SYMBIOTIC PROPERTIES

The present invention concerns a gluten free dry food, in particular a biscuit, having symbiotic properties and a process for the production thereof.

The invention refers to the field of preparation of food with high health power, indicated for consumption even by people who show symptoms of intolerance to wheat proteins. In this field, cookies play an important role in the daily diet of consumers, since they can be eaten as a snack at various times of the day.

It is known that, over the years, the nutritional awareness among consumers has led to the demand for products characterised by a low intake of calories and a high content of dietary fiber.

It is also known that celiac disease is an inflammatory disease of the small intestine triggered by wheat, barley and rye gluten proteins (Kaur et al., 2014). People with celiac disease must adhere to a gluten free diet for life. Therefore, gluten-free products and their marketing has grown at an annual rate of 28% in recent years (Glover, 2009).

It is particularly important, therefore, the need to formulate new foods studied in such a way as to provide important nutrients, including a high intake of dietary fiber, proteins, vitamins and mineral salts through the use of numerous cereals chosen among the gluten-free ones, including rice and millet.

The rice flour is naturally gluten free, rich in easily digestible carbohydrates and low in fat, and is often used for the preparation of gluten-free bakery products, such as bread and pastries, which are traditionally made with wheat flour (Man et al., 2014).

The use of millet flour is becoming increasingly common in baked goods, especially in bread, biscuits, crackers intended for gluten sensitive or diabetic consumers (Lovis, 2003). Millet appears to be a rich source of fibers and micronutrients, so much as to be called a nutri-cereal (Desikachar, 1977). Foods with a high content of dietary fiber, both total and soluble, have been associated with the reduced occurrence of a wide spectrum of diseases and can promote the correct functioning of the digestive system, bringing a sense of natural satiety (Chakraborty et al., 201 1 ).

Furthermore, the more and more increasing nutritional awareness among consumers has led to the demand for products made without yeast, milk and eggs, considered as food often involved in food intolerances, and with a low fat content, in particular to limit the intake of saturated fatty acids.

Another important aspect to which the search for new foods with high health power has been concentrated is the addition of probiotics and prebiotics. The probiotics are particular cultures of bacteria able to tenaciously overstep the gastric barrier, resisting the attacks of gastric juices and bile, improving digestion and absorption of microelements, and consequently favoring a better functioning of the intestine. In recent years the beneficial effect of intestinal microflora on human health is increasingly recognized and it is equally important that the diet can influence the relative quantity of microbial species and strains of the gastrointestinal flora (Holzapfel et al., 1998).

The bacteria belonging to the genera Bifidobacterium and

Lactobacillus turn out to be the microorganisms most used as probiotics supplements for food. They exert beneficial properties on human health, such as the inhibition of the growth of harmful bacteria, the stimulation of immune functions, additionally possess anti-tumor properties, promote cholesterol reduction, improve digestion and absorption of food/mineral ingredients and the synthesis of vitamins (Gibson, 1998). In analogy with probiotics, as living bacteria that, when added to foods, are able to cross the upper gastrointestinal tract and colonize the intestine, Delzenne & Roberfroid (1994) have proposed prebiotics, including non-digestible oligosaccharides (NDO), such as food ingredients to modify the composition of the endogenous intestinal microflora. Prebiotics are non- digestible food ingredients that can positively influence intestinal well- being by selectively stimulating the growth and/or activity of a limited number of colon bacteria that can improve health (Gibson & Roberfroid, 1995). Prebiotics include some oligosaccharides, especially fruit- oligosaccharides, which preferentially promote the growth of bifidobacteria in the large intestine.

To date, a great scientific interest has focused on the nutritional benefits of oligofructose and inulin (Hartemink & Rombouts, 1997; Roberfroid & Milner, 1999). Prebiotic fibres that are scientifically documented, and therefore usable in safety, for human use are: inulin, galacto-oligosaccharides (GOS) and fruit-oligosaccharides (FOS), which show a high strain-specificity towards bifidobacteria (Callegari et al., 2009). Consumption of appropriately selected probiotics, as well as prebiotics, may increase the beneficial effect of both (Bielecka et al., 2002).

A further possibility in the management of intestinal microflora is the use of symbiotics, a combination of probiotics and prebiotics (Indrio et al., 2009). A symbiotic has been defined as a mixture of probiotics and prebiotics that beneficially influences the host, improving the survival of live microbial food supplements in the gastrointestinal tract, selectively stimulating growth and/or activating the metabolism of one or a limited number of bacteria health promoters, and thus improving consumer welfare (Gibson & Roberfroid, 1995). A product having symbiotic properties is therefore able to bring the benefits of both probiotic and prebiotic approaches to consumer health. The probiotic treatment, recognized as functional and promising, has however its greatest problem in the vitality of the strains used in the finished formula (Callegari et al.,

2009).

The literature reports attempts to fine-tune wheat biscuits enriched with probiotics. However, these are always enrichment operations carried out after cooking, by applying on the biscuit an outer coating made of filling creams or covering icing based on milk or chocolate, in which the probiotic is inoculated. In fact, probiotics are very poorly resistant to high temperatures, so that their addition at a stage following that of coking the biscuit serves to ensure their vitality. On the other hand, such filling creams or covering icings can be the source of various forms of food intolerance.

Some attempts to add probiotics before cooking, by inoculation directly into the mixture, were made using sporogenic probiotics, which by nature are more heat-resistant than non-sporigens, but which, on the other hand, present the problem of contaminating the production plant with whom they come in contact.

In this context is inserted the solution according to the present invention, which proposes a dry food, and in particular a biscuit with healthy and functional characteristics that can contribute positively to a balanced diet for the consumer. The characteristics of this biscuit are numerous, since it is rich in important nutrients such as dietary fiber, proteins, vitamins and minerals, but is also gluten-free, being particularly indicated for consumption by people who show symptoms of intolerance to wheat protein and therefore are affected by celiac disease; as well as enriched with probiotics, which improve digestion and absorption of micro- nutrients. Furthermore, the biscuit proposed according to the present invention are low fat.

Since bacterial cultures do not withstand the process temperatures normally used for the production of this type of oven products, a new production process is proposed, not based on traditional cooking but capable of giving the product not only high nutritional properties but also the desired sensory quality, ie the right crunchiness, the desired color and a pleasant taste.

These and other results are obtained according to the present invention by proposing a dry food, in particular a biscuit, being gluten free and having symbiotic properties, in which prebiotics and probiotics, of a type not necessarily sporigenous, are present in the formulation of the dry food itself and not inside creams applied to the dry food, as well as a process for the production of said dry food, in which the cooking step of the mixture is replaced by a drying step under controlled conditions.

The purpose of the present invention is therefore to provide a dry food, in particular a biscuit, being gluten free and having symbiotic properties and a process for the production thereof that allow to overcome the limits of the solutions according to the prior art and to obtain the previously descrive technical results.

A further purpose of the invention is that said dry food and said process can be carried out at substantially reduced costs.

It is therefore a first specific object of the present invention a dry food, in particular a biscuit, comprising flour, water, living and vital probiotic microorganisms and prebiotic fibres, in addition to honey and/or sugar and that preferably additionally comprises cereals, obtainable by means of a process comprising a preliminary step of roasting flour, a step of mixing of the ingredients and a step of drying at a temperature tolerated by the probiotic microorganisms.

In particular, according to the invention, said flour and said cereals are gluten free.

Additionally, according to the present invention, said gluten free flour is chosen amongst conventional rice flour, red rice flour, Venere rice flour, buckwheat flour, carob pulp flour, and mixtures thereof and preferably is conventional rice flour mixed with carob pulp flour; whereas said gluten free cereals are chosen amongst puffed rice, puffed millet, puffed buckwheat and corn flakes.

Always according to the invention, said probiotic microorganisms are chosen amongst bacteria belonging to the genus Bifidobacterium and preferably are Bifidobacterium breve BBR8 and Bifidobacterium animalis subsp. lactis BI1 ; whereas said prebiotic fibres are chosen amongst inulin, galacto-oligosaccharides (GOS) and fruit-oligosaccharides (FOS).

It is additionally a second specific object of the present invention a process for the production of the previously defined dry food, comprising a preliminary step of roasting flour, a step of mixing of the ingredients, comprised the puffed cereals, probiotic microorganisms and prebiotic fibres, a step of conferring a desired shape and a step of drying at a temperature tolerated by the probiotic microorganisms.

Preferably, according to the invention, said drying temperature is lower than 30 °C and said probiotic microorganisms and said prebiotic fibres are added in pre-mixed form into the water of the formulation.

The invention will be described below by way of illustration, but not limitation, with particular reference to some illustrative examples.

In particular, the present invention concerns a formulation of biscuit based on flours and gluten free cereals, enriched with lyophilized probiotics strains and added with selected prebiotic fibres. More specifically, such probiotics can be selected from Bifidobacterium breve BBR8 and Bifidobacterium animalis subsp. lactis BI1 , while said prebiotics can be chosen from FOS, GOS and Inulin.

With regard to the production process of dry food according to the present invention, a roasting step of the raw materials is provided and then a drying step of the product for appropriate time and to an appropriate temperature tolerated by the probiotic microorganisms.

The particular innovation proposed by the solution of the present invention resides in having developed a product such as a biscuit, which is gluten-free and symbiotic, that is with living and vital prebiotics and probiotics. These characteristics of the final product are due to the production process forming an object of the present invention, because it is only thanks to the combination of certain process variables that an oven derivative can be produced that has the characteristics of a cooked product but that in reality has not been subjected to a heat treatment by means of traditional cooking.

Consequently, the present invention allows the realization of a final product that is not cooked but dried, so as to maintain intact all its nutritional properties, guarantee a contribution of vitamins and minerals that is not altered by the cooking processes and ensure the vitality of the same probiotic microorganisms. The application to the product of a drying process instead of a cooking process is of enormous importance for the realization of the dry food according to the present invention, since the low drying temperatures (<30 °C) allow to safeguard the delicate survival of probiotic strains within the finished product.

In particular, the direct inoculation of the probiotic strain in a lyophilized form in the biscuit formulation makes it possible to obtain a simple recipe, rich in cereals and crispy without the addition of any filling creams or covering icings.

The dry food according to the present invention has the following properties:

- use of gluten-free flour, in particular conventional rice flour and carob pulp flour, which make the product particularly suitable for people suffering celiac disease and who show symptoms of intolerance to wheat proteins;

- intake of dietary fiber, proteins, vitamins and minerals through the use of gluten-free cereals, such as puffed rice, puffed millet, puffed buckwheat and corn flakes;

- absence of yeasts, milk and eggs;

- low fat content, given the absence of butter and oil, which allows for limiting the intake of saturated fatty acids;

- symbiotic properties, resulting from the combination of strains of probiotics and prebiotic fibers, by exploiting the selective synergy between the components, capable of positively influencing the intestinal flora and consequently to improve the state of health of the consumer.

Furthermore, the formulation has the following preparation steps:

- preparing a mixture of gluten-free cereals and flours;

- inoculating of the lyophilized probiotics strains (1 %) and addition of the prebiotic fibres (1 %) in the recipe water;

- mixing all the ingredients to get the biscuit dough;

- forming of individual biscuits;

- drying of the biscuits at a temperature of 27 for 16 hours.

The application of the drying (not cooking) process at low temperatures (27 °C) allows to preserve the very delicate survival of the probiotics strains, guaranteeing their vitality in the finished product.

The experimentation involved the insertion of the inoculum in a lyophilized form directly into the biscuit matrix already in the preparation phase, without resorting, therefore, to the use of filling creams or covering icings, which are generally based on milk or chocolate, and in which generally the inoculation is carried out after the biscuit is fired to ensure its vitality but which consequently involves a greater number of preparation steps and longer execution times. In addition, the formulation of a dry biscuit, devoid of toppings or icings could overcome frequent problems of food intolerance (milk and dairy products, egg, etc.).

Example 1 . Materials and methods. Raw materials

Conventional rice flour, red rice flour, Venere rice flour, buckwheat flour and carob pulp flour were supplied by local producers; puffed rice, puffed buckwheat, puffed millet, corn flakes, sugar cane, wildflower honey and vanilla flavor were purchased at local supermarkets.

The lyophilized probiotics strains: Bifidobacterium breve BBR8 and Bifidobacterium animalis subsp. lactis BI1 , were provided by CSL (Centra Sperimentale del latte Sri, Lodi, Italy). The prebiotic fibres: FOS P95 (fruit- oligosaccharides), GOS L95 (galacto-oligosaccharides) and Inulin HP were provided by Beneo Orafti (Mannheim, Germany).

Example 2. Preparation of cookies

In the first experimental phase of the research the recipe for gluten- free biscuit was optimized. To this end, four different formulations based on conventional rice flour were made, to which 1 .7% (w/w) of red rice flour (R+RR), Venere rice flour (R+RV), buckwheat flour (R+GS) and carob pulp flour (R+C), respectively was added. The main differences between the formulations concerned the organoleptic aspects of the biscuit, evaluated through descriptive sensory analysis. Through the sensory analysis it was possible to select the optimal formulation that turned out to be the one containing the carob pulp flour (R+C). This formulation was made using: conventional rice flour (25.53%); carob pulp flour (1 .7%); water (25.53%); wildflower honey (17%); sugarcane (12.77%); puffed rice (4.26%); puffed millet (4.26%); puffed buckwheat (4.26%); corn flakes (4.26%); vanilla flavoring (0.43%). All formulations and specifically the optimized recipe (R+C), were prepared with the same procedure. Before mixing the ingredients, the rice flour was toasted in an electric oven (Europa Forni, Vicenza, Italy) at a temperature of 200 °C for 20 min. Subsequently, the dry ingredients such as rice flour, the carob pulp flour, the sugar cane, the puffed rice, the puffed millet, buckwheat and the puffed corn flakes were thoroughly mixed in a kneader (Conti Impastatrici, Verona, Italy) for 30seconds at low speed (60rpm), then the wildflower honey, the water and the vanilla flavoring were added, the mixture was mixed for 2min at a higher speed (l OOrpm). After kneading all the ingredients, the individual biscuits were formed, weighing 19g each, using a circular mold (5.5cm in diameter and 1 cm in thickness). The biscuits were formed on a tray previously coated with greaseproof paper and were placed to dry in a dryer (SG600, Namad, Rome, Italy) at a temperature of 27 °C for 16 hours. After the drying step, the biscuits were stored at a controlled room temperature (25 °C) for 1 hour and then subjected to sensory and microbiological analysis.

Example 3. Preparation of biscuits with addition of probiotics and prebiotics

In the second experimental phase, to make the biscuit become a product having symbiotic properties, the lyophilized probiotics strains (1 % w/w) Bifidobacterium breve BBR8 and Bifidobacterium animalis subsp. lactis BI1 were respectively added to the optimized recipe (called CTRL); and the prebiotic fibres (1 % w/w) FOS (fruit-oligosaccharides), GOS (galacto-oligosaccharides) and Inulin, respectively. Specifically, two different methods of insertion of probiotics and prebiotics in the recipe were used, in a first phase both were added in the form of powders, then this method was optimized by inserting both probiotics and prebiotics in pre-mixed shape in the recipe water. The following formulations were examined:

CTRL: reference biscuit (made with 1 .7% carob pulp flour)

B. breve: biscuit with 1 % of B. breve BBR8

B. breve+F : biscuit with 1 % of B. breve BBR8 and \ °/o of FOS (fruit- oligosaccharides)

B. breve+G: biscuit with 1 % of B. breve BBR8 and 1 % of GOS

(galacto-oligosaccharides)

B. breve+\: biscuit with 1 % of B. breve BBR8 and \ °/o of Inulin B. a. lactis : biscuit with 1 % of B. animalis subsp. lactis BI1 B. a. lactis+F : biscuit with 1 % of B. animalis subsp. lactis BI1 and 1 % of FOS (fruit-oligosaccharides)

B. a. lactis+G: biscuit with 1 % of B. animalis subsp. lactis BI1 and 1 % of GOS (galacto-oligosaccharides)

B. a. lactis+\ biscuit with 1 % of B. animalis subsp. lactis BI1 and

1 % of Inulin

The experimental formulations were prepared with the same method of preparing the control sample. All percentages have been calculated on the total weight of the dough.

Example 4. Sensory Analysis

The biscuit samples prepared according to the four different formulations of Example 2 were subjected to sensory evaluation using the QDA ^® (Quantitative Descriptive Analysis) test, in which more evaluations of the products have been suggested to exploit the ability of the rapporteurs in making judgments with a high degree of precision. Before proceeding with the sensory analysis, a meeting was organized with the judges to discuss the type of product and the identification of the relevant sensorial attributes (descriptors) of the product. Subsequently, a precise list of the selected descriptors and of the evaluation scale of the same was defined to guarantee objective judgments by the panelists and the correct conduct of the test.

Prior to sensory evaluation, the eight judges were trained using commercial cookies to become familiar with the use of the evaluation method, the terminology of each attribute and the sensory characteristics. The judges have evaluated the following organoleptic characteristics: appearance, color, aroma, flavor, sweet taste, bitter taste, crispness, friability, adhesiveness, hardness and overall quality of each sample, assigning a score (included in a scale from 1 to 5) on the degree of intensity of each descriptor and finally specifying a personal order of preference among the samples submitted for evaluation. The judges were instructed to clean up the palate with cold water before tasting each sample. Sensory evaluation was performed in isolated booths inside a sensory laboratory. For the biscuits related to the second experimental phase, described in Example 3, the sensory analysis of the biscuit control samples, inoculated and added with prebiotic fibres, was carried out according to the method described by Kaur et al. (2014) with some changes. Each sample was assigned a three-digit number in random order, and each judge was given a representative biscuit for each sample analysed. In addition, each panelist was provided with a glass of cold water to clean the palate between the different tastings. The judges were asked to evaluate the appearance, color, aroma, flavor, texture, crispness, friability, adhesiveness, hardness and overall quality of each sample. The samples were evaluated for acceptability using a score from 1 to 9. The definitions corresponding to each score were: 1 , extremely unwelcome; 2, not much appreciated; 3, moderately appreciated; 4, slightly appreciated; 5, neither appreciated nor unwelcome; 6, little appreciated; 7, appreciated; 8, very appreciated; 9, extremely appreciated. The evaluation was conducted under daylight and in isolated booths inside a sensory laboratory.

Example 5. Microbiological Analysis

The microbial count of the lyophilized strains Bifidobacterium breve BBR8 and Bifidobacterium animalis subsp. lactis BI1 was evaluated by vital count on plate. Subsequently, the lyophilized probiotics strains were respectively inoculated (1 % w/w) in the food matrix (raw biscuit) and the residual survival of the inoculated microorganisms in the analysed samples (raw and dried biscuit) was also determined by vital count on plate.

Both for the individual probiotics strains under examination and for the biscuit control samples (raw and dried), inoculated and supplemented with prebiotic fibres, the conducted microbiological analysis was as follows: 10g of each sample were weighed in a sterile plastic bag and homogenized in a Stomacher (Interscience, France) with 90 ml of diluent

(physiological solution containing 0.9% w/v sodium chloride) for 180 seconds, obtaining a suspension (1 /10 dilution of the sample, called mother dilution) that was subsequently placed at rest for 30 min at room temperature and homogenized for another 180 sec. The analyses were performed in duplicate on each sample. Serial dilutions in physiological solution were performed from the mother dilution and inoculated on appropriate medium. The medium and the incubation conditions used were the following: De Man, Rogosa, Sharpe (MRS) Agar (Oxoid, Milan, Italy) added with 0.5 g/L of L-cysteine HCI (Sigma-Aldrich Sri, Milan, Italy) and incubated at 37 °C for 72 hours in anaerobiosis.

Example 6. Measurement of the pH value

The pH measurement of the examined strains and of the biscuit control samples (raw and dried), inoculated and added with prebiotic fibres was performed in duplicate on each sample, taking 1 0g of sample and mixing it with 90ml of saline solution using a Stomacher (Interscience, France), the pH of the homogenate was measured with a pH meter (Crison Instrument, Barcelona, Spain).

Example 7. Statistical analysis of experimental data

For all the analyses, three repetitions were performed, of which the average values were calculated. The sensory, microbiological (log-iollFCg ^{" 1} ) and pH data of all the samples examined were submitted to One Way Variance Analysis (ANOVA). The Duncan Multiple Test with the option of homogeneous groups (p<0.05) was used to determine the significant differences. For this purpose the STATISTICA 7.1 program for Windows was used (StatSoft, Inc., Tulsa, OK, USA).

Example 8. Search results. Evaluation of sensory parameters

The results obtained from the QDA ^® sensory test regarding the comparison between the four proposed biscuit formulations, are shown in tables 1 .1 -1 .2.

Table 1 .1

Sweet Bitter

Sample Appearance Color Aroma Flavor

taste taste

4.13 ± 4.00 ± 4.63 ± 4.06 ± 4.25 ± 1 .06 ±

R+RR

0.64 ^a ' ^b 0.53 ^a ' ^b 0.74 ^a 0.68 ^a 0.71 ^a 0.18 ^b 3.50 ± 4.00 ± 3.44 ± 3.50 ± 1 .13 ±

R+RV 3.63 ± 0.74 ^b

0.76 ^b 0.38 ^a 0.50 ^a ' ^b 0.76 ^a 0.35 ^b

4.64 ± 4.00 ± 3.88 ± 2.88 ± 3.13 ± 2.75 ±

R+GS

0.63 ^a ' ^b _{0 27} a,b 0.35 ^a 0.23 ^b 0.35 ^a 0.71 ^a

4.75 ± 4.13 ± 4.00 ± 3.75 ± 1 .00 ±

R+C 4.88 ± 0.35 ^a

0.46 ^a 0.64 ^a 0.27 ^a 0.71 ^a 0.00 ^{b 0} Mean values in the same column followed by different letters in apex differ significantly (p <0.05)

Tabella 1 .2

⁰ Mean values in the same column followed by different letters in apex differ significantly (p <0.05)

Sensory analysis showed that, for all the samples examined, the judges perceived a different degree of intensity of the sensory attributes taken into consideration, highlighting the importance of each attribute and of its evaluation in order to compare the proposed formulations and indicate the order of preference. The visual sensory descriptors, such as appearance and color, were appreciated in the R+C sample, characterised by a light brown color typical of biscuits. In terms of taste, the aroma and flavor of the R+C and R+RR samples were perceived more intensively than the other samples. In addition, the R+RR biscuit had a sweetish taste, while a bitter note was perceived more intensely in the R+GS sample. With regard to the tactile sensations related to mastication, the R+C sample was pleasantly crisp and friable, moreover the same sample showed a lower adhesion to the palate compared to the other samples and a lower resistance to chewing or hardness. The evaluation of sensorial attributes, including overall quality, understood as the expression of the level of appreciation and general acceptability of the samples examined, highlighted a common preference of the judges for the biscuit sample made with the addition of carob pulp flour (R+C), data confirmed by the order of preference expressed by the judges who indicated the same sample for 75% as the best formulation.

From the processing of data emerged from the QDA ^® test the reference recipe on which to base the second phase of experimentation has been identified. This formulation, enriched with 1 .7% (w/w) of carob pulp flour, in the second experimental phase was indicated as a control sample (CTRL) and compared sensorially with the symbiotic formulations realized by inoculation of probiotics strains and the addition of prebiotic fibres. The sensory analysis was carried out to evaluate the acceptability of the sensory attributes of the investigated biscuit samples and the related results are illustrated in Tables 2.1 , 2.2, 3.1 and 3.2.

Table 2.1

Sample Appearance Color Aroma Flavor Texture

7.00 ± 7.63 ± 7.31 ±

CTRL 7.06 ± 0.32 ^c 7.88 ± 0.23 ^b

0.38 ^b 0.58 ^a 0.37 ^b

7.31 ± 7.06 ± 7.69 ± 8.13 ±

B. breve 8.44 ± 0.18 ^a

0.26 ^b ' ^c 0.18 ^b 0.37 ^a 0.23 ^a

7.88 ± 7.75 ± 7.75 ± 6.81 ±

B. breve+F 7.50 ± 0.00 ^c

0.35 ^a ' ^b 0.27 ^a 0.38 ^a 0.59 ^b 8.13 ± 7.25 ± 5.06 ±

B. breve+G 8.31 ± 0.37 ^a 5.63 ± 0.23 ^d

0.35 ^a 0.27 ^a 0.42 ^d

8.06 ± 7.31 ± 5.88 ±

B. breve+\ 8.25 ± 0.53 ^a 5.94 ± 0.18 ^d

0.18 ^a 0.37 ^a 0.23 ^c

^'a Mean values in the same column followed by different letters in apex differ significantly (p <0.05)

Tabella 2.2

a ^'e Mean values in the same column followed by different letters in apex differ significantly (p <0.05)

Tables 2.1 and 2.2 report the results related to the experimental formulations inoculated with the Bifidobacterium breve BBR8 and added with the respective prebiotics. The visual sensory attributes, appearance and color, came out to be appreciable for all samples, from the gustatory point of view, the sample B. breve showed the highest taste score with significant difference compared to the other samples. The data also indicated that even for mastication parameters such as texture, crunchiness and friability, statistically higher values were found for the B breve sample both with respect to the Control and to the samples added with prebiotics. The sensation of adhesiveness on the palate was perceived with less intensity in the CTRL and B. breve samples, indicating statistically higher scores and therefore better satisfaction compared to the other samples. An equally important parameter in the sensorial evaluation of the samples was found to be the hardness, attribute less perceived in the B. breve sample, that showed the highest acceptability score, with statistically significant differences. In particular, samples B. breve+G and B. breve+\ showed the lowest sensory values in terms of taste and of global quality, being barely acceptable. The sensory analysis showed that the B. breve inoculated biscuit sample was the most appreciated in terms of general acceptability, showing the statistically higher score among the samples examined.

The results related to the sensory evaluation of the samples inoculated with the Bifidobacterium animalis subsp. lactis BI1 and added prebiotics are reported in Tables 3.1 and 3.2.

Table 3.1

Sample Appearance Color Aroma Flavor Texture

8.13 ± 7.88 ± 7.56 ±

CTRL 8.19 ± 0.26 ^a 7.94 ± 0.32 ^a

0.23 ^a 0.23 ^a 0.32 ^c

8.25 ± 7.94 ± 8.44 ±

B. a. lactis 8.31 ± 0.26 ^a 7.81 ± 0.37 ^a ' ^b

0.27 ^a 0.18 ^a 0.18 ^a

B. a. 8.44 ± 8.00 ± 8.13 ±

8.50 ± 0.00 ^a 8.06 ± 0.18 ^a lactis+F 0.18 ^a 0.00 ^a 0.35 ^a ' ^b

B. a. 8.38 ± 7.81 ± 7.88 ±

8.44 ± 0.32 ^a 7.13 ± 0.23 ^c lactis+G 0.23 ^a 0.26 ^a 0.35 ^b ' ^c

B. a. 8.38 ± 0.35 ^a 8.31 ± 7.75 ± 6.94 ± 7.38 ± 0.23 ^{b c} lactis+\ 0.26 ^a 0.27 ^a 0.18 ^d

^'α Mean values in the same column followed by different letters in apex differ significantly (p <0.05)

Table 3.2

a ^' ° Mean values in the same column followed by different letters in apex differ significantly (p <0.05)

The visual organoleptic characteristics, appearance and color, showed no significant differences between the samples, all of which showed a very appreciated light brown color. Among the gustatory parameters, the inoculated sample B. a. lactis showed the significantly higher value both compared to the Control and to the other samples, being characterised by a more intense and characteristic flavor. In terms of tactile sensation during chewing, the consistency of CTRL and B. a. lactis+F was statistically higher, in addition, samples B. a. lactis, B. a. lactis+F and B. a. lactis+G were characterised by a pleasant crunchiness, showing a significant difference compared to CTRL. Among the gustatory sensations, friability is considered a very important mechanical attribute in the sensorial evaluation of a biscuit, in this case, the data indicated that sample B. a. lactis+F was pleasantly flaky and had a good ability to generate numerous fragments at the beginning of chewing, showing the highest and most significant score among the samples examined. The adhesion and hardness parameters showed no significant differences between samples, except for biscuit B. a. lactis+\, to which the lowest score was assigned, as the sample showed greater adherence to the palate and the teeth and greater resistance to chewing. Finally, the sensory analysis carried out made it possible to identify the best formulations, corresponding to samples B. a. lactis and B. a. lactis+F, which showed statistically higher values in terms of quality and general acceptability.

Example 9. Determination of vitality of probiotics strains

The lyophilized probiotics strains Bifidobacterium breve BBR8 and Bifidobacterium animalis subsp. lactis BI1 were respectively inoculated (1 % w/w) in the biscuit matrix (CTRL) in order to realize a product with probiotic properties, furthermore, these formulations were added (1 % w/w) with prebiotic fibres FOS, GOS and Inulin, respectively, to evaluate the possible symbiotic functions. Before the inoculations were performed, the vital count on plate of the lyophilized probiotics strains Bifidobacterium breve BBR8 and Bifidobacterium animalis subsp. lactis BI1 was performed, by detecting an initial title of 1 1 .01 log UFC g ^"1 and 1 1 .16 log UFC g ^"1 , respectively. The microbiological analysis was carried out both on raw biscuits following the production phase, to determine the vitality of the probiotic strain, and on the dried biscuits (27 °C for 16 hours) and stored at a controlled room temperature (25 °C), to evaluate the residual survival of the esamine microorganism. Table 4 shows the data related to the vital count on plate carried out on the Control samples, inoculated with Bifidobacterium breve BBR8 and the samples added with the respective prebiotics.

Table 4 Sample log 10 UFC g ^"1

Raw Dried

CTRL 3.88 ± 0.40 ^b 2.23 ± 0.32 ^c

B. breve 9.01 ± 0.16 ^a 8.27 ± 0.19 ^a

B. breve+F 9.10 ± 0.59 ^a 8.30 ± 0.32 ^a

B. breve+G 9.04 ± 0.60 ^a 8.25 ± 0.22 ^a

B. breve+\ 9.03 ± 0.68 ^a 7.43 ± 0.13 ^{b a'c} Mean values in the same column followed by different letters differ significantly (p <0.05)

The results concerning raw biscuits showed a significantly different microbial count between the experimental samples and the Control, in fact the latter presented a microbial charge (possible lactic bacteria able to grow under the experimental conditions adopted) of 3.88 logioUFC g ^{"1 ,} which compared to experimental probiotic and symbiotic biscuits is lower of 6 logarithmic units. In particular, sample B. breve+F showed a life count of 9.10 logio UFC g ^"1 . The same trend was found for the vital count performed on dried biscuit samples (finished product), for which the loss of 1 logarithmic unit was noted, both for CTRL (2.23 logio UFC g ^"1) and for sample B. breve+F (8.30 logio UFC g ^"1) to the corresponding raw samples. Only in the case of the B breve+\ biscuit, a loss of 2 logarithmic units was found between the raw sample (9.03 logioUFC g ^"1) and dried sample (7.43 logioUFC g ^"1) , this may be due to a lower affinity of the prebiotic fiber for the strain taken into consideration.

Data concerning counts on plate of CTRL samples, inoculated with the Bifidobacterium animalis subsp. lactis BI1 and added with the different prebiotic fibers are shown in Table 5.

Table 5

I Sample I log ₁₀ UFC g ^"1 Raw Dried

CTRL 3.52 ± 0.43 ^b 2.53 ± 0.40 ^b

B. a. lactis 9.03 ± 0.02 ^a 8.54 ± 0.17 ^a

B. a. lactis+F 9.09 ± 0.90 ^a 8.59 ± 0.53 ^a

B. a. lactis+G 9.05 ± 0.71 ^a 8.41 ± 0.98 ^a

B. a. lactis+\ 9.04 ± 0.32 ^a 8.39 ± 0.77 ^{a a'b} Mean values in the same column followed by different letters in apex differ significantly (p <0.05).

Also in this experimental test, the results of the raw samples showed a statistically significant difference in the microbial load between the biscuit CTRL (3.52 logi ₀ UFC g ^"1) and the inoculated and added cookies, specifically, as for the previous analysis, the inoculated sample enriched with FOS (B. a. lactis+F) showed the highest microbial concentration (9.09 logioUFC g ^"1) . The trend of the results was also confirmed for the dried samples, for which there was a significant difference of 6 logarithmic units between the CTRL biscuit (2.53 logi ₀ UFC g ^"1) and the experimental samples, in particular the biscuit B. a. lactis+F reported the highest life count (8.59 logio UFC g ^"1) confirming the data of the corresponding raw test. Moreover, as for the previous test, the microbiological analysis found for all the samples examined, the decrease of only one logarithmic unit in the concentration of the lyophilized strain between the raw and dried biscuits, probably attributed to the bland drying process.

The data reported in Tables 4 and 5, have allowed to confirm the survival of the probiotics strains inoculated in the experimental samples and subjected to the drying process, and also indicated the existence of a possible affinity of the three selected prebiotic carbohydrates (FOS, GOS and Inulin) with the esamine probiotics strains, confirming the high strain- specificity reported by Callegari et al. (2009). Finally, the best formulations were identified, which turned out to be B. breve+F and B. a. lactis+F, respectively, confirming the specific protective action carried out by the FOS prebiotic fiber towards the vitality of both the lyophilized Bifidobacterium strains.

Example 10. Determination of pH

In the pre-inoculum step, the pH values of the individual probiotic strains were measured, which showed a slightly acidic pH, linked to the high concentrations of the same microorganisms, to be precise were found values of 4.24 and 4.69 for the strains Bifidobacterium breve BBR8 and Bifidobacterium animalis subsp. lactis BI1 , respectively. The results concerning the measurement of the pH values of the experimental samples are shown in Tables 6-7.

The pH values of the CTRL samples, inoculated with the Bifidobacterium breve BBR8 strain and added with the prebiotic fibres are reported in Table 6.

Table 6

a ^'c Mean values in the same column followed by different letters differ significantly (p <0.05) The results for the raw samples indicated a less acidic pH (5.59) for the CTRL biscuit, with significant differences compared to the experimental samples, moreover, a statistically significant difference was also noticed between the B. breve sample (5.49) and the samples added with prebiotics, which reported a more acidic pH value, probably due to the greater fermentative action of the same microorganisms against prebiotic carbohydrates. A similar trend was also found for the dried samples, for which the same significant differences were recorded between the experimental samples.

Table 7 shows the pH values related to CTRL samples, inoculated with the Bifidobacterium animalis subsp. lactis BI1 and samples enriched with prebiotic fibres.

Table 7

a ^'b Mean values in the same column followed by different letters in apex differ significantly (p <0.05)

With reference to the raw samples, a statistically higher pH value for the CTRL was recorded, as for the previous analysis, compared to the other experimental samples, while no significant difference was found between the dried samples. As can be seen from the data reported in Tables 6-7, for both analyses, all the samples examined showed a stable pH value and not particularly influenced by the fermentative activity of the microorganisms present.

Example 1 1 . Optimal formulation

The optimal formulation includes: conventional rice flour (25.53%); water (25.53%); wildflower honey (17%); sugar cane (12.77%); puffed rice (4.26%); puffed millet (4.26%); puffed buckwheat (4.26%); corn flakes (4.26%); carob pulp flour (1 .7%); vanilla flavor (0.43%); probiotics (1 % w/w) respectively Bifidobacterium breve BBR8 and Bifidobacterium animalis subsp. lactis BI1 - (CSL, Centro Sperimentale del latte Sri, Lodi, Italy) and prebiotic fibres (1 % w/w) respectively FOS (fruit- oligosaccharides), GOS (galacto-oligosaccharides) and Inulin.

Specifically, both probiotics and prebiotics were dissolved in the recipe water before being added to the mixture.

Example 12. Production procedure referred to the optimal formulation

Before mixing the ingredients, the rice flour was toasted in the oven at a temperature of 200 °C for 20 min. After the ingredients have been thoroughly mixed, for 30 sec at low speed (60 rpm), adding first the dry ingredients such as rice flour, the carob pulp flour, the sugar cane, the puffed rice, the puffed millet, the puffed buckwheat and the corn flakes; after mixing the ingredients well with each other, honey, water with probiotics and prebiotics and the vanilla aroma were added. After kneading all the ingredients for a few minutes at a speed of about 100 rpm, the individual biscuits were formed using a circular mold (5.5 cm in diameter and 1 cm thick) which gave biscuits weighing 19g each. The biscuits were formed on a tray previously coated with parchment paper and were placed to dry in a dryer at 27 °C for 16 hours. After the drying step, the biscuits were left at room temperature (25 °C) for 1 hour and were then ready to be consumed or packaged.

The present invention has been described by way of illustration, but not by way of limitation, according to its preferred embodiments, but it is to be understood that variations and/or modifications may be made by those skilled in the art without departing from the relative scope of protection, as defined from the attached claims. Bibliographical references cited in the text

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