest production of the estrogen-like substance [117]. Action and effects of probiotics on numerous diseases that have been reported by in vivo analysis are shown in Table 1.



**1.**Effectandactionmechanismsofprobioticstopreventdiseasesreportedinvarious


60

AD = Alzheimer's disease.

#### *3.1. Action Mechanism of Probiotics in Reduction of Obesity*

Obesity is due to the union of microflora in the rectum area due to excessive absorption. Several new studies have confirmed that human intestinal micro-organisms play an important role in obesity through energy production and absorption of nutrients, and it has been shown that the duodenal microbiota is different in obese compared to lean persons. The upsurge of some gut microbial taxa such as *Escherichia coli*, *Staphylococcus aureus*, and other general bacterial gut fungi with *Bifidobacterium* has been shown to impact obesity [135].

*Firmicutes* are used directly as healing adjuvants, sold as named probiotics, prebiotics, or, usually, functional foods. In the United States, these products are characterized by the Food and Drug Administration and are generally recognized as safe. It is assumed that the gut of a fetus throughout the intrauterine period is deprived of any bacterial groups, i.e., it is thoroughly germ-free; however, during parturition, the transfer of micro-organisms from the mother to the fetus gut seems to occur and basic microbiota were found in it [136].

The mechanism credited with managing an upsurge in body fat was the capability of microbiota in extracting energy from food and controlling the host's energy balance. Deprivation of dietary polysaccharides and fiber by *Firmicutes* and *Bacteroides* in the gut resulted in the manufacture of SCFAs, such as acetate, propionate, and butyrate. Propionate is a significant energy source for the host from a mixture of glucose and lipids in the liver. A survey of 61 primary studies indicated differences in the microbial formation between overweight and non-obese subjects and the potential mechanisms involved [137].

Alterations are made homeostasis during the utility of dietary consumption and storehouse of lipids due to the balance of the abdominal microbiota. The microbiota is higher on metabolic pathways connected to obesity, including intonation with probiotics and prebiotics. This rise in *Bifidobacteria*, generally followed by weight gain and the increase in parameters linked to obesity [138].

Probiotics play a role in stopping the spread of bacteria and maintaining the gut environment to restore its normal flora. The ecology of the gut can be disturbed by numerous antibiotics plus some dietary products. Mostly probiotic strains come from gram-positive bacteria to treat gut disorders. To reduce tumor necrosis factor in the host, *L. reuteri* 6475 is important, and it also plays a vital role in promoting bone health and limiting bone absorption. Probiotic *Limosilactobacillus reuteri* has anti-inflammatory properties, particularly anti-TNFα properties [139].

Results demonstrated that these species reduce (LDL) low-density lipoprotein and (TC) total cholesterol, resulting in decreased coronary heart disease. A low level of leptin decreases obesity. LAB supplements also decrease LDL-C, aspartate aminotransferase (AST), alanine transaminase (ALT), high density lipoprotein (HDL), glucose, lipase, and triglycerides when a high fecal count is reported. LAB supplements also reduce intestinal microflora (tryptophanase, β-glucuronidase, and β-glucosidase). *Lactobacillus acidophilus* probiotics help ovariectomized subjects, intensify the microstructure of cortical and trabecular bone, expand the mineral density and diversity in bones, and prevent obesity [140].

Obesity levels has increased from 1980 to 2014 [141]. Obesity has an association with the high availability of energy and ambient temperature. An imbalance between intake and expenditure of energy is one of the significant causes of obesity. Gut microbiota influences the metabolism of the whole body by disturbing energy balance. They cause inflammation and barrier function in the gut, assimilating regulatory signals of central and peripheral food intake, thus enhancing body weight. Several physiologic functions are linked with probiotics that contribute to gut microbiota health and affect appetite, composition, food intake, metabolic functions, and body weight through gut bacterial community modulation and gastrointestinal pathways [142]. Figure 2 explains the anti-obesity mechanism of probiotic *bifidobacterium* spp., which is supplemented in the form of LAB supplement including *B. pseudocatenulatum* SPM 1204, *B. longum* SPM 1205, and *B. longum* SPM 1207.

**Figure 2.** Probiotics (*Bifidobacterium* spp.) against obesity (adapted from [143]).

#### *3.2. Action Mechanism of Probiotics in Minimizing T1D, T2D, GDM*

A current major health problem is diabetes mellitus [144]. Failure in insulin secretion and insulin action leads to a state of hyperglycemia, which is a metabolic disease that ultimately results in diabetes mellitus [145]. Between 5 and 10% of people have type 1 diabetes, also known as term insulin-dependent diabetes or juvenile-onset diabetes, due to the cellular-arbitrated autoimmune disorder in these β-cells of the pancreas [146].

Diabetes mellitus type 2, also known as insulin resistance, is when the patient does not utilize the insulin correctly. As a result, the pancreas generates additional insulin to compensate, but it cannot impel the production of sufficient insulin to sustain the appropriate level of blood glucose. Gut-microbiota contours in diabetic patients are not regular. A spatial association of human metagenome investigation revealed an essential relationship between metabolic pathways in type 2 diabetes subjects, specific gut microbes, and bacterial genes the level of *Lactobacillus* spp. that were very different than that of the non-diabetic subject [147].

Glycated hemoglobin (HbA1c) values, fasting glucose level, and concentration of *Lactobacillus* species are positively correlated. *Clostridium* species concentration in the gut is positively correlated with high-density lipoprotein (HDL) cholesterol level and negatively related to fasting glucose, HbA1c, insulin concentration, C-peptide, and plasma triglyceride levels [148]. Gram-positive bacteria and coagulase-negative staphylococci were prevalent in diabetic subjects in higher proportions, primarily in diabetics with retinopathy [149].

In the development of T2D, many cytokines are included. In the intestinal immune system, there are fundamental signals that are involved in suppressing the physiological state of action in the gut and affect the probiotics in glucose metabolism [150]. There is enough research to recommend that focalization of diabetes plays an important role in oxidative damage and anti-oxidative ability. In diabetic rats, inhibition of lipid peroxidation and increases in the antioxidant content of glutathione, superoxide dismutase, catalase, and glutathione peroxidase due to probiotics decreases oxidative damage. Probiotics also can prevent insulin resistance due to their anti-diabetic properties, which, in turn, increases the liver's natural T killer cells [151].

Probiotics also can improve inflammation by accentuating TNF-α expression, insulin stability, and overcoming NF-kB tying activity. Probiotics increase the bioavailability of gliclazide, which improves glucose metabolism and also helps repress the intestinal assimilation of glucose and modulate the action of the autonomic nervous system. If the colonial microbial diversity increases, it helps with upholding the probity of the gastrointestinal lining, improving glucose homeostasis, reducing inflammation, sustaining insulin production, and accumulating nutrients from the diet [152].

Adverse effects are associated with gestational diabetes mellitus (GDM) for both mother and infant during pregnancy. It is difficult, but GDM can be prevented by adopting a healthy lifestyle. The bacteria composite, e.g., the gut microbiome is available in intestines that alter the pathways for glucose and lipid metabolism, and in some host-inflammatory settings, gut microbiome alteration was shown to affect responses. Probiotics are one of the ways to modify the gut microbiome. They cause alterations in the gut microbiome but also modify the pregnancy metabolic environment [153].

*Lactobacillus* has an immunomodulatory effect on the host immune system. Subdued expression of osteoclastogenic factors (IL-6, IL-17, TNF-α, and RANKL) and enhanced expression of anti-osteoclastogenic factors are due to the *Lactobacillus acidophilus*. *Lactobacillus acidophilus* also has therapeutic, osteo-protective action in bone well-being (via tweaking Treg-Th17 cell balance) in postmenopausal osteoporosis as well as preventing diabetes mellitus [154]. Figure 3 illustrates the mechanism action of probiotics (*Bacteroides dorei*, *Bifidobacterium*, and *Lactobacillus rhamnoses*) against type-1-diabetes (T1D), type-2-diabetes (T2D), and gestational diabetes mellitus (GDM).

**Figure 3.** Mechanism action of probiotics (*Bacteroides dorei, bifidobacterium* and *Lactobacillus rhamnoses*) against type-1-diabetes, type-2-diabetes, and gestational diabetes mellitus adapted from [125,154–156].

#### *3.3. Mechanism of Action of Probiotics against CKD*

Probiotics play a vital role in reducing the risk and prevention of chronic diseases. There is an increasing concern in probiotics and prebiotics addition in the cases of chronic kidney disease (CKD) [157]. CKD increases at fluctuating rates, conditional on etiology and state of uremia. CKD is associated with consistent fatal disease build-up, and finally requires expensive treatments including peritoneal dialysis, kidney transplantation, and hemodialysis, which may prolong life [158].

The risk factor in the development of renal stones is known as hyperoxaluria, which can cause major damage to kidney function. The formation of oxalates primarily occurs. To ameliorate this condition, lactobacilli are given in the form of supplements that may decrease the formation of stones and reduce the chances of urolithiasis. *Lactobacillus casei* HY2743 and *L. casei* HY7201 may prevent the formation of oxalate [159].

Probiotics help the gut to maintain the luminal pH, produce antimicrobial peptides, influence function blockage by greater production of mucus and intonation of the host immune system, and transform the gut microflora. Gut microbiota plays an important role in the regulation of bone mass. The gut microbiota's effect on bone mass is propitiated through impacts on the immune system, whereas a change controls osteoclasto-genesis. In

normal conditions bone-forming osteoblasts and bone-resorbing osteoclasts are constantly remodeled; when an imbalance occurs in this process, osteoporosis occurs [160]. Figure 4 represents the mechanism action of *Lactobacillus* spp. against oxalate stones.

**Figure 4.** Prevention of kidney stones (oxalate) by probiotic (*Lactobacillus* spp.). Adapted from [161].

#### **4. Probiotics for Animal Health**

Sturdy pathogens such as Campylobacter and Salmonella can have a detrimental effect on the wellness and health of animals, as well as their development and reproductive abilities. These harmful bacteria can be transported via the food chain and into the human digestive organs, where they can cause serious human illnesses [162]. Numerous investigations on farm animals and normal or stressed humans have been conducted, which have proven the impact of various feed additives, and/or putative probiotics on good bacteria and decreased pathogen load in chickens, pigs, and ruminating animals [163].

#### **5. Safety of Probiotics**

A bacterial strain's safety, its source, antibiotic-resistant characteristics, and absolute lack of pathogenicity associated with virulent cultures all contribute to the safety profile's primary foundation; the rest is performance [164]. Probiotic performance promotes a variety of pathways, including adhesion to epithelial cells, decrease in gastrointestinal permeability, and immunoregulatory impacts [165]. Probiotics are not metabolized, have no potential for transference to animal-derived foods, and so do not result in the creation of residues. Due to the absence of their explicit and/or indirect transit from the gut into the animal body, they do not affect metabolic activities and therefore have no adverse effect [166].

#### **6. Conclusions**

Awareness regarding beneficial bacteria in the human gut originated in the 20th century, and by that time probiotics were considered an important player in intestinal and oral health. These gut microbes are associated with higher longevity. This associated effect between probiotics and long life is the basis of present research trends which are exploring the health-promoting properties and action mechanisms of gut microbes in cases of several prevalent diseases. Probiotics have anti-inflammatory properties because they can interfere with the working of cytokines and chemokines. Due to this vital property, they have been investigated in various diseases, especially in the case of chronic inflammatory diseases and GIT disorders. These gut microbes have a direct link with the overall nutritional status of a person as they help in the digestion and absorption of various nutrients, and

therefore help regulate cholesterol levels and pancreatic functions. Due to their interference with cytokines and inflammation stimulation factors, they also tend to affect the immune system. Probiotics also tend to affect the gut barrier and thus also play a defensive role in human health.

#### **7. Future Recommendations**

More molecular studies are needed to ensure the safe use of some strains of bacteria, as insufficient data are available on the safety aspects of the probiotic strains. Not all microbes are beneficial and some of them can result in severe outcomes. A thorough study of the strain type used, its impact on health, and its interactions should be studied. Metabolomics exploration of human microbiome functionality with the involvement of bioinformatics may be a new horizon of research, without strong evidence the use of microbes as probiotics should not be encouraged.

**Author Contributions:** All authors have contributed to the final manuscript by division of responsibilities, i.e., literature search, compilation, write-up, final version development, English language editing, figures and table formation, formatting and reference styling, and proofreading. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

