Production method to increase bioavailability of sugars from natural complex polysaccharides for human, animal and agricultural purposes

Abstract

A fermented food product or food supplement comprising a vegetable substrate fermented by a probiotic blend is disclosed. The fermented food product or food supplement according to the invention is useful in particular for improving natural maternity, lactation and baby weaning. The invention also discloses a process for the preparation of the fermented food product or food supplement comprising the fermentation of a vegetal substrate with the probiotic blend.

Claims

1. A fermented product obtained by: generating short chain sugars, free peptides and amino acids by performing a first fermentation, by inoculating a plant substrate with Lactobacillus rhamnosus LMG P-31000; adjusting pH of the plant substrate to 6.0 to 7.0; and performing a second fermentation by inoculating the plant substrate with a probiotic blend comprising Lactobacillus gasseri LMG P-30998 and Bifidobacterium breve LMG P-30999.

2. The fermented product according to claim 1, wherein the plant substrate includes vegetables and at least one of a fruit, a nut, a grain, a cereal, and a pseudo-cereal.

3. The fermented product according to claim 1, wherein the plant substrate is oat.

4. The fermented product according to claim 1, wherein the plant substrate is oat in combination with an oil selected from the group consisting of: corn oil, sunflower oil, and olive oil.

5. The fermented product according to claim 1, wherein a CFU ratio of said L. rhamnosus inoculated in the first fermentation, said L. gasseri inoculated in the second fermentation, and said Bifidobacterium breve inoculated in the second fermentation is 1:1:1.

6. The fermented product according to claim 1, wherein the probiotic blend further comprises L. rhamnosus.

7. The fermented product according to claim 1, wherein the plant substrate is combined with donkey or sheep milk.

8. A method of manufacturing a fermented product comprising: generating short chain sugars, free peptides and amino acids by performing a first fermentation by inoculating a plant substrate with Lactobacillus rhamnosus LMG P-31000; adjusting pH of the plant substrate to 6.0 to 7.0; and performing a second fermentation by inoculating the plant substrate with a probiotic blend comprising Lactobacillus gasseri LMG P-30998 and Bifidobacterium breve LMG P-30999.

9. The method of manufacturing the fermented product according to claim 8, further comprising spray-drying by feeding the fermented plant substrate to a spray drier, wherein an inlet temperature and an outlet temperature of the spray drier are different.

10. A food item comprising the fermented product according to claim 1.

11. The food item according to claim 10 being in form of drink, cream, spread or powder.

Description

(1) FIG. 1 shows the growth of bifidobacteria in stage-1 liquid formula milks added with 1% fermented ingredient of the invention, expressed as log 10 CFUs, normalized as difference between the growth of the microbial blend in formula added with the new ingredient versus the commercial formula.

(2) The presence of the fermented ingredient allows bifidobacteria to growth better in milk in comparison to formula milk as acquired from the market (basic recipe). FIG. 1 shows that, independently from the commercial brand considered, bifidobacteria grow better in the presence of the fermented ingredient. Growth gain was in the range between 0.31 and 0.95 log 10 CFUs, depending on the blend used for the fermentation. The ability of the fermented ingredient to support the growth of bifidobacteria (bifidogenic power) was therefore confirmed in all of the tested experimental conditions.

Example 3Evaluation of the Bifidogenic Potential by Fecal Fermentation Model (FMF)

(3) Fermented powders of the invention were also checked for their bifidogenic power by means of FMF fecal fermentation. A laboratory model of gut fermentation was adopted to study how the ingestion of the fermented oat ingredient could impact the composition of gut microbiota by promoting beneficial bacteria so as to antagonize pathogenic bacteria or potentially harmful microbes.

(4) The ingredient obtained as in Example 1 was added at 1% final conc. to a standardized pooled fecal sample in the presence of fresh growth medium, simulating the food assumed by the consumer. After incubation in appropriate conditions (anaerobiosis at 37? C. for 24 hours, the growth of main bacterial groups was monitored by quantitative PCR, comparing their concentration at time zero and following incubation in the presence of the fermented powder. Fluctuations in main bacterial groups were therefore evaluated to estimate the potential beneficial and/or detrimental effect of the new ingredient on human gut microbiota.

(5) The ratio Firmicutes/Bacteroidetes (F/B) represents the first line of evaluation of the effect of the ingredient on human gut microbiota. Alterations of this ratio are associated to severe dysbiotic conditions such as those associated with obesity, gut chronic diseases, etc. Normal values are around 10, while significant altered ratios (e.g., values higher than 100) indicate an abnormal prevalence of Firmicutes, frequently associated to some diseases.

(6) The fermented food of Example 1 was checked in FMF model in order to exclude its influence on the F/B ratio. The safety of the ingredient was confirmed also by the fact that the F/B ratio was not altered in the course of the assay, independently from the composition of the blends used for the fermentation of the natural substrate. Firmicutes were quantified in 8.0 log 10 CFUs in feces of healthy human donors at the beginning of the assay and 7.6 after assay in the presence of glucose. The presence of the fermented ingredient as carbon source lead to a range of values 7.4-7.7 log 10 CFUs. Bacteroidetes were found to be present at time zero in 7.8 log 10 CFUs. Following the completion of the assay, Bacteroidetes were 6.5 log 10 in the presence of glucose and in a range between 6.4-6.8 log 10 in the presence of the fermented ingredient.

(7) F/B ratio was therefore calculated to be 1.03 in average at the beginning of the assay, increasing to 1.16 following incubation with glucose. The presence of the fermented ingredient lead to values 1.11-1.17. the ratio was confirmed not to be altered by the presence of the fermented ingredient.

(8) The effect of the fermented ingredient on total bacteria was also assessed in order to exclude potential detrimental effect generated on bacterial groups other than bifidobacteria. A slight increase in total bacteria was observed, as expected in the presence of glucose with 0.3 log 10 increase in total bacteria. The fermented ingredient determines 0.3-0.4 log 10 increase in the growth of total bacteria, similarly to what was determined by glucose (reference carbon source).

(9) The bifidogenic effect of the new fermented ingredient was confirmed by direct quantification of bifidobacteria in FMF model, as described above. Data of quantification of bifidobacteria by quantitative real-time PCR are provided in Table 4.

(10) TABLE-US-00004 TABLE 4 Quantification of bifidobacteria in FMF model by comparing glucose as conventional carbon source and the new fermented ingredient. Time zero concentration was also measured as well as value obtained in the presence of non-fermented natural ingredient. Average Count Growth copies per gr gain Carbon source number FD of FMF log (log10) Glucose 5.05E+06 46.9 4.74E+08 8.7 1.1 NF powder 1.67E+06 46.8 1.57E+08 8.2 0.6 Blend A 2.48E+06 44.5 2.21E+08 8.3 0.7 Blend B 3.40E+06 46.5 3.17E+08 8.5 0.9 Blend C 3.91E+06 57 4.45E+08 8.6 1.0 Blend D 3.55E+06 61.4 4.36E+08 8.6 1.0 T0 3.66E+05 53 3.88E+07 7.6 \

(11) Bifidobacteria are stimulated to a significant extent following exposition to the fermented ingredient. Similar results of growth gain just slightly lower than glucose were obtained.

(12) The effect of the fermented ingredient was also checked on a large range of final concentrations in order to demonstrate that its efficacy is dose-dependent and in which manner this association is expressed.

(13) TABLE-US-00005 TABLE 5 Quantification of bifidobacteria in FMF model by comparing different final concentrations of the fermented ingredient. Glucose was checked as positive control as conventional carbon source. Time zero level of bifidobacteria was also measured as well as the value obtained in the presence of non-fermented ingredient (used as negative control). CFU/ml Log10 Growth gain (log10) T0 4.20E+07 7.6 \ T48 NF powder 5.90E+07 7.8 \ T48 glucose 1.30E+09 9.1 1.3 T48 0.5% ingr 8.70E+08 8.9 1.2 T48 1% ingr 1.00E+09 9 1.2 T48 1.5% ingr 1.30E+09 9.1 1.3 T48 2% ingr 1.50E+09 9.2 1.4 T48 3% ingr 2.10E+09 9.3 1.5 T48 5% ingr 1.60E+09 9.2 1.4

(14) The stimulation of bifidobacteria by the fermented ingredient was confirmed in a range of final concentration from 0.5 to 5%. The best result in terms of activation of bifidobacteria was achieved by the final concentration of 3% that can be considered as the best dose to be assumed daily by consumers to induce positive bifidogenic effect.

Example 4Improved Digestibility Compared to the Same Natural Non-Fermented Substrate

(15) The content in antinutrients of the food ingredient of the invention was evaluated. Phytic acid is the primary source of inositol and storage phosphorus in plant seeds contributing at 70% of total phosphorus. The abundance of phytic acid in cereal grains and food of vegetable origin is a concern in the food and animal feed industries because the phosphorus in this form is unavailable to monogastric animals due to a lack of endogenous phytases, enzymes specific for the dephosphorylation of phytic acid. In addition, the strong chelating characteristic of phytic acid reduces the bioavailability of other essential dietary nutrients such as minerals, e.g. Ca.sup.2+, Zn.sup.2+, Mg.sup.2+, Mn.sup.2+, Fe.sup.2+/3+, proteins and amino acids. The assay was conducted by the Megazyme? kit for the determination of phytate compounds.

(16) Levels of antinutrients of food matrices, like phytates, decrease after the fermentation by the selected blend, as shown in Table 6. Different vegetable substrates were compared, by comparing non-fermented substrate with the fermented food according to the invention. The lowest concentration of phytate was detected in fermented ingredients oat-based, with values of 1.34 and 1.17 g per 100 g of fermented ingredient. Also in the rice, the levels of phytate decrease following fermentation according to the invention.

(17) TABLE-US-00006 TABLE 6 Decrease in the final concentration of phytates in fermented ingredient compared to the non-fermented natural substrate, with reference to different type of vegetables. Substrate Phytate (g/100 g) Non-fermented oat 2.98 Fermented oat blend A 1.34 Fermented oat blend B 1.17 Fermented oat blend C 1.44 Fermented oat blend D 1.71 Non-fermented rice 1.79 Fermented rice blend A 0.86 Fermented rice blend B 0.02 Fermented rice blend C 0.70 Fermented rice blend D 1.10 Non-fermented carrot 0.02 Fermented carrot blend A 0.01 Fermented carrot blend B 0.00 Fermented carrot blend C 0.02

(18) As expected, carrot was found to contain low level of phytates that almost disappeared following fermentation. Significant reduction was achieved in other substrates, such as cereals, naturally carrying a significant amount of phytates. Fermentation of the substrates with the selected blend led to the production of fermented powders naturally lowered in these antinutrient compounds. Lowering was of significant entity since phytate were halved with some of the tested microbial blends.

(19) Example 5 Comparative tests An oat aqueous substrate was fermented with an organism blend according to EP 1169925 and with an organism blend according to the invention.

(20) The tests have been carried out either according to the method disclosed in US 2010098805 or according to the method of the invention

(21) Two Steps Fermentation According to US 2010098805

(22) The aqueous substrate based on oat was incubated for 24 hours at 37? C. in order to let the naturally present microorganisms to develop inside the substrate. After incubation, the naturally fermented biomass is pasteurized in order to inactivate viable bacteria. The resulting product form first fermentation was then inoculated with strains L. casei ATCC334, L. acidophilus ATCC4356, B. breve ATCC15700 B. longum ATCC15707, B. longum sub. infantis ATCC15697 (Blend B) mixed together at the same time (second fermentation step). Viable counts on different selective media were performed to precisely quantified the inoculum size. MRS agar for the total Lactobacillus spp. viable counts. MRS supplemented with 10 mg/ml of vancomycin for the enumeration of L. casei ATCC334, and MRS supplemented with 0.1 and 10 ?g/ml of clindamycin and ciprofloxacin for the enumeration of L. acidophilus ATCC4256. The TOS propionate agar base medium (Sigma-Aldrich Merck) supplemented with mupirocin 50 mg/i for the enumeration of Bifidobacterium genus.

(23) The fermentation bulk was incubated for 24 h at 37? C. under microaerophilic conditions. Following incubation, viable counts were performed on the above-mentioned selective media to evaluate the final concentration reached by the microorganisms. The fermented biomass was then inactivated as previously described. The same procedure was repeated using in the second fermentation a blend consisting of Lactobacillus gasseri L6, Lactobacillus rhamnosus L13b and Bifidobacterium breve 2TA (Blend A).

(24) Two Steps Fermentation According to the Invention

(25) The process of example 1 has been repeated using blend A. Blend B was also subjected to a similar process, using L. paracasei ATCC334 in the first fermentation step in substitution of L. rhamnosus. L. paracasei has in fact the closest phylogenetical similarity to L. rhamnosus.

RESULTS

(26) The growth (in log 10 CFUs) of the two blends by the methods of the invention and of US 2010098805 s reported in Table 7. The data have been obtained by decimal count difference in plate between the growth time T24 and time TO (start of fermentation) for each strain under examination. The three bifidobacteria strains of blend B have been measured by means of quantitative PCR.

(27) TABLE-US-00007 Method of Method of US201098805 the invention Strains/blend Plate count qPCR Plate count qPCR Blend A B. breve 2Ta ?0.09 \ 1.29 \ L. gasseri L6 ?0.04 \ 2.97 \ L. rhamnosus L13b 2.89 \ 2.64 \ Blend B L. paracasei ATCC 334 2.63 \ 2.88 \ L. acidophilus ATCC 4356 ?0.02 \ 3.03 \ B. breve ATCC 15700 0.47 0.13 2.03 0.60 B. longum subs infantis 0.15 0.67 ATCC 15697 B. longum ATCC 15707 0.19 0.76

(28) The data in Table 7 show that the fermentation method of US 2010098805 does not allow the growth of 2 out of 3 strains of blend A.

(29) On the contrary, the two steps fermentation method of the invention enables the growth of the three strains of blend A as well as of the strains of blend B disclosed in EP 1169925 to the same extent to that of blend A for the species L. rhamnosus (phylogenetically similar to L. paracasei) and L. gasseri (phylogenetically similar to L. acidophilus).

(30) In addition, the method of the invention enables a more efficient fermentation of Bif breve in comparison to the other bifidobacteria included in blend B.

(31) Blend B could not replicate the performances of blend A, when subjected to the same fermentation methods and conditions.

REFERENCES

(32) 1. Lichtenberger, L. M. THE HYDROPHOBIC BARRIER PROPERTIES OF GASTROINTESTINAL MUCUS. Annu. Rev. Physioi (1995). doi:10.1146/annurev.physiol.57.1.565 2. Duary, R. K., Rajput, Y. S., Batish, V. K. & Grover, S. Assessing the adhesion of putative indigenous probiotic lactobacilli to human colonic epithelial cells. Indian J. Med. Res. (2011). doi:10.4103/0971-5916.90992 3. Deepika, G., Karunakaran, E., Hurley, C. R., Biggs, C. A. & Charalampopoulos, D.

(33) Influence of fermentation conditions on the surface properties and adhesion of Lactobacillus rhamnosus GG. Microb. Cell Fact. (2012). doi:10.1186/1475-2859-11-116 4. Rousseaux, C. et al. Lactobacillus acidophilus modulates intestinal pain and induces opioid and cannabinoid receptors. Nat. Med. 13, 35-37 (2007). 5. Wang, L. et al. Activation of epidermal growth factor receptor mediates mucin production stimulated by p40, a Lactobacillus rhamnosus GG-derived protein. J. Biol. Chem. (2014). doi:10.1074/jbc.M114.553800 6. Wong, B. S. et al. Pharmacogenetic trial of a cannabinoid agonist shows reduced fasting colonic motility in patients with nonconstipated irritable bowel syndrome. Gastroenterology (2011). doi:10.1053/j.gastro.2011.07.036 7. Hermanson, D. J. & Marnett, L. J. Cannabinoids, endocannabinoids, and cancer. Cancer Metastasis Rev. (2011). doi:10.1007/s10555-011-9318-8 8. Johansson, M. L. et al. Administration of different Lactobacillus strains in fermented oatmeal soup: In vivo colonization of human intestinal mucosa and effect on the indigenous flora. Appl. Environ. Microbiol. 59, 15-20 (1993). 9. El-Abasy, A. E., Abou-Gharbia, A., H., Mousa, H. M. & Youssef, M. M. Mixes of Carrot Juice and Some Fermented Dairy Products: Potentiality as Novel Functional Beverages. Food Nutr. Sci. (2012). doi:10.4236/fns.2012.32034 10. Daneluti, A. L. M. & Matos, J. do R. Study of thermal behavior of phytic acid. Brazilian J. Pharm. Sci. (2013). doi:10.1590/S1984-82502013000200009 11. Marshall E. & Mejia D. (2012) Traditional fermented food and beverages FAO Diversification booklet n. 21. 12. FAO. 2000a. Fermented Grain Legumes, Seeds and Nuts: A Global Perspective, FAO Agricultural Services Bulletin No. 142, Rome. 13. FAO.2000b. Grain legumes, seeds and nuts: rationale for fermentation. Fermented Grain Legumes, Seeds and Nuts: A Global Perspective, FAO Agricultural Services Bulletin No 142, Rome. 14. FAO. 1999. Fermented CerealsA Global Perspective, FAO Agricultural Services Bulletin No 138, Rome. 15. FAO. 1998. Fermented Fruits and VegetablesA Global Perspective, FAO Agricultural Services Bulletin No. 134, Rome. 16. Selhub E M, Logan A C, Bested A C. Fermented foods, microbiota, and mental health: ancient practice meets nutritional psychiatry. J Physiol Anthropol. 2014; 33(1):2. Published 2014 Jan. 15. doi:10.1186/1880-6805-33-2 17. Tamang J P, Watanabe K, Holzapfel W H. Review: Diversity of Microorganisms in Global Fermented Foods and Beverages. Front Microbiol. 2016; 7:377. Published 2016 March 24. doi:10.3389/fmicb.2016.00377 18. Joint FAO/WHO Working Group Report on Drafting Guidelines for the Evaluation of Probiotics in Food London, Ontario, Canada, April 30 and May 1, 2002 19. Scholtens, Petra A. M. J., Alles, Martine S., Bindels, Jacques G., van der Linde, Esmeralda G. M., Tolboom, Jules J. M., Knol, Jan. Journal of Pediatric Gastroenterology and Nutrition: May 2006Volume 42Issue 5p 553-559 doi: 10.1097/01.mpg.0000221887.28877.c7