The Inoculation of Probiotics In Vivo Is a Challenge: Strategies to Improve Their Survival, to Avoid Unpleasant Changes, or to Enhance Their Performances in Beverages

The inoculation of probiotics in beverages (probiotication) requires special technologies, as probiotic microorganisms can experience stress during food processing (acid, cold, drying, starvation, oxidative, and osmotic stresses) and gastrointestinal transit. Survival to harsh conditions is an essential prerequisite for probiotic bacteria before reaching the target site where they can exert their health promoting effects, but several probiotics show a poor resistance to technological processes, limiting their use to a restricted number of food products. Therefore, this paper offers a short overview of the ways to improve bacterial resistance: by inducing a phenotypic modification (adaptation) or by surrounding bacteria through a physical protection (microencapsulation). A second topic briefly addressed is genetic manipulation, while the last section addresses the control of metabolism by attenuation through physical treatments to design new kinds of food.


Introduction
Beverages containing viable cells of probiotics are generally milk-based products, for instance, yogurts [1]. However, in the last decade, the demand for nondairy probiotics has increased for a wide variety of issues (increasing incidence of lactose intolerance and/or worry about the high cholesterol content of some products, new lifestyles etc.).
The inoculation of probiotics in nondairy beverages (cereal, fruit, and vegetable juices) has a key benefit: the possibility of combining probiotic microorganisms with prebiotics or prebiotic-like compounds, thus producing synbiotics [13]. Considering the fact that a probiotic is essentially active in the small and large intestines, and the effect of a prebiotic is observed mainly in the large intestine, the combination of the two may have a synergistic effect [14]. There are two possible modes of action of synbiotics: (i) the improved viability of probiotic microorganisms; ii) the provision of specific health effects [13,15]. Table 1. Stresses encountered by food-grade microorganisms during food production and storage [19][20][21].

Stress Description
Acid Acid-stress could be self-imposed (production of lactic acid or other acids because of fermentation) or environmental stress (juices, dairy beverages, gastrointestinal tract, etc.). Acidification of cytosol is genotoxic and causes the denaturation of proteins, with a deleterious effect on overall metabolism (energy depletion and death).

Bile
Bile salts can cause disruption of membranes, DNA damage, misfolding and/or denaturation of proteins, and chelation of iron and calcium.

Oxidative
Lactic acid bacteria are sensitive to aerobic environments; moreover, some strains can produce ROS (reactive oxygen species) by themselves.

Cold and chilled
Starters and probiotic cultures are stored in a frozen or freeze-dried form; in addition, storage generally occurs under refrigerated conditions. Viability decreases during storage because of certain factors (glass transition temperature, light, relative humidity, etc.).
Heat shock Starter cultures could be exposed to reheating or high temperatures, mainly in dairy products.

Osmotic
NaCl is added to inhibit spoilers and pathogens.

Starvation
Starvation could occur as a side-effect of stress (for example, auto-acidification interferes with membrane carriers).

High-pressure/Homogenization/Ultrasound
There are several alternative approaches to thermal treatments; the most used one is HHP (high hydrostatic pressure). It can induce physiological changes, changes in gene expression and protein translation, and cell damage.
Injuries to cells also occur in the case of homogenization, while ultrasound can cause the formation of pores on the membranes Ethanol Ethanol stress is important for yeast and lactic acid bacteria involved in the production of alcohol-containing beverages.

Antimicrobial compounds
Several beverages contain natural antimicrobials, such as phenols in juices or lysozyme and lactoferrin in dairy products. They act on different targets (membranes, cell wall, etc.) in cells and may cause lethal or sublethal injuries.
Survival in harsh conditions is an essential prerequisite for probiotic bacteria before reaching the target site where they can exert their health-promoting effects [22], but several probiotics show a poor resistance to technological processes, limiting their use to a restricted number of beverages. Therefore, this paper offers a short overview of the most important approaches to counteract this challenge; namely, the first two sections address the issue of improving bacteria resistance by inducing a phenotypic modification (adaptation) or surrounding bacteria through a physical protection (microencapsulation) A second challenge briefly addressed in this paper is the genetic manipulation, while the last section addresses another kind of problem: the need of controlling the metabolism of probiotics in several beverages in order to reduce or eliminate unfavorable changes in sensory scores (attenuation) or to improve their performances (modulation) through physical treatments to design new kinds of food ( Figure 1).
Beverages 2020, 6, x FOR PEER REVIEW 3 of 18 Survival in harsh conditions is an essential prerequisite for probiotic bacteria before reaching the target site where they can exert their health-promoting effects [22], but several probiotics show a poor resistance to technological processes, limiting their use to a restricted number of beverages. Therefore, this paper offers a short overview of the most important approaches to counteract this challenge; namely, the first two sections address the issue of improving bacteria resistance by inducing a phenotypic modification (adaptation) or surrounding bacteria through a physical protection (microencapsulation) A second challenge briefly addressed in this paper is the genetic manipulation, while the last section addresses another kind of problem: the need of controlling the metabolism of probiotics in several beverages in order to reduce or eliminate unfavorable changes in sensory scores (attenuation) or to improve their performances (modulation) through physical treatments to design new kinds of food ( Figure 1).

Adaptive Evolution
Phenotyping is one of the most commonly used methods to modify the properties of probiotics and food-grade microorganisms; this approach has been referred to by many authors as "adaptive evolution" or "pre-adaptation or cross-protection" and has some applications for the inoculation of probiotics in fruit-juices [19,21,23].
Bacterial cells possess a multitude of defense mechanisms, such as chaperones to assist folding of misfolded proteins, proteases which degrade proteins which are irreversibly damaged, transport systems, catalases, and superoxide dismutases to counteract ROS, as well as proton pumps, decarboxylases, and transporters to combat decreases in intracellular pH [19,21,23]. Isolation and identification of naturally-tolerant strains can ensure robustness during processing and storage (thermal treatments, freeze-drying, chilled or cold storage, presence of antimicrobial compounds such as phenols, etc.), and gastrointestinal transit. Alternatively, exploiting the inherent probiotic stress response to improve tolerance capacities of existing probiotic strains could make them amenable to large-scale production and storage [21].
Pre-adaptation or adaptive evolution or "habituation" [19] consists in treating a microorganism to a sublethal stress for a limited time; this treatment would act on strain resistance when exposed to a higher level of stress or to another stress. The mechanism behind pre-adaptation is not well understood; however, it is known that bacteria have two ways to counteract stresses: cross-protection

Adaptive Evolution
Phenotyping is one of the most commonly used methods to modify the properties of probiotics and food-grade microorganisms; this approach has been referred to by many authors as "adaptive evolution" or "pre-adaptation or cross-protection" and has some applications for the inoculation of probiotics in fruit-juices [19,21,23].
Bacterial cells possess a multitude of defense mechanisms, such as chaperones to assist folding of misfolded proteins, proteases which degrade proteins which are irreversibly damaged, transport systems, catalases, and superoxide dismutases to counteract ROS, as well as proton pumps, decarboxylases, and transporters to combat decreases in intracellular pH [19,21,23]. Isolation and identification of naturally-tolerant strains can ensure robustness during processing and storage (thermal treatments, freeze-drying, chilled or cold storage, presence of antimicrobial compounds such as phenols, etc.), and gastrointestinal transit. Alternatively, exploiting the inherent probiotic stress response to improve tolerance capacities of existing probiotic strains could make them amenable to large-scale production and storage [21].
Pre-adaptation or adaptive evolution or "habituation" [19] consists in treating a microorganism to a sublethal stress for a limited time; this treatment would act on strain resistance when exposed to a higher level of stress or to another stress. The mechanism behind pre-adaptation is not well understood; however, it is known that bacteria have two ways to counteract stresses: cross-protection and GSR (general stress response) [23]. Cross-protection relies on the principle that interrelated responses are generated by different stress conditions; different stimuli (heat, oxygen, low pH, etc.) might produce similar responses. GSR is the acquired resistance to some conditions when a population enters the stationary phase [23].
Adaptive responses are generally based on epigenetics mechanisms, because regrowth of adapted cells under optimal conditions reduces or eliminates any resistance phenotype after a few generations [19]. However, stress-induced mutations could also occur, and thus, cells exposed to some stresses enter a hypermutable state [19]; some evidence of this phenomenon has been found for lactic acid bacteria [24,25].
The most commonly used approach for stress adaptation is the modification of the growth medium and/or incubation conditions with different strategies: (a) pH modification to trigger acid adaptation [21,[26][27][28]: Probiotics are cultured in media adjusted to suboptimal pH (4.5-5.5) for several passages before inoculation in acid matrix (for example, fruit juices); (b) Osmoadaptation [29,30]: Microorganisms are grown in hyperosmotic media (for example, hyperconcentrated sweet whey) to trigger adaptation to drying; (c) Media supplementation with sugars (mannose, trehalose, sucrose) [31,32] to improve viability during freeze-drying; (d) Media supplementation with protective compounds (arginine, Tween 80, aspartate, gluthatione) [19,21] to improve acid resistance; (e) Media supplementation with prebiotics [33,34] generally increases viability throughout storage; (f) Starvation [28] for a general increase of viability during storage: Probiotics are cultured in media with a few nutrients (amino acids and/or sugar) before inoculation in food. This practice could increase viability during refrigerated storage; (g) Cold-adaptation [35]: Probiotics and/or starter strains are cultured at suboptimal temperatures (15-20 • C), thus increasing their technological performances in food; (h) Media supplementation with phenols (for example, vanillic acid) to improve strain viability in juices [26]; (i) Strain culturing in presence of increasing amount of the final food matrix [26]: Probiotics could be cultured in the presence of increasing amounts of juices (from 10% to 50%), thus increasing their viability in the product.
Another kind of pretreatment is heat adaptation based on microbial exposure to sublethal temperatures (for example, 52 • C for 15 min) [36]; this pretreatment can increase resistance to low or alkaline pHs, ROS, ethanol, spray-drying, etc.
There are different molecular mechanisms beyond adaptation and bacterial resistance; Table 2 shows the most important phenomena.
There are several examples of application of cross-adaptation to improve the performances of probiotics mainly in juices. For example, Perricone et al. [26] addressed the issue of viability of Lactobacillus reuteri in juices produced from red fruits; they used two different kinds of protocols: strain cultivation in lab media acidified to pH 5.0 or containing a phenolic compound (vanillic acid) or a sequential protocol based on strain growing in presence of increasing amount of red-juices. Thus, they increased the viability of the probiotic by 9-11 days.
A different approach was proposed by Shah et al. [37] to protect Lactobacillus rhamnosus, Lactobacillus paracasei and Bifidobacterium lactis from oxygen; they supplemented juices with antioxidants (vitamin C, grape extract, and green tea extract); after 6 weeks, the probiotic was at 4-6 log cfu/mL, while in the control, it was below the detection limit.
The supplementation of threalose, combined with a sublethal homogenization at 25-150 MPa, improved the viability of Lb. reuteri in clementine juice and throughout the transit into the gut and its antimicrobial activity against Helicobacter pylori [38].
Da Costa et al. [39] also proposed supplementation with certain active ingredients (ascorbic acid and oligofructose); the effect was not protective of the probiotic (Lb. paracasei), but a positive modulation of color and turbidity throughout refrigerated storage at 4 • C.

Encapsulation
Microencapsulation has recently been suggested as a convenient tool to improve probiotics' survival not only under gastrointestinal conditions, but also in functional foods such as milk derivatives or novel functional beverages. As is known, microcapsules are generally formed to include sensible compounds (solid, liquid or gaseous) within protective matrices (food-grade biopolymers) by entrapping or surrounding them [40]. An optimal encapsulation process can provide protection against unfavorable environmental conditions, but allowing a controlled release of the encapsulated core and ensuring all main diffusion processes (oxygen and nutrients effluxes, but also waste product expulsion) [40].
In literature, different methods are suggested for the encapsulation of probiotics, such as spray drying, extrusion, emulsion or phase separation, freeze drying, ionotropic gelation [55], but vibrational extrusion is suggested as the better technique in term of easiness, low cost, higher cell recovery (80-95%), and effectiveness in protection under stress conditions [47,56]. During extrusion, capsules are obtained by simply dropping an aqueous solution of probiotics into a gelling bath: The beads have sizes and shapes in the range of 2-5 mm and depend on the diameter of the needle used [57].
With regard to entrapping materials, polysaccharides (alginate, plant/microbial gums, chitosan, starch, k-carrageenan, cellulose acetate phthalate), as well as proteins (gelatin, milk proteins) and fats are proposed, but the use of gum or biopolymeric matrices is preferred [58][59][60][61]. Good reviews about probiotic microencapsulation can be found in the papers of Mitropoulou et al. [62], De Prisco and Mauriello [63], and Terpou et al. [64]. Apart from the kind of material, the performances of probiotic entrapment could be successfully improved through the use of some coadjutants and/or protectants [65][66][67].
There is literature concerning the use of microencapsulation for the inclusion of probiotics in functional beverages, mainly in functional fruit juices where the low pH (2.5-3.7) and the presence of phenolic acids (benzoic acid), lactones, and other compounds might affect probiotics' viability. Table 3 shows a brief synopsis of the most recent research on this application, with some details on the technology used, the entrapped probiotics, the juices/beverages where the beads were loaded in, and the most important achievements. Most of these studies focused on the impact of microencapsulation on probiotics' viability and functionality, whereas few have been performed on the evaluation of sensorial properties. Another concern is linked to the experimental temperature, since most of the studies were performed at refrigeration temperature, even if functional beverages are often stored and marketed at room temperature; thus, it might be interesting to evaluate capsules' performances at 20-25 • C.

Engineering
The performance of probiotic strains can be improved by bioengineering, that is, the manipulation of a gene to improve the tolerance to technological stress, including but not limited to temperature extremes, oxygen and acidification, during food production, and/or survival of the probiotic in the gut, to confer beneficial effects to the host [68].
Bioengineering of probiotics is not an entirely new field, and genetically engineered microorganisms have been shown to efficiently produce and secrete various proteins as well as be capable of treating obesity, diabetes, and colitis in animal models (Table 4). LAB, Saccharomyces spp., Escherichia coli Nissle 1917, and some Bacillus species are the prospective species whose efficiency and utility should be improved for them to be used as probiotics [69]. For example, recent reports support the use of recombinant probiotic yeast Saccharomyces boulardii to synthesize and deliver therapeutic biomolecules during gastro-intestinal tract colonization [70].
One of the main drawbacks of working with bioengineered probiotics is that they are classified as genetically modified organisms (GMOs) [68]. Engineered probiotics contain additional elements for inducing antigenicity and immunomodulation; however, these changes could also affect metabolic pathways and safety [69]. For instance, UV mutagenesis for S. boulardii is not limited to the URA3 gene only, and many other unknown mutations might occur during UV treatment. These unknown mutations may lead to altered phenotypes related to probiotic traits, including undesirable ones [71].
Other than the issue of biocontainment, interactions between synthetic probiotics and the commensal microbes in the human body remain poorly understood. Although significant results are observed in various in vivo models, similar effects may not be observed in humans as the human enteric microbiome is far more complex than that of animal models [72]. Therefore, large, well-designed, randomized controlled clinical trials along with culture-independent metagenomic analyses should be meticulously carried out [69].
Another approach related to bioengineering is the pangenome approach, based on the complete genome sequences of a number of members of the same species [73]. The pangenome is the global gene repertoire of a bacterial species, composed of a core genome (pool of genes shared by all strains of a species) and dispensable genome (the genes of some strains) [74]. Often, the probiotic functions are described and encoded by dispensable genes; therefore, knowledge of the full genome is a prerequisite for a proper selection of functional microorganisms [75], as well as for their improvement through engineering.

Physical Treatments
The performances of probiotics in food could be significantly improved and/or modulated using preliminary physical treatments able to exert a stimulus or a metabolic delay. This section addresses the use of two emerging nonthermal technologies (US, ultrasound; HPH, high pressure of homogenization).

Ultrasound
Bevilacqua et al. [76] reported that US can affect microorganisms through a lethal effect or through the stimulation of growth, depending on the intensity and the frequency: High-intensity US impairs the membranes, leading to loss of viability, whereas low-intensity US stimulates bacterial metabolism. Concerning probiotics, the growth and stimulation of metabolism is desirable, and therefore, US is applied by opportunely modulating intensity and frequency.
Several studies have focused on dairy products, but nondairy beverages have also been investigated. In 2011, Yeo and Liong [77] studied the effect of US on the growth of Bifidobacterium FTDC 8943, B. longum FTDC8643, Lactobacillus sp. FTDC2113, and Lb. casei ATCC393 inoculated in soymilk, and they reported that the growth of probiotics was significantly decreased (p < 0.05) immediately after the treatment, as a result of membrane permeabilization, cell lysis, and membrane lipid peroxidation. US also caused alteration at the acyl chain, polar head, and interface region of the probiotic membrane phospholipid bilayers. However, the effect was transient, because cells repaired injury and showed a growth kinetic similar to the control.  The mixture probiotic/alginate was dispensed by a pipette into a solution of pure corn oil at room temperature. The emulsion was broken through the addition of calcium chloride and the beads were recovered by centrifugation.
Microencapsulation improves probiotic viability, during storage at 4 • C for 42 days. [84] Kefir B. animalis Extrusion Sodium alginate The mixture probiotic/alginate was allowed to drip slowly through a needle into a solution of calcium chloride at room temperature.
Encapsulation improved significantly the survival of bifidobacteria during exposure to nisin, during the storage period, and in simulated gastric juice. [85] Longan juice Lb. acidophilus LA5, L. casei 01 Emulsion Alginate. Coating with sodium alginate.
The mixture probiotic/alginate was dispersed into a solution of peanut oil at room temperature. The emulsion was broken through the addition of calcium chloride, and the beads were recovered by centrifugation and coated with sodium alginate.
Encapsulated probiotics could survive in the acidic environment of the stomach and small intestine, while the free cells were completely eliminated. [86]

Mango juice
Lb. plantarum Gelation Calcium alginate-soy protein isolate Different mixtures with alginate, soy protein isolates, and probiotic cells were dropped into a gelation bath containing calcium chloride using a needle at a constant flow rate of 3.6 mL/min through a peristaltic pump, at room temperature.
The application of encapsulated beads in mango juice showed successful resistance to thermal conditions. [87] Orange and peach juices Lb. paracasei L26 Extrusion Sodium alginate. Coating with chitosan or dextran The alginate/culture mixture was extruded using a microincapsulator with a 0.5 mm orifice, a nitrogen pressure of 0.4 bar, and an extrusion rate of 4 mL/min. The microcapsules were left in a solution containing calcium chloride for 30 min at room temperature. Once recovered by gravity filtration, the beads were single-or double-coated with chitosan or dextran.
Free cells have a greater metabolic activity than microencapsulated ones. [88] Orange juice Lb. acidophilus LA5 and Lb. casei 01 Extrusion Sodium alginate and galactooligosaccharides (GOS) or inulin. Coating with chitosan.
The solution containing alginate and GOS or inulin was mixed with probiotic suspension and injected through a needle into a gelation solution containing calcium chloride for 30 min at room temperature. Then, the beads were coated with chitosan.
Beads improved cell survival. [89]  The probiotic/alginate solution was extruded as droplets through a needle using a peristaltic pump into a solution containing calcium chloride, at room temperature.
The immobilized cells endured the adverse effects of tomato juice, maintaining high viable counts.

Lactobacillus
Lb. paracasei Improved thermotolerance Increased resistance to some solvents Inhibition of adhesion of Listeria to host cells [68] Lb. salivarius Increased resistance to several stresses [68] Lb. jensenii Inhibition of HIV in CD4 + cells and macrophages [69] Lb. reuteri Binding of some enterotoxins and prevention of their toxicity in a mouse model [68] Lb. acidophilus Reduction of attachment of Escherichia coli ETEC to porcine intestinal brush border [68] Lb. gasseri Alleviation of diabetes Mellitus in rat model [93] Lb. plantarum Decreased systolic blood pressure in rats [72] Lactococcus

Lc. lactis
Enhanced resistance to gastric acid damage Enhanced efficient internalization in human intestinal cell line Caco-2 Inhibition of E. coli and Salmonella Overall reduction of inflammation and colitis Prevention of colitis in murine models Protection against rotavirus infection [68] Lc. lactis Prevention of allergen-induced airway inflammation by induction of specific mucosal immune tolerance [69] Lc. lactis Improved repair of gut epithelial damage in Hamster model [69] Other Microorganisms B. longum Reduction of colitis inflammation [94] Bacillus subtilis Prolonged colonization of recombinant B. subtilis in GI tract of mice, significant reduction in H. pylori (84%) [69] S. boulardii Secretory expression of biologically active IL-10 [69] A treatment at 100 W for 2 and 3 min also enhanced (p < 0.05) intracellular and extracellular β-glucosidase activity of probiotics, leading to increased (p < 0.05) bioconversion of glucosides to aglycones in the prebiotic-soymilk.
Recently, Gholamhosseinpour and Hashemi [95] used US (100 W, 30 kHz, 25% amplitude for 5, 10, and 15 min) to improve the metabolism and growth of Lb. plantarum AF1 during milk fermentation at 37 • C. The results showed an increase on the quality and antioxidant activity of milk, probably related to an increased cell concentration (8.7 log CFU/mL in the control; >9 log CFU/mL in US-treated samples) and a reduced lag phase.
Niamah [96] studied the effect of US at 40 kHz for 0, 5, 10, 15, and 20 min on the growth of Lb. acidophilus LA-5, Lb. casei LC, Lb. reuteri LR-MM53, B. bifidum Bb-12 and B. longum BB-536 in fermented milk. A US-treatment for 10 min improved chemical properties of fermented milk by probiotics. A further increase of exposure time caused a reduction of viability of probiotic bacteria strains and increased β-galactosidase activity.
Costa et al. [97] inoculated Lb. casei NRRL B442 in a sonicated pineapple juice (sonication at 376 W cm −2 for 10 min with a 1.3 cm probe tip and at a constant ultrasonic frequency of 19 kHz); the treatment enhanced the performance of the probiotic, which then was able to survive for at least 21 days at 4 • C. In addition, sonication reduced the impact of browning throughout storage.
Bevilacqua et al. [7,76] and Racioppo et al. [98] studied a different use of US, the possibility of a modulation or an attenuation of the metabolism of probiotics with a strong reduction of the post-acidification occurring during storage. This treatment (50-80 W) was applied on the probiotic (Lb. reuteri, Lb. plantarum, Lb. casei, bifidobacteria, and propionibacteria) before the inoculation in an organic rice beverage or in model systems and assured the maintenance of pH and sensory scores for at least 7 days.

High-Pressure Homogenization
HPH action depends mainly on microbial and physiological parameters, process parameters, and characteristics of fluids [76]. On the other hand, HPH can also exert a positive effect by modulating the metabolism of microorganisms: in fact, HPH can control the fermentation kinetics of starters and modify their metabolic activity with an enhancement of sensorial properties [76].
Patrignani et al. [99] reported that HPH could be used in fermented milk for several reasons: (1) to modulate the sensorial characteristics without harmful effects on shelf life and safety; (2) to improve the technological performances of probiotics; and (3) to change the functional features of lactic acid bacteria.
An exhaustive discussion on the actual scenario concerning HPH treatment of functional dairy beverages was reported by Patrignani et al. [99]. In particular, the authors mentioned a previous study on the effect of HPH on nonfat milk solids and milk fat on the technological performances of Lb. paracasei BFE 5264 for the production of probiotic fermented milks [100] and reported that the use of treatments at 20-100 MPa could improve the sensorial properties of fermented milk. With nonfat milk solid <3%, firmness, viscosity index, and consistency of probiotic fermented milk increased with the increase in pressure level; the content of diacetyl and acetaldehyde increased, too. Moreover, Lb. paracasei BFE 5264 coagulation time was significantly affected by the increase of pressure depending on the addition phase of milk fat (before or after the pressure treatment): When the addition of milk fat was performed before HPH treatment, the strain fermentation rate decreased, and its viability during the refrigerated storage was reduced.
Optimal results were also obtained when the authors studied the effect of HPH for the production of probiotic fermented milk containing Lb. paracasei and Lb. acidophilus. In fact, compared to heat-treated milk, HPH-milk favored the viability of starter cultures (Streptococcus thermophilus and Lb. delbrueckii subsp. bulgaricus) and the probiotic strains (Lb. acidophilus and Lb. paracasei). In addition, higher values of firmness, consistency, cohesiveness, and viscosity indices were also observed.
Finally, Bevilacqua et al. [7] used HPH for the attenuation of lactic acid bacteria in an organic rice beverage and found that the effect of multiple passes at 100 MPa delayed acidification by 4 days and reduced the maximum extent of the acidification; thus, the authors proposed this approach as a tool for attenuation to counteract the acidification of promising probiotics and to avoid the post-acidification of the rice beverage throughout storage. Apart from HPH, other possible approaches to achieve attenuation are mild heat treatment (for example, 46 • C for 1 h for Lb. rhamnosus) [101], or random mutagenesis [102].

Conclusions
Probiotication of beverages is one of the increasing trends for research and industry; however, the inoculation of probiotics in some beverages is a challenge, as some issues should be addressed. First, probiotics have to resist certain technological stresses, such as low pH, presence of antimicrobial compounds, high osmotic pressure, and oxygen, and researchers have to face the threat of the effect of probiotics on some sensory attributes.
This review discussed several approaches to counteract these phenomena. The resistance of probiotics could be increased by either cross-adaptation/adaptive evolution or by bioengineering, while a solution for sensory traits is attenuation (physical treatments). Each protocol has benefits and limits, but some issues must be clarified and the lack of data on them is a challenge, that is: (a) The effect of each treatment on consumer perception; (b) Safety and metabolic profiles of treated microorganisms, in order to assess that these approaches do not promote risk profile and do not modify desired metabolic pathways; (c) The effect of each treatment on the functional and probiotic patterns of probiotic microorganisms; (d) The effect of each treatment on the costs.
A final and important point relates to strain specificity of the treatments, as each approach is strongly affected by the strain, while for an industrial application, it is important to design general processes that might be applied to a large number of microorganisms.