Process for producing a probiotic powder from by-products of wine production

Abstract

The present invention relates to a process for producing a probiotic powder from grape marc, comprising the steps of: a) mixing 75-85% of grape marc and 15-25% of an inoculum, said inoculum consisting of Lactobacillus casei 431 and Bifidobacterium BB12; b) keeping the mixture from step a) under aseptic conditions for a time interval in the range of 24-168 hours; c) drying the mixture from step b) at a temperature in the range of 60-65°C until it reaches a constant weight; and d) grinding the dry mixture from step c). The invention pertains to the field of production of probiotic products.

Description

[0001] DESCRIPTION

[0002] "PROCESS FOR PRODUCING A PROBIOTIC POWDER FROM WINE PRODUCTION BY-PRODUCTS"

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to a process for producing a probiotic powder from by-products of wine production.

[0005] BACKGROUND OF THE INVENTION

[0006] Currently, consumers are showing a growing interest in a healthy and sustainable diet, valuing the origin of the products they consume, and often demanding that suppliers consider the reuse and valorization of agri-food by-products.

[0007] The wine sector is a very representative sector in Portugal and of extreme importance to the Portuguese economy. However, it is also recognized that wine production generates large quantities of by-products with a possible environmental impact, but which, if valorized, can also be seen as having high economic and innovation potential. These agro-industrial by-products are rich in bioactive compounds with health benefits, such as strengthening the immune system, anti-cancer effects, and preventing cardiovascular diseases (Cimino et al., 2007; Çetin and Saõdyç, 2009; Xia et al., 2010; González-Centeno et al., 2012).Thus, by implementing technologies to add value to these by-products, eliminating their potential harmful effects, they can be recycled in the food chain as functional additives for different products and applications, ensuring sustainability, promoting a circular economy, and reducing by-products and waste resulting from winemaking.

[0008] The study of chemical composition, in general, reveals a richness in bioactive compounds, which are scientifically recognized for their beneficial effects. This fact is one of the reasons why the chemical composition of residues / by-products, and in particular those of wine products, is already extensively known.

[0009] Overall, the residues / byproducts obtained from wine production represent a valuable source of polyphenolic compounds (flavonoids, tannins, and benzoic acid derivatives), which are the main phytochemicals in these materials. These secondary metabolites may exhibit antioxidant, antimicrobial, anti-inflammatory, anticancer, and cardiovascular protective activities, activities that promote human health and reduce the risk of various diseases, especially chronic ones (Cimino et al., 2007; González-Centeno et al., 2012; Katalinic et al., 2010; Ky et al., 2014; Miljic' et al., 2016; Soleas et al., 1997; Yilmaz et al., 2011). They also have high nutraceutical interest due to their most notable bioactivity – their antioxidant potential – which consists of protecting the body by scavenging free radicals and inhibiting lipid oxidation (Çetin and Saõdyç, 2009; Xia et al., 2010; Yoo et al., 2010).Several studies have been developed focusing on the use of powdered fruit pulp as a probiotic functional food (Moreira, 2011; Albuquerque, 2021). Similarly, processes for producing probiotic functional beverages from fruits are also known, making them suitable for lactose intolerant and vegan individuals.

[0010] (https: / / uf cinova. uf c. br / pt / nectar-de-f rutas-probiotico / ) .

[0011] Dias et al. (2018), "Lactic Acid Bacteria as a Bio¬

[0012] The article "Preservative for Grape Pomace Beverage," published in Frontiers in Sustainable Food Systems, September 2018, Volume 2, Article 58, refers to the use of lactic acid bacteria to preserve a beverage / juice obtained from grape byproducts. In addition to their biopreservative potential, which improves the stability of the final product, the fermented juice, the lactic acid bacteria used as a starter culture also function as probiotics.

[0013] Silva et al. (2022). 75(2):249-260, August 1, 2022, evaluates the potential of a byproduct derived from the Isabel grape variety (IGF, Isabel Grape Flour) – flour – to stimulate the growth of different probiotics, as well as to assess the beneficial effect it may provide on the human gut microbiota under in vitro conditions. In summary, the study investigates how IGF flour can act as a prebiotic, promoting the growth of beneficial bacteria in the gut and improving gut health. Anghel et al.(2023), "Dried grape pomace with lactic acid bacteria as a potential source for probiotic and antidiabetic value-added powders", Food Chemistry: X 19 (2023) 100777, evaluates the probiotic and beneficial potential in diabetes control of the wine by-product - dried grape pomace, in powder form. To confer probiotic properties to the final product, a strain of lactic acid bacteria was used. The study also involved the analysis of two drying methods (convection and infrared), using temperatures of 40, 45 and 50 °C, and it was found that the drying methods impact the characteristics of the powders, affecting the enzymatic activity.

[0014] Patent application CN115851554 refers to a method for fermenting grape pomace using Lactobacillus plantarum YDB22. The described method allows obtaining freeze-dried powders and chewable tablets of fermented grape pomace.

[0015] Therefore, there is a need to provide a simple process for producing a probiotic powder from byproducts of wine production.

[0016] SUMMARY OF THE INVENTION

[0017] The present invention provides a process for producing a probiotic powder from grape pomace comprising the steps of: a) Mixing 75-85% grape pomace and 15-25% of an inoculum, said inoculum consisting of Lactobacillus casei 431 and Bifidobacterium BB12; b) Maintaining the mixture from step a) under aseptic conditions for a time interval in the range of 24-168 hours; c) Drying the mixture from step b) at a temperature in the range of 60-65 °C until it reaches a constant weight; d) Grinding the dried mixture from step c).

[0018] In one aspect of the invention, the mixture in step a) comprises 78-82% grape pomace, preferably 80% grape pomace.

[0019] In another aspect of the invention, the mixture in step a) comprises 18-22% inoculum, preferably 20% inoculum.

[0020] In yet another aspect of the invention, the time for step b) varies in a range of 36-120 hours, preferably 48-72 hours. More preferably, 72 hours.

[0021] In one aspect of the invention, the inoculum of step a) consists of 6xl0 9 CFU / mL of Lactobacillus casei 431 and 4.6 xlO 9 CFU / mL of Bifidobacterium BB12.

[0022] In another aspect, the drying temperature for step c) is 60 °C.

[0023] The present invention also relates to a probiotic powder obtained by the process described above, comprising a concentration of Lactobacillus casei 431 and Bifidobacterium BB12 in a range between 1x10 8 CFU / mL el,3xl0 10 CFU / mL.

[0024] DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention relates to a process for producing a probiotic powder from grape pomace comprising the steps of: a) Mixing 75-85% grape pomace and 15-25% of an inoculum, said inoculum consisting of Lactobacillus casei 431 and Bifidobacterium BB12; b) Maintaining the mixture from step a) under aseptic conditions for a period of time in the range of 24-168 hours; c) Drying the mixture from step b) at a temperature in the range of 60-65 °C until it reaches a constant weight; d) Grinding the dried mixture from step c).

[0026] In the present invention, "probiotic" means live microorganisms that, when administered in appropriate amounts, provide benefits to the body, namely: reduction of constipation, whether of bacterial or viral origin; increased defense of the intestinal flora against pathogenic organisms; reduction of excessive growth of the small intestine; prevention of colon cancer; reduction of allergic reactions and blood pressure; limitation of Helicobacter pylori infection (European Commission, 2008). Probiotics can be formulated in different forms, including foods and dietary supplements. Furthermore, for a product to be included in the probiotic product category, it must have a value >10 7 CFU / mL of probiotic bacteria.

[0027] Still within the context of the present invention, "inoculum" means the component of the mixture from step a) formed by lactic acid bacteria consisting of Lactobacillus casei 431 and Bifidobacterium BB 12.

[0028] In the context of this description, the term "comprising" should be understood as "including, among others." As such, the term should not be interpreted as "consisting only of."

[0029] Note that any value X presented throughout this description should be interpreted as an approximate value of the actual value X, since such an approximation to the actual value would be reasonably expected by someone skilled in the art due to experimental and / or measurement conditions that introduce deviations from the actual value.

[0030] Unless otherwise indicated, the value ranges presented in this description are intended to provide a simplified and technically acceptable way of indicating each individual value within the respective range. For example, the expression "1 to 2" or "between 1 and 2" means any value within this range, for example 1.0; 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9; 2.0. All values ​​mentioned in this description should be interpreted as approximate values, for example the reference to "60 °C" means approximately 60 °C.

[0031] Grape pomace is a byproduct obtained from wine production. In particular, in the context of the present invention, grape pomace consists of residual pulp, seeds, and skins of grapes. The pomace used in the process of the present invention has not undergone any prior fermentation step, therefore presenting a greater potential for the proliferation and development of the organisms to be added. The action of adding lactic acid bacteria to grape pomace can also be referred to as "inoculating".

[0032] In the context of the present invention, all values ​​referred to as percentages are understood to be percentages by mass (% m / m).

[0033] In the process of the present invention, the amount of grape pomace in the mixture is 75-85%, preferably 78-82%, and most preferably 80%.

[0034] The pomace used in the process of the present invention can be obtained from any type of grape. Preferably, pomace from red grapes produced for rosé wines and pomace from white grapes are used, because these products, during their production, have not undergone any fermentation process, thus presenting a greater potential for the proliferation and development of the organisms to be inoculated. Similarly, with regard to the bacteria used, they do not undergo changes depending on the type of grape used.

[0035] The inoculum present in the mixture of step a) of the invention process consists of Lactobacillus casei 431 and Bifidobacterium BB12 in a preferred manner, 6xl0 9 CFU / mL of Lactobacillus casei 431 and 4.6 xlO 9 CFU / mL of Bifidobacterium BB12.

[0036] The mixture in step a) comprises the inoculum in an amount of 15-25%, preferably 18-22%, and even more preferably 20%.

[0037] The bacteria that constitute the inoculum are widely known and, in general, associated with gastrointestinal benefits. Even though their value, in isolation, is already highly indisputable, it has been observed that symbiotic preparations with this type of probiotic bacteria enhance their probiotic behavior in a product obtained through the invention process.

[0038] Regarding the bacteria used in the invention process, they preferably have a concentration on the order of 10 9CFU / mL to ensure that the population present in the mixture of step a) of the invention process, even if it lost viability during the process, would not fall to levels that would compromise the probiotic character of the product obtained (the therapeutic effect of these probiotic bacteria known in the literature was observed with variable doses on the order of 10 6 10 9 CFU / mL). Naturally, the invention is not limited to the use of lactic acid bacteria concentrations of this order of magnitude. Regarding the ratio of Lactobacillus casei 431 and Bifidobacterium BB12 bacteria in the inoculum, these are preferably in a 1:1 ratio.

[0039] The residence time of the mixture from step a) under aseptic conditions varies within a time range of 24-168 hours, preferably 36-120 hours, more preferably 48-72 hours, and most preferably 72 hours. Although longer time intervals could be considered, the resulting product would have a significantly increased loss of viability and would therefore not meet the desired parameters for this type of product.

[0040] In the context of this description, "aseptic conditions" means any technical means or set of technical means that, in an industrial or laboratory environment, prevent or minimize contamination of the mixture from step a) by contaminating microorganisms such as, for example, undesirable fungi or bacteria, which may compromise the implementation or maintenance of the inoculum's cell viability and, thus, the probiotic character of the final product obtained by this invention. A person skilled in the art, as part of their routine activity, will know how to implement these "aseptic conditions" without need for further description, and, by way of example only, may use a sterile, sealed container in which the mixture from step a) can be kept for a period of time, as described for step b) of the invention.Other alternative methods may be implemented depending on the technical specifications required for the industrial implementation of the invention process.

[0041] In step c) of the invention process, the mixture is dried at a temperature in the range of 60-65 °C, preferably 60 °C.

[0042] Preferably, the drying method used in the process of the present invention is an oven. During the invention process, it was found that the use of an oven resulted in significant energy savings during the process and, therefore, the product was obtained ensuring a less environmentally damaging process. Those skilled in the art, within the scope of their technical capabilities, may understand that there are other suitable technical means for drying the mixture; however, its environmental contribution is highlighted alongside the effectiveness of the drying process.

[0043] The time required for the mixture to dry depends on variables such as the amount of mixture, the components of the mixture, etc., so it is important that at the end of the process and before the grinding stage, the product dries until it reaches a constant weight, i.e., until the absolute mass variation between consecutive weighings is less than 0.1%. At this point, it is understood that the sample has already lost the water present at the beginning of the drying step.

[0044] Surprisingly, it was found that using a drying temperature in the range of 60-65°C gives the final product the desired characteristics for a probiotic product, both in terms of appearance and organoleptic properties. Furthermore, the use of this temperature proved effective against contamination of the product obtained, as it eliminated the development of fungi and bacteria normally associated with the use of lower temperatures than those used in the present invention.

[0045] At the same time, the process of the present invention has proven very easy to implement on a large scale, at an industrial level, since it eliminates the complexity associated with this type of industrial process. The equipment used in the invention's process is well known to those skilled in the art and easily found in any ordinary laboratory.

[0046] Furthermore, the process of the present invention is simple and does not require the use of specialized equipment, which, from the outset, allows for its replicability in a wine cellar environment.

[0047] It is worth noting that the invention process uses by-products from wine production as raw materials, resulting in a reduction of waste generated by this industry as well as the valorization of these by-products, contributing to a Circular Economy.

[0048] It should be noted, however, that although the process is simple, the product obtained is not of lower quality than that required for the class of probiotic products. The product obtained by the process of the present invention is a probiotic powder comprising a concentration of Lactobacillus casei 431 and Bifidobacterium BB12 in a range between 1x10 8 CFU / mL el,3xl0 10 , which surprisingly proved to be higher than the reference value (>10 7 (CFU) for a probiotic product. Furthermore, it should be noted that final products with optimal appearance and organoleptic characteristics were obtained, and that they meet the legal requirements for probiotic products.

[0049] In addition to all these benefits, there is the advantage that the product obtained through the invention process covers various consumer niches (healthy lifestyle, athletes, vegans, lactose / gluten intolerant, etc.) and can be consumed in different ways (cold infusion, mixed in yogurts, cream cheese, herbal teas, etc.).

[0050] In this way, the process of the present invention makes it possible to obtain a dry extract of a probiotic powder.

[0051] EXAMPLES

[0052] For the sake of simplicity, although several other concentrations of Lactobacillus casei 431 and Bifidobacterium BB12 have been successfully tested, examples with preferred concentrations are presented below, which correspond to cases of obtaining probiotic powder with excellent functional performance.

[0053] Example 1

[0054] A mixture of 75% white grape pomace and 25% inoculum consisting of 6 x 10⁻¹⁰ was placed in a reactor. 9CFU / mL of Lactobacillus casei 431 and 4.6 xlO 9 CFU / mL of Bifidobacterium BB12. The mixture was kept under aseptic conditions for 120 hours and dried at 65 °C until it reached a constant weight. The dried mixture was ground to obtain a probiotic powder with a total concentration of 7 x 10⁻¹⁰ CFU / mL. 9 CFU / mL of Lactobacillus casei 431 and

[0055] Bifidobacterium BB12. Example 2

[0056] A mixture of 85% white grape pomace and 15% inoculum consisting of 6 x 10⁻¹⁰ was placed in a reactor. 9 CFU / mL of Lactobacillus casei 431 and 4.6 xlO 9 CFU / mL of Bifidobacterium BB12. The mixture was kept under aseptic conditions for 96 hours and dried at 63 °C until a constant weight was reached. The dried mixture was ground to obtain a probiotic powder with a total concentration of 6.8 x 10⁻¹⁰ CFU / mL. 9 CFU / mL of Lactobacillus casei 431 and Bifidobacterium BB12.

[0057] Example 3

[0058] A mixture of 80% white grape pomace and 20% inoculum consisting of 6 x 10⁻¹⁰ was placed in a reactor. 9 CFU / mL of Lactobacillus casei 431 and 4.6 xlO 9 CFU / mL of Bifidobacterium BB12. The mixture was kept under aseptic conditions for 72 hours and dried at 60 °C until it reached a constant weight. The dried mixture was ground to obtain a probiotic powder with a total concentration of 1.3 x 10⁻¹⁰ CFU / mL. 10 CFU / mL of Lactobacillus casei 431 and Bifidobacterium BB12.

[0059] Example 4

[0060] A mixture of 80% white grape pomace and 20% inoculum consisting of 6 x 10⁻¹⁰ was placed in a reactor. 9 CFU / mL of Lactobacillus casei 431 and 4.6 xlO 9 CFU / mL of Bifidobacterium BB12. The mixture was kept under aseptic conditions for 24 hours and dried at 60 °C until it reached a constant weight. The dried mixture was ground to obtain a probiotic powder with a total concentration of 1 x 10⁻¹⁰ CFU / mL. 8CFU / mL of Lactobacillus casei 431 and Bifidobacterium BB12. Example 5

[0061] A mixture of 80% white grape pomace and 20% inoculum consisting of 6 x 10⁻¹⁰ was placed in a reactor. 9 CFU / mL of Lactobacillus casei 431 and 4.6 xlO 9 CFU / mL of Bifidobacterium BB12. The mixture was kept under aseptic conditions for 168 hours and dried at 60 °C until it reached a constant weight. The dried mixture was ground to obtain a probiotic powder with a total concentration of 2 x 10⁻¹⁰ CFU / mL. 8 CFU / mL of Lactobacillus casei 431 and Bifidobacterium BB12.

[0062] Example 6

[0063] A mixture of 80% red grape pomace and 20% inoculum consisting of 6 x 10⁻¹⁰ is placed in a reactor. 9 CFU / mL of Lactobacillus casei 431 and 4.6 xlO 9CFU / mL of Bifidobacterium BB12. The mixture is kept under aseptic conditions for 96 hours and dried at 60°C until it reaches a constant weight. The dried mixture is ground to obtain a probiotic powder with a concentration of 8 x 10⁻¹⁰ CFU / mL. 9 CFU / mL of Lactobacillus casei 431 and Bifidobacterium BB12.

[0064] Example 7

[0065] A mixture of 85% red grape pomace and 15% inoculum consisting of 6 x 10⁻¹⁰ is placed in a reactor. 9 CFU / mL of Lactobacillus casei 431 and 4.6 xlO 9 CFU / mL of Bifidobacterium BB12. The mixture is kept under aseptic conditions for 48 hours and dried at 65 °C until it reaches a constant weight. The dried mixture is ground to obtain a probiotic powder with a concentration of 8 x 10⁻¹⁰ CFU / mL. 8 CFU / mL of Lactobacillus casei 431 and Bifidobacterium BB12.

[0066] It should be noted that although the present invention has been described with reference to its preferred embodiments, many modifications and alternatives may be realized by a person skilled in the art without departing from the scope of the invention, which is defined by the claims.

Claims

CLAIMS 1. Production process of a probiotic powder from grape pomace characterized by comprising the steps of: a) Mixing 75-85% grape pomace and 15-25% of an inoculum, said inoculum consisting of Lactobacillus casei 431 and Bifidobacterium BB12; b) Maintaining the mixture from step a) under aseptic conditions for a period of 24-168 hours; c) Drying the mixture from step b) at a temperature in the range of 60-65 °C until constant weight is reached; d) Grinding the dried mixture from step c).

2. Process according to claim 1, characterized in that the mixture of step a) comprises 78-82% grape pomace.

3. Process according to claim 2, characterized in that the mixture of step a) comprises 80% grape pomace.

4. Process according to claim 1, characterized in that the mixture of step a) comprises 18-22% inoculum.

5. Process according to claim 4 characterized in that the mixture of step a) comprises 20% inoculum.

6. Process according to claim 1, characterized in that the inoculum of step a) consists of 6xl0 9 CFU / mL of Lactobacillus casei 431 and 4.6 xlO 9 CFU / mL of Bifidobacterium BB12.

1. Process according to claim 1, characterized in that the time of step b) varies in a range of 36-120 hours.

8. Process according to claim 7, characterized in that the time of step b) varies in a range of 48-72 hours.

9. Process according to claim 8, characterized in that the time of step b) is 72 hours.

10. Process according to claim 1, characterized in that the drying temperature of step c) is 60 °C.

11. Probiotic powder obtained by the process as defined in claims 1-10, characterized by comprising a concentration of Lactobacillus casei 431 and Bifidobacterium BB12 in a range between 1x10 8 CFU / mL el,3xl0 10 CFU / mL.