Patent Publication Number: US-2022211082-A1

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

Description:
The present invention refers to fermented food products or food supplements and to a process for their preparation. 
     BACKGROUND OF THE INVENTION 
     Fermented foods are traditionally used for human nutrition from centuries, all over the world. Different types of foods are used as substrate for the fermentation process, including vegetables, meat, milk, fish, etc. The ecological environment strongly impacts the type of matrix to be fermented based on the prevalent availability of this substrate in that niche. 
     Fermentation is known to improve the nutritional value of the original substrate as well as to extend the so-called shelf-life of the food, allowing to store it for long time periods even in harsh environmental conditions. 
     Fermentation processes are based, especially for traditional food, on the activity of spontaneous communities of bacteria, fungi and yeast that, taken together, maximize their ability to take profit of the substrate. Some of the natural microbial fermenting communities represent wonderful examples of symbiosis between genetically different microbes. Unfortunately, natural spontaneous fermenting pools of microbes have to be preserved with continuity in order to maintain their features but the production of metabolites and flavors, associated to their fermentative activity, cannot be controlled. In most cases, this uncontrolled process is however not suitable for the industrial production of fermented food due to regulatory and safety requirements. 
     Fermented foods represent historically a valuable resource for the livelihoods of rural communities as well as for traditional artisanal local productions. Besides being a tool for income generation in this small-medium scale economy, fermentation assures stronger safety requisites and allows to design a very diversified pattern of processed foods suitable for long term storage, despite seasonal variations of natural resources. (FAO, 2012). 
     Usually fermented foods come from natural and spontaneous processes of degradation of food by natural communities of bacteria, yeast, fungi and molds that act together in order to take full metabolic advantage from the substrate. Traditional production processes by spontaneous fermenting communities are usually passed down by cultural and traditional values to subsequent generations. 
     Great diffusion of fermented food based on grain, fruit and vegetables as well as fermented alcoholic beverages demonstrate the relevance of fermented food in human nutrition. A detailed and comprehensive overview of the most known fermented food in the world was provided by FAO from 1998 to 2000. 
     The main reason for promoting fermented food is due to the acidification of the substrate that assures the prolongation of its shelf-life but other benefits are usually associated to this conventional technological advantage, that promoted at the very early beginning the development of fermented food in rural communities. Besides the preservative action of fermentation, it also provides palatability to nutritional substrates as well as improves their nutritional value e.g. by reducing the concentration in antinutrients. (Selhub et al. 2014). 
     Beneficial health effects are also traditionally attributed to fermented foods through the interaction of food with human gut microbiota and the positive impact that the improvement of the intestinal functions can have on the wellbeing of consumers. However, the mechanisms underlying these beneficial effects have not been clarified yet. 
     Naturally fermenting microbial communities are composed of complex groups of bacteria, fungi, mold, and yeast, some culturable and some non-culturable, able to utilize different food substrates (Tamang et al. 2016). Numerous research projects were focused on the comprehension of the composition of these natural spontaneous communities, that vary greatly in relation to various foods and beverages across the world. Anyway, the reliable identification of the different components based on taxonomic criteria is always a very difficult issue leading to an incomplete knowledge of these ecological groups. 
     Properly controlled fermentation can assure the maximization of beneficial nutrient and the reduction of potential deleterious effect due to antinutrients and contaminants, amplifying the beneficial action of fermented foods apart from their nutritive supporting action. 
     Current approach to the selection of starter strains for controlled fermentation of food is focused on the achievement of the best fermentative performances (e.g. yeast for fermenting flour for bakery) and/or to the reduction of risk of contamination (e.g. starter lactic acid bacteria for meat) and/or the promotion of the development of special and typical flavors and metabolites (e.g. starter strains for yoghurt, wine, etc). 
     On the other hand, probiotics are living microorganisms than, when administered in adequate amount, are able to exert beneficial effect on the host (FAO, 2002). Microbial strains with proven beneficial properties are available for human consumption as food (e.g. fermented milk) as well as food supplements and even drugs. The main and necessary feature to exert their beneficial action is that they have to be consumed in viable form and maintain their viability through the passage in the gut. Anyway, the use of probiotics in some critical conditions appears to be questionable in expert&#39;s opinion. 
     Carefully selected probiotics can be associated to different food substrates, other than milk, by performing a controlled fermentation able to maximize the properties of the substrate in terms of beneficial effect for special categories of population. Pregnant women, lactating mothers, baby during the weaning period usually suffer from an endogenous lack of bifidobacteria, a key group of bacteria whose beneficial functions mainly rely on the production of short chain fatty acids essential for the colonic functions. In order to promote the selective development of bifidobacteria, some prebiotics, such as fructo- and galacto-oligosaccharides are exogenously added to weaning foods (Sholtens et al. 2006). 
     Selected starter strains are developed at the industrial level in order to promote controlled fermentation of food. However, starter strains are usually selected for their technological features e.g. for their ability to rapidly degrade the substrate, but not for their potential benefit to the consumer and/or for their ability to improve the nutritional value of the food. 
     DESCRIPTION OF THE INVENTION 
     It has now been found that, by a suitable selection of a specific bacterial blend and of specific fermentation conditions, it is possible to obtain a new fermented ingredient for food and food supplements providing a beneficial action on the host, through the accumulation of useful metabolites from microbial activity as well as the reduction of antinutrients present in the original food substrate, such as phytates in cereals and legumes. In particular, it has been found a specifically selected bacterial blend, composed of  Lactobacillus gasseri, Lactobacillus rhamnosus  and  Bifidobacterium breve  able to effectively ferment food of vegetable origin. 
     A first object of the invention accordingly comprises a fermented food product or food supplement comprising a vegetable substrate fermented by a probiotic blend consisting of  Lactobacillus gasseri, Lactobacillus rhamnosus  and  Bifidobacterium breve.    
     A second object of the invention comprises a process for the preparation of a fermented food product or food supplement comprising a first fermentation step of a vegetal substrate with  L. rhamnosus  followed by a second fermentation step with  L. gasseri  and  Bifidobacterium breve.    
     The invention also concerns the use of the obtained fermented food product or food supplement for improving natural maternity, lactation and baby weaning. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Any strain of  L. gasseri, L. rhamnosus  and  Bifidobacterium breve  may be used according to the invention, in any ratio. A ratio of 1:1:1 expressed as cell count is preferred. 
     Preferred strains have been deposited at the BCCM/LMG collection at Ghent University (Belgium) under the Budapest treaty, under the following accession numbers: 
       Lactobacillus gasseri  LMG P-30998 
       Lactobacillus rhamnosus  LMG P-31000 
       Bifidobacterium breve  LMG P-30999 
     The vegetable substrate which is subjected to fermentation by the probiotic blend according to the invention may be selected from oat, barley, corn, cereals, pseudo-cereals, gluten-free vegetables, vegetables, fruits, nuts, optionally in combination with an oil such as corn oil, sunflower oil, olive oil or the like. Oat is particularly preferred as a substrate, alone or in combination with corn oil, sunflower oil or other vegetal oils. 
     If desired, the fermented food product or food supplement of the invention may also contain an animal milk, preferably donkey or sheep milk. 
     The fermented food product of the invention may be in any form suitable for administration, e.g. in liquid, powder or cream form. Said food product may be used as food ingredient and/or food supplement, added in concentration ranging from 0.5 to 20% by weight to conventional food such as yogurt, milk, formula, fruit juices, soft-drinks, baby foods, fruit jams, and the like. 
     The probiotic blend used in the fermentation of the vegetable substrate is not viable in the food or food supplement of the invention which is submitted to a pasteurization process that assures that at the end of the production process it does not contain viable microbial cells. Depending on the final processing conditions the resulting ingredient has different composition and features depending on the inactivation and/or preservation of some microbial metabolites and catabolic/anabolic products. 
     The fermented food product or food supplement of the invention is prepared by a process comprising a first fermentation step of the vegetal substrate with  L. rhamnosus  followed by a second fermentation step with  L. gasseri  and  Bifidobacterium breve . In fact, due to the inability of bifidobacteria to use oat drink as growth medium, and the only partial performances observed for  L. gasseri , the fermentation is carried out by inoculating the medium with  L. rhamnosus  first and then, after correcting pH to 6.0, by adding  L. gasseri  and  Bif. breve . By this strategy (2-steps fermentation) the substrate is firstly degraded by  L. rhamnosus , producing short chain sugars and free peptides and amino acids able to better support the growth of bifidobacteria. 
     As soon as the fermentation is complete, the substrate is subjected to thermal treatment to inactivate bacteria. The inactivated fermentation product can be directly used for animal or human consumption. Otherwise, the product is powdered by different technological approaches, e.g. by lyophilization, spray-drying or similar techniques. 
     In case of lyophilization, the inactivated fermented substrate is frozen to −20° C.-−80° C. and then dried in a lyophilizer. Timing and drying conditions depends on the size of the apparatus. Final powder has a humidity content between 3.0 and 4.5%. A fine powder, white and with sweet taste is obtained. The % of recovery of the powder from the liquid fermented substrate is between 10 and 30%. 
     In case of spray-drying, the inactivated fermented substrate is directly fed to a spray drier and treated at different inlet and outlet temperatures (from 80 to 180° C.) and different fluxes (in ml per min). Final powder has a humidity content between 2.0 and 4.0%. The appearance of the powder is fine, from white to yellowish with sweet taste. The % of recovery of the powder from the liquid fermented substrate is between 20 and 60%. The assays conducted in order to set the optimal production conditions suggested that the low temperature of spraying gave higher % of recovery of the dried powder. 
     The two-step fermentation process allows to maximize the ability of the selected blend to improve the quality of the original food substrate by accumulating useful metabolites, by reducing the concentration of potentially dangerous substances and by increasing the digestibility of natural foods, particularly of vegetables. 
     The fermented food product of the invention provides a number of beneficial effects, including the stimulation of bifidobacteria, the decrease of anti-nutrients such as phytates, the improvement of natural maternity, lactation and baby weaning, the modulation of abdominal pain by acting on the endo-cannabinoid system of human gut mucosa, the promotion of the digestibility of natural vegetable food and increase in the bioavailability of some nutrients. By promoting the growth of bifidobacteria, improving thereby the composition of gut microbiota, the fermented food of the invention effectively antagonizes pathogenic bacteria or potentially harmful microbes. The invention accordingly enriches and improves the diet of special category of consumers with very restricted food choice, such as weaning babies and elderly people. 
     The efficacy of the new fermented food for the natural improvement of maternity, lactation and weaning was evaluated by means of several in-vitro tests finalized to measure the bifidogenic power (ability to act as a prebiotic substance, determining an increase of bifidobacteria) by direct inoculation of milk and by fecal fermentation model (FMF), to modulate abdominal pain by acting on the endo-cannabinoid system of human gut mucosa and to reduce the amount of antinutrients contained in natural matrices of vegetable origin, promoting the digestibility of natural vegetable food and increasing the bioavailability of nutrients. The obtained results are reported in the following examples. 
     Example 1—Preparation of the Fermented Food 
     The following blends of different strains obtained have been used and compared either according to the process of the invention, by performing first the fermentation with  L. rhamnosus  and then with the two other species, as reported above (two step fermentation), or by fermenting the substrate with the three species contemporaneously, in the same conditions reported for fermentation 1. 
     Blend A 
       Lactobacillus gasseri  L6 
       Lactobacillus rhamnosus  L13b 
       Bifidobacterium breve  2TA 
     Blend B 
       Lactobacillus gasseri  20243 
       Lactobacillus rhamnosus  GG 
       Bifidobacterium breve  BB03 
     Blend C 
       Lactobacillus gasseri  LG36 
       Lactobacillus rhamnosus  Sp1 
       Bifidobacterium breve  M16V 
     Preparation of mother cultures: preparation of washed pure cultures of the 3 strains composing the blend by culturing the three bacterial species in the culture medium, centrifugation to eliminate spent supernatant, washing with pure water and resuspension with fresh medium or pure water. Mother cultures are counted on plate to check viability. 
     Fermentation No. 1 (one-step fermentation): inoculation of the food substrate with 1% final concentration of  L. rhamnosus, L. gasseri  and  Bif. breve  washed cultures (10{circumflex over ( )}5-10{circumflex over ( )}8 CFU/ml of medium). 
     Incubation for 18-24 hours at 37° C. in microaerophilic conditions, static or under slow agitation. 
     Final count of viable  L. rhamnosus, L. gasseri  and  Bif. breve  at least 3×10{circumflex over ( )}8 CFU/ml each, pH between 3 and 4. 
     Inactivation of viable cells of bacteria by thermal treatment e.g. pasteurisation at 65° C.×30 min. 
     Correction of the pH to 6.0-7.0 by sodium hydroxide (sodium bicarbonate and/or other alkaline reagents are also suitable). 
     Fermentation No. 2 (Two-Step Fermentation) 
     Phase 1—inoculation of the food substrate with 1% final concentration of  L. rhamnosus , washed culture (10{circumflex over ( )}5-10{circumflex over ( )}8 CFU/ml of medium). 
     Incubation for 18-24 hours at 37° C. in microaerophilic conditions, static or under slow agitation. 
     Final count of viable  L. rhamnosus  at least 3×10{circumflex over ( )}8 CFU/ml each, pH between 3 and 4. 
     Inactivation of viable cells of bacteria by thermal treatment e.g. pasteurisation at 65° C.×30 min. 
     Correction of the pH to 6.0-7.0 by sodium hydroxide (sodium bicarbonate and/or other alkaline reagents are also suitable). 
     Phase 2-Inoculation of the substrate fermented in Phase 1 with 1% final concentration of  L. gasseri  and  Bif. breve  washed cultures (10{circumflex over ( )}5-10{circumflex over ( )}8 CFU/ml of medium). 
     Incubation for 18-24 hours at 37° C. in microaerophilic conditions, static or under slow agitation. 
     Final count of viable  L. gasseri  and  Bif. breve  at least 3×10{circumflex over ( )}8 CFU/ml each, pH between 3 and 4. 
     Inactivation of viable cells of bacteria by thermal treatment e.g. pasteurisation at 65° C.×30 min. 
     Correction of the pH to 6.0-7.0 by sodium hydroxide (sodium bicarbonate and/or other alkaline reagents are also suitable). 
     The final concentrations in viable cells and the growth gain obtained with the species indicated in the blends A, B and C above in the one- or two-steps fermentations are reported in Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Comparison of the growth of the different blends (composed 
               
               
                 of three bacterial species) composing the blend in one-step 
               
               
                 fermentation process VERSUS two-step fermentation process. 
               
               
                 Total count of viable bacteria was performed at the end of the 
               
               
                 fermentation for both tested process, one-step, based on the 
               
               
                 simultaneous inoculum of the three bacterial species in the food 
               
               
                 substrate, and the two-step, based on a first fermentation with 
               
               
                 the species  L. rhamnosus  and, following complete inactivation of 
               
               
                 this species, by the fermentation with  L. gasseri  and  Bif. Breve . 
               
            
           
           
               
               
               
            
               
                   
                 Final concentration in viable cells (CFU/ml) 
                 Growth gain 
               
            
           
           
               
               
               
               
            
               
                 Blend 
                 one-step ferm 
                 two-step ferm 
                 (log10 CFUs) 
               
               
                   
               
               
                 BLEND A 
                 8.72 
                 8.88 
                 0.16 
               
               
                 BLEND B 
                 8.65 
                 9.11 
                 0.46 
               
               
                 BLEND C 
                 8.46 
                 9.24 
                 0.77 
               
               
                   
               
            
           
         
       
     
     The results reported in Table 1 show that the two-step fermentation secured an overall better performance of the blend and an increase in the final content in viable cells. Even if the obtained fermented food does not contain viable cells but only metabolic products from microbial fermentation, the concentration in viable microbial cells at the end of the fermentation process assures the final quality expected for the fermented food of the invention. It is evident that different strains provide, based on the same composition of the blend in terms of species, different results but all of them are able to assure, in a two-step fermentation process better results than in one-step fermentation. 
     The final concentrations of the single bacterial species composing the blends measured by selective counting of viable cells on laboratory growth media are reported in Table 2. Again, the two-step fermentation assures an improved growth of  Bif. breve  and of  L. gasseri  (from 3.32 log 10 CFUs and 0.7 log 10 CFUs, respectively) compared to one-step process in which the three species are added simultaneously to the substrate. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Comparison of the growth (as average of 3 different experiments) 
               
               
                 of the three bacterial species composing the blend in one-step 
               
               
                 fermentation process VERSUS two-step fermentation process. 
               
            
           
           
               
               
            
               
                   
                 Final concentration in viable cells (log10 CFUs) 
               
            
           
           
               
               
               
            
               
                 Strain 
                 one-step ferm. 
                 two-step ferm. 
               
               
                   
               
               
                 
                   B. breve 
                 
                 5.00 
                 8.32 
               
               
                 
                   L. rhamnosus 
                 
                 8.23 
                 8.86 
               
               
                 
                   L. gasseri 
                 
                 7.81 
                 8.51 
               
               
                   
               
            
           
         
       
     
     Example 2—Evaluation of the Bifidogenic Potential (Growth Assays in Milk) 
     The bifidogenic activity, i.e. the property to selectively support the growth of bifidobacterial, a bacterial genus naturally present in human gut producing beneficial metabolites such as short chain fatty acids, essential for the survival of colonocytes, was tested by adding fermented powders obtained as in Example 1 to commercial milk formulas (Chinese and Korean Ofmom® liquid formula). The modified milk was then inoculated with washed cultures of reference bifidobacteria (strains  Bif. breve  LMG P-30999 and  Bif. breve  LMG S-29966) and their growth was checked and recorded in 24 hours. Results of modified milk were compared with the results previously achieved with the same normal milk formulas. Tests were performed on Stage 1 formula milk. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Growth of  bifidobacteria  (reference strains  Bif. breve  LMG S-29966 and  Bif. breve  LMG P-30999) in formula 
               
               
                 milk stage 1 (for 0-6 months babies) added with 1% final concentration of powdered fermented ingredient. 
               
            
           
           
               
               
            
               
                   
                 Stage 1 formula 
               
            
           
           
               
               
               
            
               
                   
                 brand A 
                 brand B 
               
            
           
           
               
               
               
               
               
            
               
                 Reference 
                 Normal 
                 1% fermented ingredient 
                 original 
                 1% fermented ingredient 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 
                   bifidobacteria 
                 
                 formula 
                 BLEND A 
                 BLEND B 
                 BLEND C 
                 recipe 
                 BLEND A 
                 BLEND B 
                 BLEND C 
               
               
                   
               
               
                   Bif. breve  LMG 
                 7.64E+07 
                 2.12E+08 
                 2.61E+08 
                 1.96E+08 
                 4.73E+07 
                 1.18E+08 
                 9.64E+07 
                 2.66E+08 
               
               
                 S-29966 
               
               
                   Bif. breve  LMG 
                 4.09E+07 
                 3.64E+08 
                 1.29E+08 
                 3.45E+08 
                 2.55E+07 
                 1.47E+08 
                 1.39E+08 
                 1.06E+08 
               
               
                 P-30999 
               
               
                   
               
            
           
         
       
     
       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. 
     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 3—Evaluation of the Bifidogenic Potential by Fecal Fermentation Model (FMF) 
     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. 
     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. 
     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. 
     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 110 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. 
     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. 
     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). 
     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. 
     
       
         
           
               
             
               
                 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 
                 \ 
               
               
                   
               
            
           
         
       
     
     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. 
     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. 
     
       
         
           
               
             
               
                 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 
               
               
                   
               
            
           
         
       
     
     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 4—Improved Digestibility Compared to the Same Natural Non-Fermented Substrate 
     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 2+ , Zn 2+ , Mg 2+ , Mn 2+ , Fe 2+/3+ , proteins and amino acids. The assay was conducted by the Megazyme kit for the determination of phytate compounds. 
     Levels of antinutrients of food matrices, like phytates, decrease after the fermentation by the selected blend, as shown in Table 5. 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. 
     
       
         
           
               
             
               
                 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 
               
               
                   
                   
               
            
           
         
       
     
     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. 
     Example 5 Comparative Tests 
     An oat aqueous substrate has been fermented a blend according to the invention and with a blend according to EP 1169925 and with the blend according to the invention. 
     The tests have been carried out either according to the method disclosed in US 2010098805 or according to the method of the invention 
     Two Steps Fermentation According to US 2010098805 
     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 μg/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/l for the enumeration of  Bifidobacterium  genus. 
     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). 
     Two Steps Fermentation According to the Invention 
     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 
     The growth (in log 10 CFUs) of the two blends by the methods of the invention and of US 2010098805 s reported in Table 1. 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. 
     
       
         
           
               
               
               
            
               
                   
                   
               
               
                   
                 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 
               
               
                   
               
            
           
         
       
     
     The data in Table 1 show that the fermentation method of US 2010098805 does not allow the growth of 2 out of 3 strains of blend A. 
     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 ). 
     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. 
     Blend B could not replicate the performances of blend A, when subjected to the same fermentation methods and conditions. 
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