Compositions for improving the dispersion of probiotics in oily suspensions

Abstract

The present invention relates to a composition for oral use, in the form of an oily anhydrous suspension, comprising probiotics and the related preparation process. This composition is also suitable for paediatric subjects, or subjects suffering from swallowing difficulties or from dysphagia.

Description

TITLE: "Compositions for improving the dispersion of probiotics in oily suspensions”DESCRIPTIONFIELD OF THE INVENTIONThe present invention falls in the technical field of preparations for oral use, comprising probiotic microorganisms, in the form of oily suspensions; their uses and the processes for their preparation.STATE OF THE ARTProbiotics are “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host' (FAO / WHO definition). Present in some foods and dietary supplements, probiotics are gaining considerable interest for their potential in promoting overall wellness. They are based on the concept of maintaining a healthy balance of microorganisms in the digestive system, where trillions of microorganisms known as gut microbiota reside. These play a vital role in overall health, metabolism, digestion and immunity. In particular, one of the main functions of probiotics is to compete with harmful microorganisms present in the intestine, inhibiting the growth and activity of pathogenic bacteria and thus promoting a healthier microbial environment. This competition helps to preserve the health of the gastrointestinal system and supports the body's different metabolic processes.Probiotics are known to have multiple functions to benefit health. For example, they can positively affect the metabolism and absorption of nutrients, including the digestion of dietary fibre. Furthermore, they can help to improve the body's resistance to toxic substances and strengthen the barrier function of the intestine, preventing the passage of harmful substances into the blood stream. In addition, probiotics have the ability to modulate the immune system, promoting a balanced and effective immune response.In this context, it is important to consider that specific health benefits can vary depending on the probiotic strain and dosage [Hill, C., Guamer, F., Reid, G. et al. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol 11, 506-514 (2014). Sanders, M. E., Merenstein, D. J., Ouwehand, A. C., Reid,G., Salminen, S., Cabana, M. D., Paraskevakos, G., & Leyer, G. (2016). Probiotic use in at-risk populations. Journal of the American Pharmacists Association: JAPhA, 56(6), 680-686.Ouwehand, A. C., Salminen, S., & Isolauri, E. (2002).Probiotics: an overview of beneficial effects. Antonie van Leeuwenhoek, 82(1-4), 279-289. FAO / WHO, “Evaluation of health and nutritional properties of probiotics in food including powdermilk with live lactic acid bacteria. Expert consultation report ” Food and Agriculture Organization of the United Nations and World Health Organization, Cordoba, Argentina, October 2001],The probiotic microorganisms commonly used in food belong mainly to the families I.aclobacillaceae. Bifidobacteriaceae , Enterococcaceae, Streptococcaceae, Saccharomycetaceae . These kinds of probiotics in turn comprise a wide range of strains, each with specific properties and potential health benefits.Probiotics are often given as dietary supplements, available in different formulations, including capsules, tablets, powders and liquids. The liquid form of administration of probiotics has considerable advantages: liquids are generally easier to consume, particularly for people who have difficulty swallowing solid dosage forms (capsules or tablets), as seen in infants or people with dysphagia. Furthermore, liquid probiotics have a certain versatility, allowing direct consumption or easy incorporation into beverages and food matrices, thus expanding their application in various dietary contexts. However, despite these advantages, liquid preparations containing probiotic microorganisms are more sensitive to environmental and industrialization factors. These factors can potentially lead to the formation of aggregates and / or cake with consequent impact on the usability, efficacy and shelf life of the product. Therefore, from an application and industrial point of view, for probiotic products to be efficient in providing their benefits, it is essential to develop stable probiotic compositions.Over the years, different approaches have been used by researchers and producers to preserve the functionality of liquid probiotic formulations. Some of the most widely used strategies include: micro-encapsulation of strains, the use of stabilizing additives, and the development of new delivery or packaging systems. In particular, micro-encapsulation is a technique which involves the creation of a protective coating around the probiotic cells which, used subsequently in the product, helps to prevent aggregation and cake formation [Chavarri, M.; Maranon, I.; Villaran, M. C. Encapsulation Technology to Protect Probiotic Bacteria,' Rigobelo, E. C., Ed.; IntechOpen: Rijeka, 2012; p Ch. 23. https. / / dgi.org / l 0.5772 / 50046. Sultana, K., Godward, G., Reynolds, N., Arumugaswamy, R., Peiris, P., & Kailasapathy, K. (2000). Encapsulation of probiotic bacteria with alginate -star ch and evaluation of survival in simulated gastrointestinal conditions and in yoghurt. International journal of food microbiology, 62(1-2), 47-55. h tps: / / d0i .0rg / l 0. 1016 / s0168-1605(00)00380-9] .Alternatively, additives are added to liquid-based probiotic products in order to improve their stability. These stabilizing additives can comprise ingredients such as prebiotic fibres, polysaccharides or emulsifiers. [Makinen, K.; Berger, B.; Ananta, E. Science and Technology for the Mastership of Probiotic Applications in Food Products. J. Biotechnol. 2012, 162 (4), 356-365. Jang,Joeng Su, Liquid stabilizer composition for probiotics, Patent no. KR100693764B1, Publication date: 06-03-2007. Ziar, H.; Riazi, A. Polysorbate 80 Improves the Adhesion and Survival of Yogurt Starters with Cholesterol Uptake Abilities. Saudi J. Biol. Sci. 2022, 29 (8), 103367], This approach is frequently used in the production of fresh probiotic-based foods such as yoghurt, milk and other food products.Other solutions known in the state of the art are listed below:W02011035093A1 discloses a nutritional composition comprising probiotic microorganisms, characterized by containing a stabilization mixture capable of guaranteeing the survival of the strains under processing and storage conditions, and until the product is taken (par.

[0010] ). The stabilization mixture comprising a hydrolyzed mammalian protein (par.

[0034] ); preferably, one or more carbohydrates or dietary fibres such as inulin (par.

[0039] ); optionally, a gelling agent (par.

[0040] ); and possibly other ingredients aiding stabilization such as pectin, resistant starches, high amylose starches, guar gum, locust bean gum, agar, xanthan gum, carrageenans, glucans, FOS, polyfructose, maltodextrins (par.

[0041] ). According to an embodiment, the composition can contain lipids in order to be nutritionally complete (par.

[0053] ).W02010103374A2 discloses a composition in the form of an oily suspension comprising probiotic microorganisms in solid form (p. 3), enclosed in a coating comprising at least one vegetable lipid having a melting point comprised between 35°C and 75°C (p. 5). The composition further comprises stabilizing ingredients, suitable for food use, such as silica dioxide (p. 7, Table 2) or natural gums (p. 7). Fibres and / or carbohydrates can be present, which offer a prebiotic and thickening / stabilizing function respectively (p. 8).WO2017 / 223150A1 discloses a composition in the form of an oily suspension (par.

[0086] -

[0087] ) containing a probiotic in powder form (par.

[0016] ,

[0024] ,

[0060] ); an edible thickening additive selected for example from gums, polysaccharides, collagen, starches, silica (par.

[0019] ,

[0067] ) or an edible wax which increases the viscosity of the preparation so that the dosage is repeatable and reliable (par.

[0009] ,

[0015] ).WO2018 / 142346A1 discloses a composition containing probiotics in the form of oily anhydrous suspension (p. 4, lines 5-10). The oily vehicle comprises a solid vegetable fat at room temperature having a saturated fatty acid content of at least 25%, which has the function of thickening the continuous phase of the suspension, thus overcoming the problems of the prior art related to the formation of indissoluble aggregates of probiotics (p. 4, lines 5-17).W02011 / 016070A1 discloses a nutraceutical composition containing probiotics in an oily vehicle, characterized in that it contains polysorbates, ascorbyl palmitate and silica (p. 3, lines 17-20). Thecombination of sorbitan ester and ascorbyl palmitate forms a surfactant mixture which, following the addition of an aqueous phase, allows the formation of an extemporaneous emulsion by simple mixing (p. 9, lines 11-16). The presence of silica makes it possible to dehydrate the oily vehicle (page 9, lines 16-17), thus stabilizing the preparation.In “The effects of pectins on survival of probiotic Lactobacillus s.p.p. in gastrointestinal juices is related to their structure and physical properties -Science Direct” Larsen N. , 1 September 2018 (2018- 09-01) pages 1-22-XP93187050, the ability of pectins to improve the survival of the following probiotic species was investigated: Lactobacillus fermentum PCC, L. reuteri, L. rhamnosus LGG, and L. paracasei F-19 in simulated gastric solutions.Problems o f the background artLiquid formulations containing probiotic microorganisms, despite the aforementioned dosing advantages, are susceptible to a number of factors which can compromise their stability. Environmental conditions, such as temperature and humidity variations, are capable of negatively affecting not only the viability of probiotics, but also their organoleptic stability. During industrial processes, these microorganisms can undergo mechanical stress, changes in temperature and can interact with the primary packaging, further contributing to their instability. These combined effects can generate a negative impact on the use of the product by the final consumer and lead to a significant reduction in the shelf life and effectiveness of the product itself.During the storage of liquid formulations, a significant problem is represented by the tendency of probiotics to form aggregates. This aggregation process can cause difficulties during resuspension or even lead to the formation of insoluble precipitates, commonly known as “cake”. These deposits can have negative effects on the overall quality of the finished product, changing the consistency and organoleptic appearance, and leading to inaccuracies in the dosage due to the inability to evenly distribute the probiotic within the solution.To counteract the aggregation process of probiotics in liquid products, one potential strategy is the use of stabilizing additives. These additives play a key role in slowing down the aggregation phenomenon of probiotics, allowing to keep the solution in a homogeneous state and preventing the formation of insoluble deposits for a longer period. This helps to preserve the integrity of the product and extend the shelf life thereof.Generally, aqueous solutions containing probiotics and stabilizing additives are widely used in different sectors, including food products. However, when dispersing probiotics in oil-based formulations, the options and availability of stabilizing additives are often more limited, especially for formulations intended for foods for children in paediatric age, as for example happens in the caseof what are called foods for special medical purposes (Dietary foods intended for special medical purposes for infants and young children, as defined by Directive 1999 / 21 / EC, and special foods for infants, section 13.1.5 [EU REGULATION 1333 / 2008 of the European Parliament and of the Council and subsequent updates].The limited availability derives from the different nature of oil-based formulations and compliance with current regulations related to products intended for children of paediatric age. These factors make the choice of additives more complex and limited with respect to aqueous formulations. In more specific terms, due to differences in the chemi cal -physical composition of oil-based formulations, stabilizing additives, not specially designed for such systems, may not be effective in preventing sedimentation.The use of waxes and / or thickening additives, for example, only partially solves this problem, allowing to create a solid suspension which does not require stirring before intake. The use of solid vegetable fats at room temperature, speeding up the precipitation of probiotics, allows the formation of voluminous solid aggregates and therefore sediments which can be easily dispersed. However, that has not yet been proven in the long term. Added to this is the fact that the use of these additives, as well as the use of solid vegetable fats, certainly requires the use of hot product preparation processes to promote the solubilisation thereof. This step would not make the process appealing from an economic point of view. In some cases, moreover, the oily suspensions thus obtained would regardless suffer from product usability problems since the additional use of emulsifiers and inorganic fillers such as silica is foreseen, so as to achieve the desired dispersibility and viscosity. However, the use of such additives could affect the product drip-dosing characteristics.Therefore, the need is felt to identify a formula which, in accordance with current regulations, simplifies the production process, to obtain products in which the probiotic microorganisms are easily resuspended inside the oily vehicle. This formula must also guarantee the organoleptic stability of the product in the long term without negatively affecting the viability of the probiotics and, in addition, the correct functioning and consistent dosage of the product.SUMMARY OF THE INVENTIONThe Applicant has identified a probiotic-based composition, in the form of an oily suspension, which solves the problems of the prior art. Furthermore, during the development of the inventive composition, the Applicant has also identified a process for its preparation, which is particularly advantageous with respect to the methodologies known in the state of the art, since it is carried out at low temperatures, close to room temperature.Therefore, an object of the present invention is a composition, for oral use, in the form of an oily suspension comprising(a) active ingredients comprising or consisting of- at least one probiotic microorganism, not encapsulated, in powder form;(b) at least one oily liquid vehicle for food use, and(c) a stabilizing mixture.The stabilizing mixture (c) is characterized in that it consists of:(c-1) at least one emulsifying agent in a concentration comprised between about 0.001% and about 5% by weight, on the total weight of the composition, said emulsifying agent being selected from at least one of:(c-1-1) at least one substance of plant origin for food use comprising or consisting of a mixture of glyceryl phosphatidic acid, and relative esters with inositol and choline.(c-1-2) mono- and / or diglycerides of fatty acids for food use and / or mono- and / or diglycerides of fatty acids with a C2-C6 mono- or poly-carboxylic acid, optionally substituted with one or more hydroxyl groups, for food use;(c-1-3) at least one sucrose ester with fatty acids for food use, and(c-2) - optionally, one or more carbohydrates.A further object of the present invention is a process for the preparation of the composition, comprising the steps of:(i) dispersing at least one emulsifying agent in the oily liquid vehicle for food use (b), until a first, clear solution is obtained;(ii) optionally, adding one or more carbohydrates (c2) to the first solution;(Hi) dispersing at least one probiotic microorganism in the solution, coming from stage (i) or from the dispersion obtained in stage (ii), preferably in a gradual manner, and stirring until a uniform suspension is obtained, wherein all the steps constituting the method are carried out under conditions of temperature < 25 ± 3 °C and of relative humidity < 30 ± 3%.In fact, the Applicant has found that the composition which is the subject of the present invention, unlike the other compositions of the state of the art, can be prepared with a process carried out at room temperature, with considerable savings in energy and set-up time.Advantages o f the inventionThe composition identified by the Applicant has the following technical benefits:It complies with the regulations in force in the paediatric field, since it does not require - for the purpose of product stabilization - the use of inorganic stabilizing agents / additives (such as silica, silica gel, silicates);The dosage of the product is easy to achieve consistently over time, since the composition does not contain stabilizing agents / additives of a fatty nature which affect the product viscosity / density.It is easy to make, since it does not contain encapsulated probiotics, nor does it require the use of high temperatures during preparation.Based on the stability analyses conducted by the Applicant (Examples 7, 8 and 9) it is noted, in fact, that the presence of the stabilizing mixture is able to guarantee a substantial stability of the particle size distribution and of the optical density value (turbidity), as indirect measurements of the aggregation of probiotics over time.DESCRIPTION OF THE FIGURESFigures 1-6'. Microscopic images (40x) of the formulations of Examples 1, 2, 3, 4, 5, and 6 and the respective comparisons, when tested for natural (25°C) or accelerated (40°C) stability.Figure 7 Graph related to the trend of turbidity (T%) or optical density (OD%) of the formulation according to Example 1 (above) and of a comparison formulation, at 25°C up to 24 months.Figure 8 is a graph of the trend of the optical density (OD%) at 25°C of the composition of Example 4 and the comparison at 25°C up to 3 months.DETAILED DESCRIPTION OF THE INVENTIONFor the purposes of the present invention, the definition comprising does not exclude the presence of further components or stages beyond those expressly listed after such a definition.For the purposes of the present invention, the presence of further components not expressly mentioned is excluded with the definition constituted by or consisting of.Therefore, with the definition of stabilizing agents consisting of an emulsifying agent (cl) and optionally of (c2) it is intended to state that the composition which is the subject of the invention does not contain thickeners such as solid vegetable fats at room temperature, for example those disclosed in WO2018 / 142346A, technological additives such as silica or derivatives thereof or mixtures of the aforesaid thickeners, while it can contain other conventional additives such as antioxidants, preservatives, flavouring, etc.The emulsifying mixture (cl) is contained in concentrations comprised preferably between about 0.001% and about 5%, preferably between about 0.005% and about 3.0%, preferably between 0.01% and about 2.5%, preferably between 0.01% and about 2.0% by weight.In the composition which is the subject of the invention, the at least one substance (c-1-1) employed as at least one of the possible emulsifying agents (cl) is lecithin of vegetable origin, preferably from sunflower, which comprises a mixture of glycerylphosphatidic acid, glycerylphosphatidyl inositol and glycerylphosphatidylcholine.As emulsifying agent (c-1-2) a mixture of mono- and di -glycerides of fatty acids and preferably the mixture identified with the code E471 as reported in EU Regulation no. 1333 / 2008 is used in the composition which is the subject of the present invention.Another type of component to be used optionally alternatively or in combination with the mixture of mono- and / or diglycerides of fatty acids are the relative esters of mono- and diglycerides of fatty acids with other C2-C6 mono- or poly-carboxylic acids possibly substituted with hydroxyl groups such as acetic, lactic, citric or tartaric acid, of mono- and diglycerides of fatty acids.Preferably, for the purposes of the present invention, the esters of the mono- and di-glycerides of fatty acids with citric acid identified with the symbol E472c reported in the aforesaid Community Regulation are used.Preferably as emulsifiers and belonging to class (c-1-3) in the oral composition which is the subject of the present invention is a mixture of esters, called sucresters, identified with the abbreviation E473 as reported in the aforesaid Community Regulation.The carbohydrates possibly present as component (c2) in the stabilizing agent (c) which is the subject of the present invention are preferably polysaccharides and in particular are chosen from pectin, starches and derivatives, cellulose and derivatives, gums and fibres for food use.Preferably the carbohydrates (c2), when present in the stabilizing mixture (c) of the composition which is the subject of the present invention have a concentration comprised between about 0.001% and about 10% by weight, based on the total weight of the composition.They can be present at concentrations preferably comprised between about 0.01% and about 8.0%, preferably between about 0.1% and about 6.0% by weight, on the total weight of the composition.The at least one probiotic (a) present in the composition which is the subject of the present invention is preferably chosen from the families of microorganisms: Lactobacillaceae, Bifidobacteriaceae , Enter ococcaceae, Streptococcaceae, Saccharomycelaceae. or mixtures of the foregoing.The concentration of said at least one probiotic (a) is preferably comprised between about 0.01% and about 25% by weight, on the total weight of the composition according to the present invention.The definition of oily vehicle (b) means either a vegetable oil as such or the related derivatives obtained by synthesis, such as medium-chain fatty acid triglycerides obtained for example from palm oil, the preparation of which comprises esterification with glycerol. These triglycerides are particularly suitable as vehicles for probiotics.The oily liquid vehicle for food use (b) is therefore preferably selected from: medium chain triglycerides, preferably palm oil, and / or a vegetable oil selected from: corn oil, sunflower seed oil, linseed oil, canola oil (Canadian oil low acid or Canadian brassica), avocado oil, grape seed oil, sesame seed oil, soybean oil, olive oil, rapeseed oil (Brassica napiis), hempseed oil, borage seed oil, amaranth oil, palm oil, coconut oil and mixtures of the foregoing.Preferably, the concentration of the oily liquid vehicle (b) in the composition which is the subject of the present invention is preferably comprised between about 65% and about 99% by weight, on the total weight of the composition.The composition which is the subject of the present invention can contain at least one further active ingredient selected from the group consisting of: Vitamins, preferably vitamins E, D3, A and K, plant extracts, oleoresins, essential oils or related mixtures.The composition which is the subject of the invention is especially suitable in subjects suffering from swallowing difficulties or from dysphagia or in paediatric subjects.In any case, depending on the probiotics present, the oral composition can be employed for many uses: to maintain the health of the intestinal, oral and vaginal microbiota, to stimulate the immune system, to counteract the effects of ageing and to promote mental well-being.The composition which is the subject of the invention is in particular suitable for the maintenance of gastrointestinal health or in the treatment of altered states of the intestinal barrier.The composition for oral use which is the subject of the present invention is preferably in the form of a dietary supplement.For the purposes of the present invention, dietary supplement is understood as: "'foodstuffs intended to supplement the common diet and constituting a concentrated source of nutrients, such as vitamins and minerals, or of other substances having a nutritional or physiological effect, in particular, but not exclusively, amino acids, essential fatty acids, fibres and extracts of plant origin, both mono- and multi-compound” , based on what is established by Directive 2002 / 46 / EC, implemented in Italy with Legislative Decree no. 169 of 21 May 2004.Examples of embodiments of the composition according to the invention are shown below, by way of non-limiting illustration.EXAMPLESExample 1Components % w / wB. / ac7 / .s HNO I 9 8.89Vitamin E 1.94Vitamin D3 0.04Lecithin 0.10Pectin 1.00Medium chain fatty acid triglyceride 88.03The composition was obtained by the following process, at a temperature < 25 ± 3 °C and relative humidity < 30 ± 3%. i) The lecithin is dissolved in the triglycerides of medium chain fatty acids, stirring continuously until completely dissolved, obtaining a clear and uniform solution; ii) Vitamin E and Vitamin D3 are added one after the other, under stirring, until completely dissolved. This phase produces a clear solution; iii) The pectin is added to the solution during stirring, producing a weakly visible suspension; iv) B. lactis HN019 is added to the suspension in aliquots, keeping the preparation under continuous stirring until a uniform suspension is obtained.The controlled working conditions ensure the consistency and stability of the preparation of the composition.Example 2Components % w / wB. Gc7 / .s HNO I 9 6.35L. rhamnosus HN001 8.47Vitamin D3 0.10Lecithin 0.10Com oil 84.98The composition was obtained by the following process, at a temperature < 25 ± 3 °C and relative humidity < 30 ± 3%. i) The lecithin is dissolved in com oil, stirring continuously until completely dissolved, obtaining a clear and uniform solution; ii) Vitamin D3 is added, under stirring, until completely dissolved. This phase produces a clear solution; iii) B. lactis HNO 19 and L. rhamnosus HN001 are added to the suspension in aliquots, keeping the preparation under continuous stirring until a uniform suspension is obtained.The controlled working conditions ensure the consistency and stability of the preparation of the composition.Example 3Components % w / wL. rhamnosus GG (FLORGG) 9.72Vitamin D3 0.09Lecithin 0.10Sunflower oil 90.09 The composition was obtained by the following process, at a temperature < 25 ± 3 °C and relative humidity < 30 ± 3%.i) The lecithin is dissolved in sunflower oil, stirring continuously until completely dissolved, obtaining a clear and uniform solution; ii) Vitamin D3 is added, under stirring, until completely dissolved. This phase produces a clear solution; iii) L. rhamnosus GG (FLORGG) is added to the suspension in aliquots, keeping the preparation under continuous stirring until a uniform suspension is obtained.The controlled working conditions ensure the consistency and stability of the preparation of the composition.Example 4Components % w / wB. GC7 / .S HN0 I 9 8.89Vitamin E 1.94Vitamin D3 0.04Lecithin 0.10Pectin 1.00Com oil 88.03The composition was obtained by the following process, at a temperature < 25 ± 3 °C and relative humidity < 30 ± 3%. i) The lecithin is dissolved in corn oil, stirring continuously until completely dissolved, obtaining a clear and uniform solution; ii) Vitamin E and Vitamin D3 are added one after the other, under stirring, until completely dissolved. This phase produces a clear solution; iii) The pectin is added to the solution during stirring, producing a weakly visible suspension; iv) B. lactis HN019 is added to the suspension in aliquots, keeping the preparation under continuous stirring until a uniform suspension is obtained.The controlled working conditions ensure the consistency and stability of the preparation of the composition.Example 5Components % w / wVitamin E 1.94%Vitamin D3 0.04%Lecithin 3.00%Pectin 1.00%Com oil 85.13%The composition was obtained by the following process, at a temperature < 25 ± 3 °C and relative humidity < 30 ± 3%. i) The lecithin is dissolved in corn oil, stirring continuously until completely dissolved, obtaining a clear and uniform solution; ii) Vitamin E and Vitamin D3 are added one after the other, under stirring, until completely dissolved. This phase produces a clear solution; iii) The pectin is added to the solution during stirring, producing a weakly visible suspension; iv) B. lactis HN019 is added to the suspension in aliquots, keeping the preparation under continuous stirring until a uniform suspension is obtained. The controlled working conditions ensure the consistency and stability of the preparation of the composition.Example 6Componentso / o w / wL. rhamnosus HN001 9.63%Lecithin 2.00%Medium chain fatty acid triglyceride 81.15%The composition was obtained by the following process, at a temperature < 25 ± 3 °C and relative humidity < 30 ± 3%.i) The lecithin is dissolved in the triglycerides of medium chain fatty acids, stirring continuously until completely dissolved, obtaining a clear and uniform solution; ii) B. lactis HN019 and L. rhamnosus HN001 are added to the suspension in aliquots, keeping the preparation under continuous stirring until a uniform suspension is obtained. The controlled working conditions ensure the consistency and stability of the preparation of the composition.Analysis of the compositions containing c) according to the present invention, obtained with respect to the relative comparison compositions in the absence of c)Table 1. Formulation of Example 1 -6 vs corresponding formulations without the technologyExample 7 -Visual evaluation of the probiotic aggregationAnalysis methodOptical microscopy is a valuable technique which allows to directly view and measure the particle sizes in a sample, providing a quick and convenient method to assess the particle size distribution ina sample. To quickly determine the aggregation process, the analysis was performed under a 40x light microscope.The three formulations according to Examples 1-3 were tested, (see Fig.1-3) and compared with identical formulations, lacking stabilizing agents. The microscope images were acquired on the sample at time TO (at the moment of preparation); at time T1 (1 month after preparation) in the case of stability under accelerated conditions, i.e., where the formulations are kept at 40°C; at time T15 (15 months after preparation) at time T24 (i.e., after 24 months) in the case of stability under natural conditions, i.e., at 25°C. In all cases, the images were acquired after vortex resuspension of the sample to simulate the use of the product in a standardized manner.The compositions of Examples 4-6 were also tested, of which:- those in Figure 4 related to the compositions of Example 4 and its comparison at TO or at the moment of preparation and at 3 months at 25°C;- those in figure 5 related to the compositions of Example 5 and its comparison at TO or at the moment of preparation and at 3 months at 25°C and at 40°C for 1 month;- those in Figure 6 related to the compositions of Example 6 and its comparison at TO or at the moment of preparation and at 9 months at 25°C and 40°C for 1 month.ResultsThe most significant microscopic images (40x) of the particle size for each formulation tested are shown in Figures 1-6. The difference in particle size between probiotics in oil and probiotics in oil with stabilizing additives can be observed.As shown in Figures 1-6, in the accelerated stability conditions the “visual” differences are more evident and suggest that the compositions of Examples 1-6 have a better dispersibility than the respective Comparisons 1-6. It should also be considered that, in these accelerated stability conditions, the formulations of Comparisons 1-6 exhibited the formation of cakes (indicated with * in the respective figures) which cannot be resuspended even after resuspension, which is not highlighted in the corresponding Examples 1-6 due to the presence of inventive technology. The resulting images exhibit the presence of large inhomogeneous aggregates (Figure 1-6, sections Tl, 40°C). Therefore, in order to obtain images related to the formulations of Comparisons 1-6, the cake was resuspended after manual rupture of the sedimentation.The presence of the stabilizing additives has a substantial impact on minimizing the aggregation process, thus maintaining the particle size distribution stable over time.These results provide valuable insight into the effectiveness of stabilizing additives in preventing aggregation and maintaining the desired particle size distribution, ultimately contributing to product stability and quality.Example 8 - Particle size analysis of probiotics in oilAnalysis methods - Measurement of particle size (PS) and imageThe measurement of particle sizes plays a crucial role in understanding the physical properties of probiotic suspensions and in assessing their tendency to aggregate. In this study, the chosen technique was microscopy, which allows direct visualization, image recording and particle size measurement. This approach offers the advantage of providing detailed information not only on the size of the particles but also on their shape and morphology.By accurately measuring the particle size, the degree of aggregation and the stability of the suspension can be evaluated. In this study, the probiotic suspensions tested are the same as those in Table 1 (Examples 1, 2, 3, 4, 5, and 6, and their Comparisons). Each probiotic suspension was vortexed for up to 2 minutes to ensure proper dispersion. Subsequently, an aliquot of the suspension was observed under a microscope. The particle sizes were measured at different times and storage conditions to assess any changes over time.ResultsAfter 24 months under standard conditions (25°C), the formulations of Examples 1-3 without stabilizing additives can have slightly larger particle sizes. However, when the probiotic in oil is subjected to accelerated stability by storage at 40°C, considerable differences in particle size are highlighted between the two formulations, as shown in Table 2. It should also be considered that, in these accelerated stability conditions, the formulations of Comparisons 1-3 exhibited the formation of non-suspendable cake even after vortexing, which is not highlighted in the corresponding Examples 1-3 due to the presence of inventive technology. Therefore, in order to obtain microscopic information of the particle size, the cake of the formulations of Comparisons 1-3 was resuspended after manual rupture of the sedimentation.The addition of the stabilizing additives plays a crucial role in keeping the probiotics separate, slowing the aggregation thereof: in the product with stabilizing additives, the particle size remains relatively small even after one month of accelerated stability, indicating that the additives effectively prevent or delay the aggregation process. In the product without stabilizing additives, on the other hand, the particle size significantly increases after one month, suggesting a greater tendency for aggregation and larger particles to form.These additives effectively inhibit the increase in particle sizes of probiotics in the dosage form. By hindering aggregation, stabilizing additives play a crucial role in maintaining the desired particle size distribution and in preventing the formation of larger aggregates with consequent reasonable cake formation. Table 2. Particle size (um) at 15 months and 24 months at 25°C and at 1 month at 40°C* Non-resuspendable products, cake formation: the sediment was manually ruptured for particle analysisThe particle size data of the compositions of Example 4 and their comparison at T3 at 25°C are shown in the following Table 3.Table 3. Particle size (um) at 3 months at 25 °C* Non-resuspendable products, cake formation: the sediment was manually ruptured for particle analysisThe following Table 4 shows the particle size of the composition of Example 5 and its comparison at 3 months at 25°C and at 1 month at 40°C.Table 4 Particle size ( m) at 3 months at 25 °C and at 1 month at 40° C* Non-resuspendable products, cake formation: the sediment was manually ruptured for particle analysis The particle size of the composition of Example 6 and its comparison at T9 at 25°C and T1 at 40°C are reported in the following Table 5.Table 5. Particle size (pm) at 9 months at 25 °C and at 1 month at 40° C* Non-resuspendable products, cake formation: the sediment was manually ruptured for particle analysisThese results highlighted for Examples 4-6 also confirm what was highlighted for longer times for formulations 1-3 according to the invention.Example 9 - Analysis of the optical density of probiotics in oilTest method - Measurement of optical densityIn this case, the measurement of optical density is used as an indirect method to evaluate the aggregation of probiotics, monitoring the sedimentation rate of the resuspended cells. The principle of this sedimentation is based on the fact that the largest and densest particles settle faster than the smallest and lightest ones. When a probiotic aggregates, it tends to deposit, while individual cells remain suspended longer.To carry out this measurement, a probiotic suspension is resuspended (up to 2 minutes of stirring) to create a homogeneous suspension, the optical density is measured at 660 nm and changes over time are observed.If the probiotic cells are well dispersed and do not aggregate significantly, the suspension will remain relatively stable and the optical density may not significantly change over time. However, if the probiotics aggregate, the larger aggregates deposit more quickly, resulting in a decrease in optical density (as the deposited aggregates no longer contribute to the turbidity of the suspension). By monitoring the optical density and observing any variations over time, the tendency of probiotics to aggregate can be deduced.A more marked decrease in optical density indicates a higher degree of aggregation, while a stable or minimal variation suggests a lower propensity for aggregation.In detail, the percentage of aggregation (Agg) was calculated according to the following formula: ioowhere Ao and Atare the suspension absorbance at 0 h after vortexing and 1 h after resuspension, respectively, from which the turbidity (T) is calculated by the following equation:T (%) = 100 - AggFor the purposes of the analysis, the composition according to Example 1 was compared with a formulation which does not use the technology disclosed in the present patent application, i.e., is free of the stabilizing mixture (Comparison 1 - see Table 1). The two compositions were maintained at 25°C and their turbidity was measured over time (0, 1, 2, 3 6, 9, 12, 15, 24 months).The turbidity data collected are shown in the graph of Figure 7.From the trend of the curves, it is noted that the presence of stabilizing additives not only helps to maintain the stability of the formulation during the initial preparation phase (TO), but also throughout its shelf life.Despite the absence of obvious sedimentation or cake formation when the formulation was stored at 25°C for 24 months, the divergent optical density trends observed between the formulations, developed with or without stabilizing additives, provide valuable information on particle aggregation processes. These results suggest that the presence of stabilizing additives can have a significant impact on product stability. The mixture of actives attenuates the aggregation of the particles and contributes to the overall stability of the product over time.As an example of the aggregation process, formulations containing the probiotic B. lactis HN019 with or without stabilizing additives (Example 1 vs Comparison 1) and the data of their optical density tests expressed as turbidity (%) were reported. Both formulations showed no sedimentation and were easily resuspendable. However, they showed distinct trends in optical density:- The formulation of Example 1 resulted in a substantially constant optical density value over time, equal to about 85%. This indicates the effectiveness of the stabilizing additives even better in the presence of a carbohydrate, such as pectin, in maintaining the stability of the desired optical density over the long term.- In the absence of stabilizing additives (Comparison 1), a lower optical density value was observed, indicating a propensity for aggregation between the probiotic microorganisms. Furthermore, the optical density value showed an inconsistent trend, characterized by an initial phase of increase, lasting up to 3 months, followed by a variable phase with a value of about 70%. At T18, there is instead a significant decrease in optical density, which anticipates the formation of a cake at T24.This behaviour suggests a lack of stability in the formulation without the presence of stabilizing additives, resulting in decreased optical density and potentially compromising the overall quality and efficacy of the product over time.The measurement of the optical density was also evaluated on the formulation of Example 4 up to 3 months and the results are reported in the following Table 6 and in the graph of Figure 8.Table 6. OD values up to 3 months at 25°CAs can be seen from both the graph and the table, the optical density of the composition according to the present invention is 90% already after 2 months, while that of comparison already collapses after 2 months due to the formation of large aggregates (cake).

Claims

CLAIMS:

1. Oral composition, in the form of an oily anhydrous suspension, comprising(a) active ingredients comprising or consisting of- at least one probiotic microorganism, not encapsulated, in powder form;(b) at least one oily liquid vehicle for food use, and(c) a stabilizing mixture, wherein: said stabilizing mixture (c) consists of:(c-1) at least one emulsifying agent in a concentration comprised between about 0.001% and about 5% by weight, on the total weight of the composition, said emulsifying agent being selected from at least one of:(c-1-1) at least one substance of plant origin for food use comprising or consisting of a mixture of glyceryl phosphatidic acid, and relative esters with choline and inositol;(cl-2) mono- and / or diglycerides of fatty acids for food use and / or esters of mono- and / or diglycerides of fatty acids with a C2-C6 mono- or polycarboxylic acid, optionally substituted with one or more hydroxyl groups, for food use;(c-1-3) at least one sucrose ester of fatty acids for food use and(c-2) - optionally, one or more carbohydrates; said anhydrous suspension not containing thickening agents selected from solid vegetable fats at room temperature, technological additives selected from silica and derivatives thereof, and mixtures of said thickening agents.

2. Oral composition according to claim 1, wherein said at least one substance (c-1-1), is lecithin of plant origin, preferably from sunflower.

3. Oral composition according to claim 1 or 2, wherein (c-1 -2) is selected from the group consisting of: a mixture of mono- and di-glycerides of fatty acids identified with the code E 471.

4. Oral composition according to any one of claims 1-3 wherein (c-1-2) is a mixture of esters of mono- and / or di-glycerides of fatty acids with a C2-C6 mono- or polycarboxylic acid optionally substituted with one or more hydroxyl groups, selected from acetic, lactic, citric or tartaric acid.

5. Oral composition according to claim 4, wherein (c-1-2) is a mixture of esters of mono- and diglycerides of fatty acids with citric acid and said mixture is identified with Code E472(c).

6. Oral composition according to any one of claims 1-5, wherein the at least one sucrose ester (c-1- 3) with fatty acids for food use is a mixture of esters identified with the code E473.

7. Oral composition according to any one of claims 1-6 wherein the polysaccharides (c2) are preferably polysaccharides, preferably selected from pectin, starches and derivatives thereof, cellulose and derivatives, gums and fibres for food use.

8. Oral composition according to any one of claims 1-7, wherein the one or more carbohydrates (c2) is in a concentration comprised between about 0.001% and about 10% by weight, on the total weight of the composition.

9. Oral composition according to any one of claims 1 to 8, wherein the at least one probiotic microorganism is selected from the families of microorganisms I.aclobacillaceae, Bifidobacteriaceae, Enter ococcaceae, Streptococcaceae, Saccharomycetaceae or mixtures of the foregoing.

10. Oral composition according to any one of claims 1-9 wherein the at least one probiotic microorganism is in a concentration comprised between about 0.01% and about 25% by weight, on the total weight of the composition.

11. Oral composition according to any one of claims 1 to 10, wherein the at least one oily liquid vehicle for food use (b) is selected from the group consisting of: medium chain triglycerides, and / or a vegetable oil preferably selected from: corn oil, sunflower seed oil, linseed oil, canola oil (Canadian oil low acid or Canadian brassica), avocado oil, grape seed oil, sesame seed oil, soybean oil, olive oil, rapeseed oil (Brassica napiis), hempseed oil, borage seed oil, amaranth oil, palm oil, coconut oil and mixtures of the foregoing.

12. Oral composition according to any one of claims 1 to 11, wherein the edible vehicle is in a concentration comprised between about 65% and about 99% by weight, on the total weight of the composition.

13. Oral composition according to any one of claims 1 to 12, comprising at least one further active ingredient selected from the group consisting of: Vitamins, preferably vitamins E, D3, A and K, plant extracts, oleoresins and essential oils.14 Oral composition according to claim 14 in the form of a food supplement.

15. Composition for oral use according to any one of claims 1-14, for a use selected from: maintenance of the health of the intestinal, oral and vaginal microbiota, stimulation of the immune system.

16. Non medical use of the oral composition according to anyone of claims 1-14 to counteract the ageing effects and to promote mental well-being.

17. Composition according to any one of claims 1-14 for use in the maintenance of gastrointestinal health or in the treatment of altered states of the intestinal barrier.

18. Oral composition for a use selected from those of claims 15 or 17 in subjects suffering from swallowing difficulties or from dysphagia or in paediatric subjects.

19. Process for the preparation of the composition according to any one of claims 1 to 14, comprising the steps of:(i) dispersing at least one emulsifying agent (cl) in the oily liquid vehicle for food use (b), until a clear solution is obtained;(ii) optionally, adding one or more carbohydrates to the first solution;(Hi) dispersing at least one probiotic microorganism in the solution, coming from stage (i) or from the dispersion obtained in stage (ii), preferably in a gradual manner, and stirring until a uniform suspension is obtained wherein all the steps constituting the method are carried out under conditions of temperature < 25 ± 3 °C and of relative humidity < 30 ± 3%.

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