CO-ENCAPSULATED PROBIOTICS AND PREBIOTIC DIETARY FIBERS IN FOOD GRADE MULTIPHASE GEL SYSTEM AND USE THEREOF IN FATTY PRODUCTS

Abstract

A food grade double gel system or bigel is disclosed. The bigel comprises an oleogel phase and a hydrogel phase. The Olcogel phase comprises vegetable oil and a gelator, and the hydrogel phase comprises collagen. The bigel further comprises co-encapsulated probiotics and prebiotic soluble dietary fibers. Disclosed are also food products comprising the bigel as well as method for preparing the bigel.

Claims

1. A food grade double-gel system or bigel, comprising an oleogel phase, comprising vegetable oil and gelator thereof, and a hydrogel phase, comprising collagen, wherein said bigel further comprises co-encapsulated probiotics and prebiotic soluble dietary fibers.

2. The bigel of claim 1, wherein the gelator of the vegetable oil in the oleogel phase is carnauba wax.

3. The bigel of claim 1, wherein the co-encapsulated prebiotic soluble dietary fibers are extracted from berry pomace.

4. The bigel of claim 3, where i n the co-encapsulated prebiotic soluble dietary fibers extracted from berry pomace, selected from a group comprising: lingonberry, cranberry, sea buckthorn, and inulin.

5. The bigel of claim 1, wherein the co-encapsulated prebiotic soluble dietary fibers are extracted from inulin.

6. The bigel of claim 1, wherein the co-encapsulated probiotics are selected from a group comprising probiotic species: L. reuteri, L. plantarum, L. paracasei.

7. A food product, comprising the food grade double-gel system or bigel of claim 1.

8. The food product of claim 7, where in the food product is butter.

9. A method of preparing the bigel of claim 1, comprising steps of: a. preparing the soluble prebiotic dietary fiber from berry pomace; b. preparing the suspension of probiotic cells; c. for the hydrogel phase, dissolving collagen and soluble dietary fiber in distilled water and incubating; d. for the oleogel phase, dissolving the gelator, such as carnauba wax, in vegetable oil and incubating; e. homogenizing the resulting oil and water phases in two stages: mixing the oil and water phases and homogenizing the mixture; cooling down the homogenized mixture to the temperature appropriate for probiotic cells, adding the pre-prepared probiotic cells suspension, and additionally homogenizing the mixture; and f. immediately after homogenization, transferring the mixture to the ice bath to induce gelation of both phases, and optionally, storing the obtained food grade double-gel system or bigel in cold conditions.

10. The method of claim 8, where in the step a) of preparing the soluble prebiotic dietary fiber from berry pomace is performed in the following substeps: fresh or defrosted berry pomace is dried to a moisture content of 7-9% by using any of drying methods: hot air of 35-40 C. during 48-72 hours, freeze-drying in 50 C., 0.5 mbar, during 24-48 hours; optionally, the dried pomace is cooled, weighed, and stored in sealed packages in a well-ventilated room with a relative humidity of no higher than 75% and an ambient temperature not exceeding 20 C. up to 4 months, or refrigerated at 4 C. up to 12 months; before usage dry pomace is milled to 0.2-0.25 mm particles; powders are mixed with water in a ratio of 1:10, stirred for 10-15 min and centrifuged at 8000-10000 rev/sec for 15 min.; the separated water soluble fraction is mixed with ethanol in a ratio of 5:95 and stirred for 5-10 min.; and after filtering, the sediments are separated and dried by using any of drying methods: hot air of 35-40 C. during 48-72 hours, freeze-drying in 50 C., 0.5 mbar, during 24-48 hours.

11. The method of claim 9, wherein in the step b) obtained probiotic cell suspension has to contain no less than 1.310.sup.11 cfu/ml of viable cells, for using it in the bigel preparation.

12. The method of claim 9, where i n the step b) of preparing the suspension of probiotic cells is performed in the following substeps: Activating the probiotic strains using MRS broth and incubating at 37 C. for 22 hours aerobically, wherein the MRS broth is for the enrichment, cultivation, and isolation of Lactobacillus species; after incubation, extracting the probiotic cells by centrifugation at 6000 rpm for 10 min at +4 C. washing the extracted probiotic cells with sterile saline water; wherein the obtained probiotic cell suspension has to contain no less than 1.310.sup.11 cfu/ml of viable cells, for using it in preparation of the food grade double-gel system or bigel.

13. The method of claim 9, where i n the step c) of preparing the hydrogel phase is performed under the following preferred conditions: 60 g/100 g of collagen and 1.34 g/100 g of soluble dietary fiber dissolved in distilled water and incubated at 85 C. for 30 min continuously mixing.

14. The method of claim 9, where in the step d) of preparing the oleogel phase is performed under the following preferred conditions: 9 g/100 g of carnauba wax as a gelator was dissolved in vegetable oil and incubated at 85 C. for 30 min.

15. The method of claim 9, where in the step e) of homogenizing the oil and water phases is performed under the following preferred conditions: mixing the oil and water phases at ratio 25:75, and homogenizing the mixture for 60 s at 15 000 rpm, while maintaining the temperature at 85 C.; cooling down the homogenized mixture until 55 C., adding pre-prepared probiotic suspension by 1 ml/100 g, and additionally homogenizing the mixture at 11000 rpm for 60 s;

16. The method of claim 9, wherein the step f) of gelating the homogenized mixture is performed under the following preferred conditions: transferring the mixture to the ice bath to induce gelation of both phases, and optionally, the obtained food grade double-gel system or bigel is stored at +4 C. or 18 C.

Description

DESCRIPTION OF DRAWINGS

[0031] The invention is explained in the drawings and diagrams. The drawings are provided as a reference to possible embodiments and experimental results and are not intended to limit the scope of the invention.

[0032] FIG. 1 depicts viability of L. reuteri cells during storage of bigels loaded with probiotics and prebiotic dietary fibers at +4 C. for 180 days with bigel loaded with probiotics alone as control.

[0033] FIG. 2a, b, c depicts appearance of bigels loaded with probiotics and bigels loaded with probiotics and prebiotic dietary fibers (a) after preparation; (b) after storage at +4 C. for 180 days; and (c) after storage at 18 C. for 360 days: AControl-bigel loaded with L. reuteri; BBigel loaded with L. reuteri and soluble fibers extracted from sea buckthorn pomace; CBigel loaded with L. reuteri and soluble fibers extracted from cranberry pomace.

[0034] FIG. 3 depicts changes in consistency index of bigels loaded with probiotics and bigels loaded with probiotics and prebiotic dietary fibers during storage at +4 C. for 180 days.

[0035] FIG. 4a, b depicts changes storage modulus (G) of (a) bigels loaded with probiotics and (b) bigels loaded with probiotics and prebiotic dietary fibers during storage at +4 C. for 180 days (b).

[0036] FIG. 5 depicts viability of L. reuteri cells during storage of bigels loaded with probiotics and prebiotic dietary fibers at 18 C. for 360 days with bigel loaded with probiotics alone as control.

[0037] FIG. 6 depicts changes in consistency index and viscosity of bigels loaded with probiotics and bigels loaded with probiotics and prebiotic dietary fibers during storage at 18 C. for 360 days.

[0038] FIG. 7a, b depicts changes storage modulus (G) of bigels loaded with probiotics and bigels loaded with probiotics and prebiotic dietary fibers (a) after preparation and (b) after storage at 18 C. for 360 days.

[0039] FIG. 8 depicts L. reuters viability at various digestion time points.

[0040] FIG. 9A,B,C,D depicts the appearance of butter spread products: [0041] Anon-probiotic butter spread product after storage at +4 C. for 150 days; [0042] Bprobiotic butter spread product after storage at +4 C. for 150 days; [0043] Cnon-probiotic butter spread product after storage at 18 C. for 150 days; [0044] Dprobiotic butter spread product after storage at 18 C. for 150 days.

DETAILED DESCRIPTION OF INVENTION

[0045] The description discloses food grade double-gel (bigel) system based on oleogel (vegetable oil and carnauba wax as gelator) and hydrogel (collagen) with co-encapsulated probiotics and prebiotic soluble dietary fibers extracted from berry pomace as structural approach for the delivery of sufficient amount of viable probiotic cells in butter spread products without changing their quality properties. Probiotic bacteria remain viable (above 10.sup.7 CFU/g) for 12 months in a bigel stored at 18 C. and 6 months in a bigel stored at +4 C. Co-encapsulation of probiotics and prebiotics in bigel is mandatory for the viability of probiotics during storage at +4 C. and optional when stored at 18 C. The distinctive feature of the developed bigel system is the capacity to protect viable probiotic cultures under upper gastrointestinal tract conditions and release them in the colonic environment where complex microbiota is residing in order to achieve the expected beneficial effect to host health.

[0046] Components of the bigel. The components listed below and their amounts compose one of the possible product embodiments developed and tested in the laboratory. However, the following components and their amounts do not limit the present invention, both in terms of the content of the materials and the proportions of the composition: [0047] Distilled water; [0048] Vegetable oil used for the oleogel formation; [0049] Carnauba wax as gelator; [0050] Collagen concentrate with 90% of protein for the hydrogel formation; [0051] Suspension of probiotic cells (containing no less then 1.310.sup.11 CFU/ml viable cells); [0052] Soluble prebiotic dietary fiber extracted from berry pomace.

[0053] Preparing the soluble prebiotic dietary fiber from berry pomace. Fresh or defrosted berry pomace is dried to a moisture content of 7-9% by using various drying methodshot air (35-40 C., 48-72 hours), freeze-drying (50 C., 0.5 mbar, 24-48 hours). The dried pomace is cooled, weighed, and stored in sealed packages in a well-ventilated room with a relative humidity of no higher than 75% and an ambient temperature not exceeding 20 C. up to 4 months, or refrigerated at 4 C. up to 12 months. Before usage dry pomace is milled to 0.2-0.25 mm particles. Pomace powders are mixed with water in a ratio of 1:10, stirred for 10-15 min and centrifuged at 8000-10000 rev/sec for 15 min. The separated water-soluble fraction is mixed with ethanol in a ratio of 5:95 and stirred for 5-10 min. After filtering, the sediments are separated and dried by using various drying methodshot air (35-40 C., 48-72 hours), freeze-drying (50 C., 0.5 mbar, 24-48 hours).

[0054] Preparing the suspension of probiotic cells. The probiotic (Lactobacillus reuteri) strains were separately activated using MRS broth and incubated at 37 C. for 22 h aerobically. After incubation, the probiotic cells were obtained by centrifugation at 6000 rpm for 10 min at +4 C. and washed with sterile saline water. The obtained probiotic cell suspension contained no less than 1.310.sup.11 cfu/ml of viable cells and was used in the bigel preparation.

[0055] Preparing the bigels: Two types of bigels (BI) are prepared: [0056] Bigel loaded with probiotics in the hydrogel phase; [0057] Bigel, loaded with probiotics and prebiotic dietary fibers in the hydrogel phase.

[0058] For this purpose, two types of water solutions for the hydrogel phase were produced: [0059] 60 g/100 g of collagen and dissolved in distilled water and incubated at 85 C. for 30 min continuously mixing (for the bigel loaded with probiotics); [0060] 60 g/100 g of collagen and 1.34 g/100 g of soluble dietary fiber dissolved in distilled water and incubated at 85 C. for 30 min continuously mixing (for the bigel loaded with probiotics and prebiotic dietary fibers).

[0061] For the oleogel phase 9 g/100 g of carnauba wax (as a gelator) was dissolved in vegetable oil and incubated at 85 C. for 30 min.

[0062] The resulting oil and water phases were homogenized in two stages. Firstly, oil and water phases were mixed at ratio 25:75 and homogenized for 60 s at 15 000 rpm maintaining the temperature at 85 C. The mixture was cooled until 55 C., 1 ml/100 g of pre-prepared probiotic suspension was added and the mixture was additionally homogenized at 11000 rpm for 60 s. Immediately after homogenization mixture was transferred to the ice bath in order to induce gelation of both phases and was stored at +4 C. or 18 C.

[0063] Both prepared bigels contained 35.0 g/100 g of protein, 35.0 g/100 g of lipids and 29 g/100 g of water. These freshly prepared bigels were further used to prepare butter spread product.

[0064] Characteristics of the bigels. The following characteristics of prebiotic dietary fibers were examined: [0065] The prebiotic activity of different probiotics paired with various soluble dietary fibers extracted from berry pomace or commercial prebiotic inulin (used as a control); [0066] Kinetics of saccharide profile of various soluble dietary fibers extracted from berry pomace or commercial prebiotic inulin (used as a control) incubated with different probiotics.

[0067] Prebiotic activity (PA), reflects the ability of a given substrate to support the growth of an organism relative to other organisms and relative to growth on a non-prebiotic substrate, such as glucose. Therefore, dietary fibers containing carbohydrates can have a positive prebiotic activity score if they are metabolized as well as glucose by probiotic strains and are selectively metabolized by probiotics but not by other intestinal bacteria. The assay was performed according to Huebner et al. (2007) by adding 1% (vol/vol) of an overnight culture of each probiotic strain (Lactobacillus plantarum F1, Lactobacillus reuteri 182 or Lactobacillus paracasei subsp. paracasei ATCC BAA-52) to separate tubes containing MRS Broth with 1% (wt/vol) glucose or 1% (wt/vol) soluble dietary fibers extracted from berry pomace or inulin (known as prebiotic and used as control). The cultures were incubated at 37 C. at ambient atmosphere. After 0 and 24 h of incubation, samples were enumerated on De Man, Rogosa (MRS), and Sharpe agar (Liofilchelm). In addition, overnight E. coli ATCC 25922 bacteria were added at 1% (vol/vol) to separate tubes containing M9 broth with 1% (wt/vol) glucose or 1% (wt/vol) prebiotic. The cultures were incubated at 37 C. at ambient atmosphere, and enumerated on Plate Count Agar (PCA, Liofilchelm) after 0 and 24 h of incubation. Each assay was replicated three times. The prebiotic activity score was determined using the following equation:

[00001]$\mathrm{PA}=\left[\frac{\begin{array}{c}(\mathrm{Probiotic}\lg \mathrm{CFU}/\mathrm{ml}\mathrm{on}\mathrm{the}\mathrm{prebiotic}\mathrm{at}24h-\\ \mathrm{Probiotic}\lg \mathrm{CFU}/\mathrm{ml}\mathrm{on}\mathrm{the}\mathrm{prebiotic}\mathrm{at}0h\end{array}}{\begin{array}{c}(\mathrm{Probiotic}\lg \mathrm{CFU}/\mathrm{ml}\mathrm{on}\mathrm{the}\mathrm{glucose}\mathrm{at}24h-\\ \mathrm{Probiotic}\lg \mathrm{CFU}/\mathrm{ml}\mathrm{on}\mathrm{the}\mathrm{glucose}\mathrm{at}0h)\end{array}}\right]-\left[\frac{\begin{array}{c}(E.\mathrm{coli}\lg \mathrm{CFU}/\mathrm{ml}\mathrm{on}\mathrm{the}\mathrm{prebiotic}\mathrm{at}24h-\\ E.\mathrm{coli}\lg \mathrm{CFU}/\mathrm{ml}\mathrm{on}\mathrm{the}\mathrm{prebiotic}\mathrm{at}0h\end{array}}{\begin{array}{c}(E.\mathrm{coli}\lg \mathrm{CFU}/\mathrm{ml}\mathrm{on}\mathrm{the}\mathrm{glucose}\mathrm{at}24h-\\ E.\mathrm{coli}\lg \mathrm{CFU}/\mathrm{ml}\mathrm{on}\mathrm{the}\mathrm{glucose}\mathrm{at}0h)\end{array}}\right]$

[0068] Kinetics of saccharide profile of various soluble dietary fibers extracted from berry pomace or commercial prebiotic inulin (used as a control) was measured during their incubation with different probiotics (Lactobacillus plantarum F1, Lactobacillus reuteri 182 or Lactobacillus paracasei subsp. paracasei ATCC@ BAA-52). The samples were subjected to quantification of oligo-and mono-and di-saccharides content after 0, 2, 4, 8, 12, 24, and 48 hours of incubation by size exclusion HPLC method. This involved dissolution of 10 mg of a freeze-dried sample in 1 mL of Millipore water (10 mg/mL). Analyses were performed in a Thermo Scientific Ultimate 3000 HPLC system coupled to a RefractoMax 521 refractive index detector (Thermo Fisher Scientific, Waltham, MA, USA). Saccharide components were separated using two size exclusion columns in series, Shodex SUGAR KS-802 and KS-801 (8.0 mmID300 mm each (Showa Denko, Tokyo, Japan)), with ultrapure water as a mobile phase. The columns were operated at 80 C. with an isocratic flow rate of 0.5 mL/min, with a detector cell temperature of 60 C. Samples were run for 60 min, and the injection volume was 10 L. The total oligosaccharides were recorded as the sum of all the detected and quantified fractions of oligosaccharides with a degree of polymerization (DP) DP 7-10, DP 5-6, DP4, DP3, while the total mono-and disaccharides were recorded as a sum of the sucrose, glucose, fructose, sugar alcohols, and galacturonic acids. Quantification was achieved with external calibration curves of available standards or response factors. All chromatograms were recorded and processed using Chromeleon 7 software (Thermo Fisher Scientific, Waltham, MA, USA).

[0069] Characteristics of the bigels. The following characteristics of the bigels during storage at +4 C. for 6 months and 18 C. for 12 months were examined: [0070] physical stability, [0071] rheological properties, [0072] viability of probiotic cells.
Viability of probiotic cells during in vitro digestion of bigels was evaluated as well.

[0073] Rheological characteristics were evaluated by shear sweep and frequency sweep tests at 25 C. using a rheometer with a plate-to-plate system (diameter 20 mm, gap 2 mm).

[0074] The flow behaviour was estimated over a shear ranging from 0.01 to 500/s. Data were analyzed using the Herschel-Bulkley model, and the viscosity coefficient (K), and flow index (n) were calculated.

[0075] The limit of the linear viscoelastic (LVE) area was confirmed by the amplitude sweep test, before the frequency sweep test, and the shear strain value of 0.1% was determined for the LVE region. In the frequency sweep test, the storage (G) and loss moduli (G) were measured, and the angular frequency was changed from 0.1 to 100 rad/s at 25 C.

[0076] Viability of probiotic cells was measured every month of the storage. The viable counts of L. reuteri were determined at all sampling points following EN ISO 15214:1998. 1 g of the bigel was weighed into a tube containing pre-warmed (37 C.) 9 ml of sterile saline water and serial dilutions were made so that the number of colonies per plate was between 15 and 300. The viable counts of L. reuteri were evaluated by the pour plating method of 1 ml of preparation in MRS Agar with Tween 80. All samples were plated in quadruplicate. The plated Petri plates were incubated in 37 C. temperature for 72 h. Viable cell numbers were calculated as log CFU/g.

[0077] Probiotic cells stability at each sampling point (upon production and throughout 180 days storage) was expressed as log CFU/g, and calculated according to the equation:

[00002]$\log \mathrm{CFU}/g=\log \mathrm{CFU}/g_{\mathrm{ti}}-\log \mathrm{CFU}/g_{\mathrm{t0}}$

where ti represents the viable cell numbers reported at each sampling time throughout storage and to represents the initial viable cell numbers reported at sampling time 0 days.

[0078] Simulated in vitro digestion was performed with the prepared bigels to evaluate the viability of probiotic cells during in vitro digestion under upper gastrointestinal tract conditions. The study was performed according to the static in vitro digestion protocol Infogest. Two test tubes one with 5 g of bigel containing co-encapsulated probiotics and prebiotics and another with the suspension of probiotic cells (as a control) were used to simulate the digestion in the mouth, stomach and small intestine. Samples to measure probiotic survival were withdrawn at the following times: initial, following the salivary phase (5 min, G5), at the end of the gastric phase (120 min, G120), the beginning of the intestinal phase (D120), mid-way through the intestinal phase (240 min, D240), and end of the intestinal phase (360 min, D360). Gastric phase samples were neutralized to pH 7.00.1, and the digestive process of intestinal phase samples was stopped by cooling in ice water to 0-4 C. After the reaction was stopped, the amount of viable probiotic cells was determined.

[0079] A 1 gram sample was diluted in 9 mL sterile saline water (0.75% NaCl) according to EN ISO 15214:1998. To ensure accurate counts, the sample and saline water were shaken with a Vortex for 5 s. Serial dilutions were done, and enumeration was completed using a pour plate method. L. reuteri were grown aerobically on MRS agar with Tween 80 at 37 C. for 72 h. Viable cell numbers were calculated as log.sub.10 cfu/1 g.

[0080] Characteristics of the probiotic butter spread product made with BI. For the characterization of the probiotic butter spread product, it was made with bigel containing co-encapsulated probiotics and prebiotics and with probiotic cells suspension (as control). The following characteristics of the probiotic butter spread product were examined: [0081] rheological properties, [0082] textural properties, [0083] viability of probiotic cells.

[0084] Rheological characteristics were evaluated by shear sweep and frequency sweep tests at 25 C. using a rheometer with a plate-to-plate system (diameter 20 mm, gap 2 mm) as described above.

[0085] The viability of probiotic cells was measured as described above.

[0086] Statistical analysis. All analyses were carried out in triplicate. The results are presented as the meanstandard deviation. A p-value of <0.05 was used to indicate significant differences between the mean values determined by an analysis of variance (ANOVA) using Statistica 12.0 (StatSoft, Inc., Oklahoma, AK, USA, 2013). For sensory evaluation, scores were submitted to the ANOVA with product, gender, and dysphagia (yes/no) as fixed factors and participants as random factor. Interactions were removed from the model as they were found to be not significant.

[0087] Results. The ability of soluble dietary fibers extracted from berry pomace to serve as prebiotics is described in several aspects: prebiotic activity score and kinetics of saccharide profile of various soluble dietary fibers extracted from berry pomace or commercial prebiotic inulin (used as a control) incubated with different probiotics. Quantitatively, the prebiotic activity score of different probiotics paired with soluble dietary fibers extracted from various berry pomace or commercial prebiotic inulin (used as a control) varied with the strains of organisms tested. Results are presented in Table 1. The highest values of prebiotic activity scores were reported to soluble dietary fibers extracted from lingonberry, cranberry and sea buckthorn pomace on L. reuteri (1.2470.013, 1.2140.029 and 1.0350.009 respectively). The prebiotic activity scores of L. plantarum and L. paracasei paired with various soluble dietary fibers extracted from berry pomace were considerably lower than those indicated for L. reuteri. However, for these cells grown in a medium supplemented with soluble dietary fiber extracted from lingonberry and sea buckthorn pomace, the prebiotic activity scores were insignificantly higher or close to those determined for cells grown with inulin. It is very important that the PA scores of all tested soluble dietary fibers on L. reuteri were significantly (p>0.005) higher than that of commercial prebiotic inulin, indicating pairing soluble dietary fibers extracted from berry pomace with L. reuteri as an effective strategy for ensuring the viability of L. reuteri cells in an encapsulation system. The prebiotic activity scores for different berry pomaces and probiotic cells species are presented in Table 1.

TABLE-US-00001 TABLE 1 Prebiotic activity score of soluble dietary fibers extracted from berry pomace. Soluble dietary Prebiotic activity score for different probiotics fibers extracted grown with soluble dietary fibers from berry pomace L. reuteri L. plantarum L. paracasei Lingonberry 1.247 0.013.sup.d 0.202 0.000.sup.c 0.210 0.001.sup.c Cranberry 1.214 0.029.sup.c 0.060 0.002.sup.a 0.029 0.005.sup.a Sea buckthorn 1.035 0.009.sup.b 0.114 0.008.sup.b 0.179 0.004.sup.b Inulin 0.193 0.003.sup.a 0.103 0.022.sub.b 0.219 0.008.sup.c

[0088] Changes in the saccharide profile of soluble dietary fibers extracted from various berry pomace or commercial prebiotic inulin (used as a control) incubated with different probiotics for 48 hours were also measured (Table 2). Very similar kinetics of the saccharide profile were observed for all samples. Namely, a decrease of mono- and di-saccharides was determined after 24 h of incubation, while the amount of oligosaccharides remained unchanged. This means that the probiotic cells primarily used the mono- and di-saccharides from soluble dietary fiber as a carbon source for their activity. The remaining oligosaccharides will provide the carbon source needed for their activity, thus prolonging their viability. This is an important proof that enriching the environment of probiotic cells with soluble fibers extracted from berry pomace will prolong cells viability due to the presence of oligosaccharides, which the probiotic bacteria will be able to use as a carbon source for their activity. The saccharide profiles for different berry pomaces and probiotic cells species are presented in Table 2.

TABLE-US-00002 TABLE 2 Saccharide profile (mg/ml) of various soluble dietary fibers extracted from berry pomace or commercial prebiotic inulin (used as a control) incubated with different probiotics. Saccharides Incubation duration profile 0 h 2 h 4 h 8 h 12 h 24 h 48 h Soluble fiber extracted from lingonberry pomace with L. reuteri oligo 28.8 27.8 27.3 28.3 24.3 27.9 27.6 mono-di 4.3 4.7 4.4 4.7 4.6 0.7 0.7 Soluble fiber extracted from cranberry pomace with L. reuteri oligo 30.8 30.0 27.6 29.6 26.0 29.4 27.9 mono-di 4.3 4.6 3.7 4.4 4.1 1.3 0.6 Soluble fiber extracted from sea buckthorn pomace with L. reuteri oligo 29.2 29.7 28.4 27.6 24.7 27.6 27.5 mono-di 3.2 3.9 3.3 3.1 3.7 0.9 0.7 Inulin with L. reuteri oligo 30.9 31.4 30.9 26.1 27.4 30.8 28.5 mono-di 2.2 2.8 3.4 2.7 3.1 0.7 0.6 Soluble fiber extracted from lingonberry pomace with L. plantarum oligo 61.6 64.3 50.9 50.6 64.7 68.4 69.7 mono-di 7.6 8.7 7.4 6.9 7.5 0.9 1.2 Soluble fiber extracted from cranberry pomace with L. plantarum oligo 68.7 69.3 60.0 61.1 63.1 69.1 68.5 mono-di 7.5 7.4 6.9 6.5 5.9 2.6 0.7 Soluble fiber extracted from sea buckthorn pomace with L. plantarum oligo 68.5 67.7 68.2 68.0 62.7 71.5 68.7 mono-di 6.2 6.0 7.8 7.4 4.8 3.2 1.3 Inulin with L. plantarum oligo 75.2 71.2 73.3 69.1 71.9 76.0 74.8 mono-di 4.6 3.0 5.6 3.6 3.9 2.4 1.5 Soluble fiber extracted from lingonberry pomace with L. paracasei oligo 66.1 63.0 68.1 62.6 63.8 70.8 71.9 mono-di 9.4 8.8 44.3 8.9 9.4 1.8 2.6 Soluble fiber extracted from cranberry pomace with L. paracasei oligo 68.2 66.4 67.0 65.1 64.1 72.4 67.3 mono-di 7.0 6.9 9.0 7.9 6.9 2.8 1.4 Soluble fiber extracted from sea buckthorn pomace with L. paracasei oligo 68.7 66.0 66.7 64.9 63.6 68.5 65.2 mono-di 6.9 6.5 7.7 6.8 6.1 3.1 1.0 Inulin with L. paracasei oligo 75.2 73.0 74.5 71.3 69.1 61.4 76.4 mono-di 3.8 4.6 3.9 4.0 3.8 1.6 1.7

[0089] According to our results, we can reasonably say that soluble dietary fibers extracted from berry pomace have a prebiotic effect and the addition of them to the environment of probiotic cells can be used as an effective strategy for ensuring the viability of cells in an encapsulation system.

[0090] To use soluble dietary fibers extracted from berry pomace loaded together with probiotic bacteria in the double gel system, the bigel containing L. reuteri and soluble fiber extracted from berry pomace in the hydrogel phase was produced. As a control the bigel containing L. reuteri in the hydrogel phase was produced. Physical stability, rheological properties of bigel and viability of loaded L. reuteri cells were examined during storage of loaded bigel at +4 C. and 18 C. L. reuteri viable cell numbers in bigels loaded with cells and prebiotic dietary fibers extracted from berry pomace during storage for 6 months at +4 C. are presented in FIG. 1. In the initial time point, the viable cell numbers of all tested samples varied between 9.15 and 9.46 log CFU/g. Throughout the storage period up to 180 days, L. reuteri viable cell numbers were found to be 7.41 and 7.61 log CFU/g (FIG. 1) when they were loaded into bigels together with prebiotic dietary fibers. Whereas, in bigel loaded only with L. reuteri cells and stored at the same conditions, the viable cells numbers were detected at the level 4.21 log CFU/g. Bigels loaded with L. reuteri together with prebiotic dietary fibers had significantly lower log cycle reduction by 180 days of storage in comparison with bigels loaded only with L. reuteri (Table 3). Meanwhile, bigels loaded with L. reuteri cells were less stable over storage registering >5 log cycle reduction by 180 days of storage. So, co-encapsulation of L. reuteri cells and prebiotic dietary fibers extracted from berry pomace in bigel system had positive effect on the viability of probiotic cells during storage for 6 months at +4 C. The results of this effect are presented in Table 3.

TABLE-US-00003 TABLE 3 Fluctuation in viable L. reuteri cell numbers during storage of bigels loaded with probiotics and bigels loaded with probiotics and prebiotic dietary fibers at +4 C. for 180 days. Bigels viable cell numbers after storage for sample 30 days 60 days 90 days 120 days 150 days 180 days 1 1.40 0.00.sup.aE 3.70 0.02.sup.aD 3.69 0.08.sup.aD 4.61 0.36.sup.aB 4.25 0.03.sup.aC 5.11 0.12.sup.aA 2 0.75 0.13.sup.bE 1.54 0.06.sup.cB 1.40 0.05.sup.bC 1.21 0.08.sup.bD 1.64 0.02.sup.cB 1.85 0.03.sup.bA 3 0.90 0.05.sup.bF 2.08 0.05.sup.bA 1.52 0.04.sup.bD 1.38 0.02.sup.bE 1.97 0.01.sup.bB 1.74 0.03.sup.bc 1Bigel loaded with probiotic cells; 2Bigel loaded with probiotic cells and prebiotic dietary fiber extracted from cranberry pomace; 3Bigel loaded with probiotic cells and prebiotic dietary fiber extracted from sea buckthorn pomace. Lower case letters indicate significant (p < 0.05) differences between different bigel samples and upper case letters indicate significant (p < 0.05) differences during storage

[0091] In order to evaluate the possibilities of using bigels loaded with probiotics and prebiotic dietary fibers in probiotic food products, their physical stability and rheological properties were determined during storage. From the pictures presented in FIG. 2 it can be seen that all bigels were able to support their own weight (did not flow when inverted). Bigel loaded with probiotics had an off-white color, bigels loaded with probiotics and prebiotic dietary fibers color was with a slightly brownish shadow depending on the soluble dietary fibers extracted from different types of berry pomace. The appearance of the bigels remained unchanged during the entire storage period. The rheological properties of bigels were explored by flow and viscoelastic behavior studies to provide insight into their utility for food application and stability during storage. The obtained results were shown in FIGS. 3 and 4. The consistency indices of bigel loaded with probiotics were lower than those of bigels loaded with probiotics and prebiotic dietary fibers and remained almost the same during the whole storage period indicating that all tested bigels displayed adequate stability. The storage modulus G of all tested bigels tended to increase with frequencies (0.1 to 100 rad/s) and the bigels were frequency-dependent during the whole period of storage. It was found that G of bigels loaded with probiotics and prebiotic dietary fibers remained similar throughout the 6 months of storage, while the G values of bigels loaded with probiotics alone differed significantly during storage. Such results testify to the positive influence of probiotic dietary fibers on the stability of bigels when they are co-encapsulated in the hygrogel phase of bigels together with probiotic bacteria.

[0092] While keeping the bigels at 18 C., we conducted their research for 360 days. As before, we studied the viability of probiotic bacteria and their rheological properties changes. FIG. 5 presents L. reuteri cells that were loaded alone or together with prebiotic dietary fibers in the bigels during storage at 18 C. for 360 days. In the initial time point, there were no significant variability in the cell numbers loaded in all bigels and they were in the range 9.5-10.0 log cycles. The counts of L. reuteri bacteria were found to be on the same line at the final sampling point (between 8.75 and 9.33 log CFU/g). Additionally, L. reuteri cells remained relatively stable during storage at 18 C. registering <1 log cycle reduction by 270 days of storage for bigels loaded with probiotics only and 360 days of storage for bigels loaded with probiotics and prebiotic dietary fibers (Table 4). So, co-encapsulation of L. reuteri cells and prebiotic dietary fibers extracted from berry pomace in bigel system had no positive effect on the viability of probiotic cells during storage for 12 months at 18 C.

TABLE-US-00004 TABLE 4 Fluctuation in viable L. reuteri cell numbers during storage of bigels loaded with probiotics and bigels loaded with probiotics and prebiotic dietary fibers at 18 C. for 360 days. viable cell numbers after storage for Bigels sample 60 days 150 days 270 days 360 days 1 0.75 0.00.sup.aB 0.40 0.06.sup.aD 0.57 0.09.sup.aC 1.27 0.00.sup.aA 2 0.32 0.01.sup.bB 0.08 0.04.sup.bD 0.44 0.03.sup.aA 0.20 0.03.sup.cC 3 0.26 0.07.sup.bB 0.06 0.03.sup.bD 0.02 0.06.sup.bC 0.68 0.01.sup.bA 1Bigel loaded with probiotic cells; 2Bigel loaded with probiotic cells and prebiotic dietary fiber extracted from cranberry pomace; 3Bigel loaded with probiotic cells and prebiotic dietary fiber extracted from sea buckthorn pomace. Lower case letters indicate significant (p < 0.05) differences between different bigel samples and upper-case letters indicate significant (p < 0.05) differences during storage.

[0093] Bigel systems encounter the problem of less structural stability during storage at low temperatures. In order to evaluate whether these systems can be used as a carrier of probiotic bacteria in food products, it is necessary to determine their structural stability during storage. We did this by comparing the flow and viscoelastic properties of bigels loaded with probiotics alone and bigels loaded with probiotics and prebiotic dietary fibers immediately after production and after storage at 18 C. for 360 days. The obtained results are shown in FIGS. 6 and 7. It was observed that Consistency index and viscosity slightly increased for bigels loaded with probiotics and slightly decreased for bigels loaded with probiotics and prebiotic dietary fibers during storage for 360 days at 18 C. The differences in storage modulus G of tested bigels were more pronounced at the very beginning of storage. After 360 days of storage the values of G were at the same range for all tested bigels. These data lead to the conclusion that regardless of the filling of the bigels, their structure remained stable during the entire period of storage at a temperature of 18 C., which was also confirmed by the appearance of the bigels. In FIG. 2c, we can see that after 360 days of storage at low temperatures, their appearance remained unchanged.

[0094] It is well established that food grade double-gel system based on oleogel and hydrogel with co-encapsulated probiotics and prebiotic soluble dietary fibers extracted from berry pomace is favourable for the delivery of sufficient amount of viable probiotic cells without changing structural stability of bigel during storage. Probiotic bacteria remain viable (above 10.sup.7 CFU/g) for 12 months in a bigel stored at 18 C. and 6 months in a bigel stored at +4 C. Co-encapsulation of probiotics and prebiotics in bigel is mandatory for the viability of probiotics during storage at +4 C. and optional when stored at 18 C.

[0095] To prove that bigel loaded with probiotics and prebiotic dietary fibers is capable to protect viable probiotic cultures under upper gastrointestinal tract conditions and release them in the colonic environment where complex microbiota is residing digestive degradation of the bigels loaded with probiotics and prebiotic dietary fibers was analysed. We evaluated the survival of co-encapsulated L. reuteri cells together with prebiotic dietary fibers throughout in vitro digestion. As a control, the suspension of L. reuteri cells was used in this experiment. Free L. reuteri cells and those encapsulated in bigel system showed similar trends in viability throughout in vitro digestion, with decreasing viability as digestion progressed (FIG. 8). In the gastric phase, the same behavior of L. reuteribacteria was recorded registering no significant variation in bacteria numbers at the beginning of the gastric stage (point G5) and around 4 log cycle reduction at the end of gastric stage (point G120), regardless of the matrix. The low survival of probiotic bacteria during the gastric phase was observed and it was obvious that the bigel system insufficiently protected the cells from degradation under acidic conditions (pH2). Although it should be stated that a slightly higher number of viable bacteria was recorded at this point in case of encapsulated L. reuteri cells. Upon reaching the intestinal phase probiotic cells numbers were higher in bigel system than that in the not protected bacteria suspension. Further in the intestinal phase (mid-way point D240 and end of the intestinal phase point D360), the viability of non-protected L. reuteri cells decreased while protected bacteria remained stable. Apparently, by encapsulating the bacteria in the bigel, it was possible to protect them from the lethal effects of the bile acids present in the intestine. Overall, the results showed there was no significant difference in L. reuteri cells viability during the gastric stage of digestion when they were uploaded into the bigel system versus non-protected bacteria. However, a trend of higher probiotic viability was recorded in bigels uploaded with L. reuteri cells and prebiotic dietary fibers compared to non-protected probiotics during the intestinal phase of in vitro digestion. Such results lead to the conclusion that the developed bigel system allows to keep probiotics viable under upper gastrointestinal tract conditions because of stable two-immiscible phase structure containing co encapsulated probiotic bacteria and prebiotic dietary fibers in hydrogel phase surrounded by oleogel phase.

[0096] In order to find out whether bigel could be used as a carrier of probiotic bacteria in the production of probiotic products, a probiotic butter spread product with the developed bigel loaded with L reuteri cells and prebiotic dietary fibers extracted from sea buckthorn pomace was produced. The probiotic butter spread product was stored under different conditions and the number of viable L reuteri cells, storage modulus G and hardness were determined monthly. Non-probiotic butter spread product (without bigel loaded with probiotics and prebiotic dietary fibers) was used as a control. Results are presented in Table 5.

[0097] The number of probiotic bacteria in the product is a crucial index for qualifying the product as a probiotic. According to the results of our experiment, the number of probiotic cells was 8.3 log CFU/g at the beginning of storage. The survival of L reuteri cells in the butter spread product depended on the conditions of storage. When the butter spread product was stored at +4 C., the viable cell number decreased gradually and was 7.1+0.2 log CFU/g after 2 months of storage thus meeting the required 6 log CFU/g for a functional probiotic food product. Further storage of the butter spread product resulted in the reduction of probiotics to a level where the product cannot be classified as a probiotic product. Different trends in bacterial viability were observed when the butter spread product was stored at 18 C. During the entire product storage period of 5 months, the number of probiotic bacteria decreased slightly to 7.4 log CFU/g. So, according to existing requirements, the product remained probiotic during the entire storage period.

[0098] The rheological and textural properties of the butter spread products stored in a refrigerator (+4 C.) and in a freezer (18 C.) were monitored at 30-day intervals for 5 months. As the storage time was increased, the results showed imperceptible changes of storage modulus G for both butter spread products regardless of the storage temperature. The trends in hardness changes during storage were also similar for both butter spread products. During the first 2 months of storage, the hardness increased and began to decrease during further storage, reaching the values determined at the beginning of storage. The results presented in Table 5.

TABLE-US-00005 TABLE 5 Changes of probiotic and non-probiotic butter spread product during storage at +4 C. and 18 C. Probiotic butter spread product made with bigel Control - butter spread product loaded with probiotics and prebiotic dietary fibers 0 1 2 3 4 5 0 1 2 3 4 5 Storage at +4 C. for months Viable cell n* n* n* n* n* n* 8.3 7.7 7.1 5.6 5.1 4.9 numbers of 0.1.sup.e 0.1.sup.d 0.2.sup.c 0.1.sup.b 0.1.sup.a 0.2.sup.a L. reuteri, log CFU/g Storage 1.64 1.28 1.81 1.78 1.85 1.85 1.49 1.55 1.48 1.55 1.57 1.52 modulus 0.04.sup.b 0.12.sup.a 0.03.sup.bcd 0.05.sup.bcd 0.17.sup.d 0.03.sup.cd 0.08.sup.ab 0.30.sup.ab 0.07.sup.ab 0.07.sup.ab 0.04.sup.b 0.01.sup.ab G, Pa .Math.10.sup.5, at 1 Hz, Hardness, 8.8 20.2 20.2 14.6 16.6 15.6 6.1 14.2 13.8 10.3 8.9 8.1 N 1.4.sup.a 1.4.sup.c 1.5.sup.c 1.6.sup.d 1.6.sup.f 1.7.sup.e 0.8.sup.a 3.2.sup.d 1.7.sup.d 1.1.sup.b 1.3.sup.ab 0.9.sup.ab Storage at 18 C. for months Viable cell n* n* n* n* n* n* 8.3 n* 8.1 n* 7.4 7.4 numbers of 0.1.sup.b 0.1.sup.b 0.1.sup.a 0.0.sup.a L. reuteri, log cfu g1 Storage 1.64 1.39 1.75 1.70 1.70 1.74 1.49 1.55 1.48 1.35 1.36 1.32 modulus 0.04.sup.bc 0.07.sup.a 0.08.sup.c 0.16.sup.ab 0.00.sup.bc 0.09.sup.bc 0.08.sup.ab 0.17.sup.b 0.09.sup.ab 0.02.sup.ab 0.09.sup.ab 0.10.sup.a G, Pa .Math.10.sup.5, at 1 Hz, Hardness, 8.8 16.2 18.4 9.7 11.5 10.5 6.1 10.0 14.7 7.8 6.9 5.3 N 1.4.sup.a 0.9.sup.c 2.1.sup.d 1.5.sup.ab 1.0.sup.b 0.8.sup.ab 0.8.sup.ab 0.8.sup.c 2.5.sup.d 0.4.sup.bc 0.3.sup.ab 0.8.sup.a Lower case letters indicate significant (p < 0.05) differences during storage.

[0099] These results confirm that bigels loaded with L reuteri cells and prebiotic dietary fibers provide protection to the probiotic bacteria and do not affect the appearance (FIG. 9) and texture of the food made with these bigels.

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