METHOD FOR THE BIOTECHNOLOGICAL PROCESSING OF MAIZE GERM FOR THE PRODUCTION OF SEMI-FINISHED PRODUCTS AND FOOD PRODUCTS

Field of the invention

The present invention belongs to the field of biotechnological processes for the production of food products.

In particular, it relates to the biotechnological processing of maize germ, which is used as an ingredient in the production of semi-finished products for the food industry and in the production of innovative food products such as sourdoughs, spreadable creams, yoghurt-like snacks and vegetable cheeses.

Background

Maize (Zea maize L.) is the third most widely grown and consumed cereal in the world, after wheat and rice. It is mainly used in the animal feed industry, but it is also an important staple food for human nutrition. In addition to food and feed, maize has a wide range of industrial applications, of which ethanol production is of particular importance.

In the United States of America, maize is one of the most important crops, producing this country over a third of the world's maize production.

Maize cultivation is widespread in the main agricultural areas of the world, although it is present in each of them with different characteristics. The US is the world's leading producer with 370 million tonnes. EU countries rank fourth with 60 million tonnes. Among the top 15 producers France is at seventh position and Italy at the tenth.

The products that can be obtained from the transformation of this cereal are numerous and, depending on the intended use of these products, the maize is subjected to different processes: dry milling, wet milling or nixtamalization.

Dry milling is mainly used for obtaining food products and most of the grain used is of the vitreous type. The grain is conditioned to 24% humidity, then subjected to a first coarse milling which serves to remove the fraction of the germ.

The maize grain is composed of four primary structures: endosperm, germ, pericarp and tip cap, which make up 83%, 11%, 5% and 1% of the maize grain respectively. The endosperm is mainly starch surrounded by a protein matrix. The germ or embryo is rich in polyunsaturated fats (33.3%) in addition to the enzymes and nutrients for the growth and development of new maize plants. The germ also contains B-complex vitamins and antioxidants such as vitamin E.

Despite these interesting nutritional aspects, the germ is rarely used for human consumption. Indeed, the high amount of unsaturated fats and the presence of hydrolytic and oxidative enzymes (Sjovall et al., 2000. Journal of Agricultural and Food Chemistry 48:3522-7) cause rapid rancidity during storage with the release of, among other compounds, off-flavors.

It also has some anti-nutritional factors (raffinose, phytic acid) that adversely affect the nutritional profile of the finished product (Rizzello et al., 2010. Food Chemistry 119:1079-89).

Finally, the germ negatively affects the technological quality of the flour and, above all, the stability of the dough (Srivastava et al., 2007. European Food Research and Technology 224:365-72).

Recently, research has made numerous efforts to stabilise and improve the shelf life of the germ. All approaches involved the inactivation of the enzymatic activities, with particular attention to lipase and lipoxygenase (Boukid et al., 2018. Trends in Food Science & Technology 78:120-33). This can be achieved directly, by using thermal processings to inactivate the enzymes, or indirectly, by creating adverse conditions for their action (e.g. by acidification, oxygen elimination, etc.). Until the 1980s, thermal processings were the only methods used to delay rancidity (Rao et al., 1980. Food Science and Technology 17:171-75). Currently, microwave baking and heating have been reported as rapid and interesting approaches for enzymatic inactivation (Matucci et al., 2004. Food Control 15:391-95). However, thermal processings can be costly and responsible for a decrease in nutritional value.

It is therefore of interest a method that allows the maize germ to be used for the preparation of food products that does not have the above drawbacks.

Recently, ad-hoc selected lactic bacteria have been used as starters to guide the fermentation of the germ in order to stabilise it and improve its nutritional and sensory profile (Rizzello et al., 2010. Food Chemistry 119:1079-89; Pontonio et al., 2019. Frontiers in Microbiology 10:561).

Studies have shown that the germ, when fermented, has a low percentage of compounds responsible for the perception of rancidity released during the lipid oxidation process, even during storage (Boukid et al., 2018. Trends in Food Science & Technology 78:120-33). Acidification by lactic bacteria is also responsible for the inhibition of endogenous lipase, delaying rancidity and prolonging the shelf-life of the germ, and the activation of endogenous phytases with hydrolysis of phytic acid and increased bioavailability of minerals and proteins. During fermentation, the lactic bacteria are also responsible for the process of proteolysis, which leads to an increase in the concentration of peptides and amino acids. Some of them are considered bioactive due to their ability to exert antimicrobial, antioxidant and/or antihypertensive activities. In addition, the hydrolysis of proteins with the release of lower molecular weight nitrogenous forms (peptides and amino acids) contributes to the improvement of protein digestibility in the intestine (Gobbetti et al., 2019. International Journal of Food Microbiology 302:103-113).

A process for processing maize germ is therefore desired that comprises a fermentation with lactic bacteria which allows to achieve the advantages set out above, in particular an improvement in the organoleptic characteristics, technological properties and protein digestibility, and a reduction in anti-nutritional compounds such as phytic acid.

Summary of the invention

It has been found that the use of two selected strains of lactic bacteria for fermenting maize germ overcomes the problems outlined above, resulting in fermented maize germ with improved technological, nutritional and organoleptic characteristics.

It is therefore an object of the invention a process for fermenting maize germ which comprises using an admixture of lactic bacteria comprising at least one strain selected from Lactobacillus plantarum which was deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures) on the date of 22nd January 2020 and which is identified by the deposit number DSM 33412 and the strain of Lactobacillus brevis which was deposited with the culture collection Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures) on the date of 22nd January 2020 and which is identified by the deposit number DSM 33413.

In particular, it is an object of the present invention a process for fermenting maize germ comprising the following steps: a. separating the maize germ from the other fractions of the maize; b. optional roasting of the maize germ; c. fermenting the maize germ by means of an admixture of lactic bacteria comprising at least one strain selected from Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413; d. optional stabilization and/or conservation of the fermented maize germ.

Advantageously, the admixture of lactic bacteria comprises said at least one strain selected from Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413 at cell densities greater than 1x10 ⁶ cfu/g and preferably between 1x10 ⁶ and 1x10 ⁷ cfu/g.

The maize germ processed in this way can be advantageously used for the production of certain food products of particular interest, such as:

- sourdough based on maize germ for the production of oven-baked leavened products;

- spreadable cream based on maize germ;

- vegetable yogurt-like drink based on maize germ;

- vegetable cheeses on the basis of maize germ.

Such food products are within the scope of the present invention as are the processes for obtaining them.

The features and advantages of the invention will best be shown by the detailed description of some preferred examples of its implementation, illustrated by reference to the accompanying drawings.

Definitions

In the context of the present invention, maize germ means the embryonic fraction of the maize caryopsis (Zea mays), i.e., the part containing the seedling and rootlets of the seed, the enzymes and the reserve substances necessary for germination.

In the context of the present invention, "starter" means one or more microorganisms used in a live and viable status for inoculating food biomass for transformation by fermentation into ingredients or foodstuffs or drink for food use. The term “started also refers to the preparation in liquid or solid form, fresh or frozen or lyophilised, containing a high cell density of the aforesaid micro-organisms in a live and viable form.

Figures

Figure 1. Diagram of production and processing of the maize germ.

Figure 2. Flow chart of the production of spreadable cream based on the processed maize germ. Figure 3. Flow diagram of the production of yoghurt-like drink based on the processed maize germ. Figure 4. Flow chart of the production of vegetable cheese on the basis of processed maize germ.

Detailed description of the invention

For the purposes of the uses described in the present invention, maize of any nature and with any technological characteristic may be used.

In particular, maize grain is used.

Preference is given to the use of FAO Class 40 (early quality) vitreous maize, preferably from a controlled supply chain, free of gluten and soya, and guaranteeing mycotoxin levels below the legal limits described in EU regulations Reg. EC 1829/2003, Reg. EC 1881/2006, Reg. EC 1126/2007.

The steps in the process of the present invention as defined above are described herein in detail.

In some embodiments, the process of the invention may comprise further steps in addition to steps a)-d) described above.

An exemplary embodiment of the process is illustrated in Figure 1.

Step a. Separation of the maize perm

In this step the maize germ is separated from the other maize fractions. This can be done using methods known in the sector.

In an embodiment of the invention, this step may comprise one or more of the following steps: a.1 cleaning the cereal by sieving or brushing or similar process; a.2 degermination, made for example by using a rotor degerminator. In this exemplary embodiment, the rotor rotates inside a casing with contrasts (blades); the maize grain passes inside the cavity, where the blades cause a longitudinal fracture thereof. Characteristically, maize breaks along the longitudinal line, releasing the germ section, which has a different density. This type of processing produces about 8% germ; a.3 separation by densimetry of the germ from the other caryopsis fractions. The separated germ is then channelled to the next technological step; a.4. optional optical sorting for the elimination of germ particles differing in colour and shape, which may be mouldy, dirty or otherwise. Non-conforming batches are re-processed further until the product is completely clean. a.5. optional grinding with a fine granulometry, i.e. <1000 pm. This step is preferably only carried out if the maize germ is not subjected to a subsequent roasting step. The product G in Figure 1 is thus obtained.

Step b. roastinp and prindinp

In some embodiments of the invention, the maize germ is roasted and possibly ground.

This step may comprise one or more of the following steps: b.1 roasting: the product is thermally processed at a temperature of between 200 and 230°C; this processing stabilises the enzyme content of the product and gives it its special colour and aroma; b.2 Sieving: once roasted, the germ passes through a sieve with a cut-off preferably of about 2000 pm to eliminate the dust formed during roasting; b3. Grinding with a fine granulometry, i.e.<1000 pm.

The product Gt in Figure 1 is thus obtained.

Step c. fermentation with selected starters

The maize germ, which may be roasted, is fermented with one or both of the above two strains.

In one embodiment of the invention, the fermentation comprises the following steps: c.1 mixing the maize germ with drinking water. The weight/volume percentage of the maize germ in the admixture is in the range from 30 to 70%, e.g. it is in the range from 55 to 60%. c.2 inoculating into the admixture thus obtained an admixture of lactic bacteria comprising at least one strain selected from Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413; c.3 fermenting the admixture at a temperature between 20 and 35°C, for example at 30°C, for a time between 8 and 48 hours until reaching a pH between 3.8 and 5.0, for example in the range 4.0-4.5, and a final cell density of the microorganisms in the range from 1 to 7 x 10 ⁹ cfu/ml.

Advantageously, the admixture of lactic bacteria comprises the at least one strain selected from Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413 at cell densities greater than 1x10 ⁶ cfu/g and preferably between 1x10 ⁶ and 1x10 ⁷ cfu/g.

The admixture of lactic bacteria comprises both strains Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413.

Step d. Stabilisation

The fermented maize germ can be stabilised by refrigeration, freezing or dehydration.

The dehydration can be carried out for example by lyophilization or evaporation at temperatures of 55-65°C.

This results in the products fG and fG _T in Figure 1 , obtained from unroasted and roasted germ, respectively.

The products thus obtained can be stored and, if necessary, packed in appropriate packages.

The strain of Lactobacillus plantarum (F.1) used in the process of the present invention was deposited with the culture collection Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures) on the date of 22nd January 2020 and is identified by the deposit number DSM 33412.

This strain of Lactobacillus plantarum DSM 33412 comprises at least one lactic acid bacterium isolated from spontaneously fermented chickpeas. It is a GRAM+, anaerobic bacterium. It can be grown in MRS medium (De Man, Rogosa, Sharpe) under the following conditions: incubation temperature 30°C, incubation time 24 hours. The pH of the medium is preferably about 6.1-6.2.

The strain of Lactobacillus brevis (F.4) used in the process of the present invention was deposited with the culture collection Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures) on the date of 22nd January 2020 and which is identified by the deposit number DSM 33413.

This strain of Lactobacillus brevis DSM 33413 comprises at least one lactic acid bacterium isolated from spontaneously fermented chickpeas. It is a GRAM+, anaerobic bacterium. It can be grown in MRS medium (De Man, Rogosa, Sharpe) under the following conditions: incubation temperature 30°C, incubation time 24 hours. The pH of the medium is preferably about 6.1-6.2.

Said strains can be left in microaerophilic conditions at about 16°C-25°C for up to 7 days. The strains of the present invention can be stored by means of methods known in the art for storing Lactobacillus strains. For example, they can be stored at -20°C in an admixture with 20% v/v glycerol. The viability of the strains can also be assessed according to what is known in the sector, e.g. by placing them in the above-mentioned medium and checking the growth thereof after 24 hours.

In a preferred embodiment, the admixture of lactic bacteria comprises both strains Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413. In said embodiment, the two strains are present in a ratio Lactobacillus plantarum DSM 33412/Lactobacillus brevis DSM 33413 between 1:1 and 1:10 or between 1 :1 and 10:1 , preferably 1:1.

The admixture of lactic bacteria may also comprise one or more bacteria belonging to one or more species selected from the group consisting of: Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus rossiae, Lactobacillus sanfranciscensis, Pediococcus pentosaceus, Leuconostoc spp.

The bacteria are inoculated in a live, viable form in a liquid, pellet or lyophilised preparation.

The bacteria are inoculated in such a way as to obtain a cell density of, for example, 1 to 5 x 10 ⁷ cfu/ml of matrix to be fermented. For inoculation, the matrix can be brought to a temperature between 20 and 35°C, for example 30°C.

In some embodiments of the present invention, the fermented maize germ obtained at the end of the fermentation step c) may be mixed with roasted maize germ obtained at the end of the separation and roasting processes b) described above.

The process of the invention may comprise further steps following fermentation, such as mixing with additional ingredients or using special processings, for example thermal processings.

These steps may also be present between the possible roasting step b) and the fermentation step c).

The strains used in the present invention have characteristics which make them particularly performing in the application conditions of interest of the present invention and better than known microorganisms previously used as starters for fermenting maize germ.

In particular, compared to data present in scientific literature and obtained under similar application conditions (see Pontonio et al., 2019. Frontiers in Microbiology, 10:561) they have the following advantages, as shown in the examples:

- an increased proteolytic activity assessed as a percentage increase in the concentration of peptides and amino acids between the unfermented and fermented matrix. In detail, the strains deposited and used in the present invention are capable of producing increases in Total free amino acids (TFAA) of up to 20% higher than the increase found under similar application conditions for previously selected strains (Pontonio et al., 2019. Frontiers in Microbiology, 10:561). In addition, increases of about 15% higher than those reported in the literature were found in terms of peptide concentration (see Example 2). The proteolytic activity is directly and indirectly related to the improvement of the organoleptic characteristics (taste and olfactory profile), the improvement of technological properties and the improvement of the digestibility of the proteins in the fermented matrix.

- an increased degradation activity of the anti-nutritional compound phytic acid. The strains included in the present invention are capable of reducing the concentration of phytic acid between 42 and 60% during the fermentation process of unroasted and roasted germ, respectively. Significantly lower reductions are reported in the literature for other bacterial strains used under similar conditions (Pontonio etal., 2019. Frontiers in Microbiology, 10:561).

The maize germ obtained by means of the processes described above is also an object of the present invention, as are the food products containing it.

This maize germ is characterised by a high concentration of dietary fibre, on average 32-33% dry matter, and proteins with a high biological value, on average 20-22%, and the absence of endogenous lipase. The processed maize germ also has a distinctive sensory profile that can be adjusted according to the intended use, i.e. the type of foodstuff to be obtained, and the preferences of the consumer.

The processed maize germ has nutritional characteristics that make it suitable, from the point of view of food technologies:

- for use as a fat matrix, made stable to rancidity thanks to the deactivation of endogenous lipases (enzymes capable of catalyzing the oxidation and rancidity processes) by means of roasting and biological acidification following fermentation with selected lactic bacteria;

- for the fortification of food formulations thanks to the high concentration of dietary fibre (on average 32-33% dry matter) and protein (on average 20-22%) with a high biological value.

The processed maize germ therefore lends itself to the fortification of food formulations of various types, such as savoury and sweet snacks, oven-baked leavened products and functional drinks.

Below is a description of certain products that can be obtained from maize germ processed by means of the method of the present invention.

Such products are also the object of the present invention.

Sourdough

Sourdough means herein the “natural yeast” obtained from maize germ.

Said sourdough can advantageously be used as an acidifying and flavouring ingredient in the production of oven-baked leavened products.

The process for producing sourdough based on maize germ comprises the process steps described above, which may be followed by direct use or a stabilisation step.

In an exemplary embodiment, the process involves the following steps: d.1 separating the maize germ from the other maize fractions; d.2 roasting the maize germ; d.3 mixing the roasted maize germ with drinking water. The (weight/volume) percentage of maize germ in the admixture is in the range from 45 to 70%, e.g. from 55 to 60%; d.4 inoculating into the admixture thus obtained an admixture of lactic bacteria comprising at least one strain selected from Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413; d.5 fermenting the admixture at a temperature between 20 and 35°C, for example at 30°C, for a time between 8 and 24 hours until reaching a pH between 3.8 and 4.5, for example in the range 4.0-4.5, and a final cell density of the microorganisms in the range from 1 to 7 x 10 ⁹ cfu/ml; d.6 optional refrigeration or freezing of the dough; d.7 optional dehydration of the dough which may be after the step d.5 or d.6. Once this step has been carried out, the sourdough based on maize germ is referred to as “dried”.

Advantageously, the admixture of lactic bacteria comprises said at least one strain selected from Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413 at cell densities greater than 1x10 ⁶ cfu/g and preferably between 1x10 ⁶ and 1x10 ⁷ cfu/g.

The sourdough obtained with the process described above is also an object of the present invention.

The sourdough based on maize germ is comparable to a conventional natural yeast obtained on the basis of wheat or rye flour. Like the latter, it is produced by means of a fermentation process involving lactic bacteria at a high cell density (such as >10 ⁷ cfu/g). It has high concentrations of organic acids (lactic and acetic) and a significant concentration of free amino acids deriving from proteolysis.

Compared to conventional natural yeast, the sourdough based on maize germ described herein has the following advantageous distinguishing features, evident from Example 4:

- a higher dietary fibre content, particularly about 33% in the dehydrated preparation;

- a higher concentration of free amino acids, in particular >1 OOOmg/kg;

- a higher concentration of protein, in particular about 20%.

The maize germ sourdough also has an optimal fermentation quotient (QF) of less than 5, a very low concentration of phytic acid (anti-nutritional factor), high protein digestibility, a significant antioxidant activity and a high concentration of essential fatty acids. The maize germ contains omega-3 and omega-6 fatty acids equal to 3 and 50% of the total fat fraction, respectively.

The characteristics of maize germ sourdough make it advantageously usable as an ingredient in oven-baked leavened products, to which it confers, depending on the dosage, organoleptic profiles typically associated with natural leavening: intensity of taste linked to free amino acids, intensity of acid taste and smell linked to high concentrations of lactic and acetic acid, dark and fragrant crust (intensification of the Maillard reaction).

Therefore, the use of such sourdough allows the advantages linked to natural leavening and the advantages linked to the use of maize germ, as defined above, to be obtained simultaneously.

In particular, this sourdough can be advantageously used to obtain a bread, e.g. from wheat, enriched with roasted and fermented maize germ.

Spreadable cream

The spreadable cream is a fluid food preparation, characterised by high cohesiveness and viscosity.

It can be used, for example, as a filling or pastry ingredient.

A process for preparing a spreadable cream based on maize germ according to the invention is shown by way of example in Figure 2.

The process for the production of spreadable cream based on maize germ comprises the process steps described above followed by a mixing step of the roasted and fermented maize germ with roasted maize germ and additional ingredients.

In an exemplary embodiment, the process involves: e.1 separating the maize germ from the other maize fractions; e.2 roasting the maize germ; e.3 fermenting the maize germ by means of an admixture of lactic bacteria comprising at least one strain selected from Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413. Fermentation is carried out at a temperature between 20 and 35 °C, for example 30 ° C, for a time between 8 and 24 hours until reaching a pH between 4.0 and 5.0, for example in the range 4.3-4.5; e.4 stabilizing by means of dehydration; e.5 mixing of the germ obtained at the end of step e.4, also referred to herein as roasted and fermented maize germ or fG _T , with germ obtained at the end of step e.2, also referred to herein as roasted maize germ or G _t , in a ratio G _T :fG _T between 1 :1 and 10:1 and with any additional ingredients; e.6 packaging followed by pasteurization or alternatively pasteurization followed by packaging under aseptic conditions.

Advantageously, the admixture of lactic bacteria comprises said at least one strain selected from Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413 at cell densities greater than 1x10 ⁶ cfu/g and preferably between 1x10 ⁶ and 1x10 ⁷ cfu/g.

The mixing step (e.5) can be carried out with an arm mixer or similar machine, preferably with a ball mill, for a time of 30-60 min, e.g. 40 min, at a temperature of 25-30°C.

In the mixing step (e.5) one or more additional ingredients may be added selected from: vegetable oils, e.g. sunflower oil, palm oil, safflower oil, rice oil, rapeseed oil and/or mixtures thereof; cacao, cocoa mass, cocoa butter, hazelnuts and derived food preparations, milk, e.g. whole milk, semi- skimmed or skimmed milk, liquid or powdered milk; sugars, e.g. sucrose, glucose, fructose, lactose, maltose, maltodextrins, in powder or syrup form; sweeteners, e.g. sorbitol, xylitol, mannitol, aspartame, saccharin, cyclamates, acesulfame; honey, pistachio and derived food preparations, soya lecithin, flavourings and other additives permitted by food regulations.

In a preferred embodiment, the following ingredients are added: seed oil, cacao, cocoa mass and sugar.

In a preferred embodiment, the spreadable cream thus obtained has the following composition:

- admixture of roasted maize germ and roasted and fermented maize germ obtained by the process of the invention in a total percentage of between 30 and 70% of the final weight of the formulation, preferably 50%; in a preferred embodiment the roasted and fermented maize germ is present at 25% by total weight of the preparation and the roasted maize germ at 25% by total weight of the preparation;

- seed oil, at a percentage with respect to the final weight of the formulation between 7 and 30%, preferably 15%;

- cacao, at a percentage with respect to the final weight of the formulation between 7 and 30%, preferably 15%;

- cocoa mass, at a percentage with respect to the final weight of the formulation between 10 and 30%, preferably 15%;

- sugar (or sucrose), preferably “cane” sugar, at a percentage with respect to the final weight of the formulation between 5 and 20%, preferably 10%.

The spreadable cream based on maize germ described herein is also an object of the present invention and has the following advantageous distinguishing features, shown for example in Example 5:

- the dietary fibre concentration is higher than in sweet spreadable creams currently on the market. Among these products, the fibre is provided by the quantity of hazelnuts, usually less than 20%, which are in any case deprived of their cuticle ("skin"), and by cacao, also present in varying percentages but usually less than 20%;

- reduced sugar content. The carbohydrate content is far lower than in most commercial spreadable creams, where it is sometimes the main ingredient, see for example Nutella™; - abundance of essential fatty acids. The spreadable cream based on processed maize germ has a high percentage of essential fatty acids (omega-3 and omega-6) deriving from the maize germ itself;

- high protein content. Considering the high protein supply of the germ and the high usage in the recipe of the invention, the total protein content is higher than in commercial spreadable creams, in particular about 2 times higher;

- minerals and vitamins. The contribution of these two categories of nutrients in the finished product can be greater than in commercial products based on cacao or hazelnuts thanks to the high quantity of these nutrients in the germ and the high quantity of inclusion in the cream.

Yoqhurt-like vegetable drink

Yoghurt-like vegetable drink means a snack that can be eaten with a spoon, that is spoonable similar to conventional yoghurt, the latter resulting from acid coagulation of pasteurised milk, due to its viscosity and high cell density of viable lactic bacteria in the product, typically >10 ⁷ cfu/ml, but obtained with alternative ingredients to milk. Like conventional yoghurt, the viability of the lactic bacteria is preserved by maintaining the cold chain (refrigeration).

A process for preparing a yoghurt-like vegetable drink based on maize germ according to the invention is shown by way of example in Figure 3.

The process for producing a yoghurt-like vegetable drink based on maize germ comprises the process steps of the invention together with further steps described below.

In an exemplary embodiment, the process involves: f.1 separating the maize germ from the other maize fractions; f.2 roasting the maize germ and subsequent grinding; f.3 mixing the roasted maize germ with flour and/or cereal starch and water at the following percentages:

- roasted maize germ: 5-15% of the final weight of the formulation, preferably 8%;

- flour and/or cereal starch: 5-15% of the final weight of the formulation, preferably 8%;

- water: 70-90% of the final weight of the formulation, preferably 84%; f.4 thermal processing by means of heating of the suspension of roasted maize germ and flours obtained in the previous step at a temperature between 75 to 90°C, e.g. 85°C, for a time between 10 to 20 minutes, e.g. 15 minutes. The aim of the treatment is to break down the contaminating microbial load and to promote the formation of a viscous structure due to the partial gelatinisation of the starch; f.5 cooling to a temperature of from 5 to 10°C, e.g. 8°C, for no longer than 15 minutes; the aim of this step is to favour the formation of a creamy-cohesive structure and avoid organoleptic alteration of the matrix; f.6 heating to 30±2°C in order to reach a temperature suitable for the inoculation of lactic bacteria; f.7 inoculating an admixture of lactic bacteria comprising at least one strain selected from Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413 and fermentation at a temperature between 20 and 35 °C, e.g. 30 °C, for a time between 8 to 24 hours until reaching a pH between 4.0 and 5.0, e.g. in the range 4.3 to 4.5; f.8 refrigeration at from 0 to 8°C, e.g. at 4°C; f.9 optional addition of additional ingredients, such as, for example, sweeteners, purees or fruit juices, dried fruit, vegetable fibres or fibre sources, such as oat, inulin, beta-glucan, cereal bran, cacao, chocolate drops, coffee, vanilla and flavourings; f.10 optionally packaging, for example, in jars for food use, and distribution in a chilled chain.

Advantageously, the admixture of lactic bacteria comprises said at least one strain selected from Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413 at cell densities greater than 1x10 ⁶ cfu/g and preferably between 1x10 ⁶ and 1x10 ⁷ cfu/g.

The flour or starch used in step f.3 may for example be from the following cereals: maize, wheat, oat, rice.

Mixing of step f.3 can be carried out with a mixer for liquid or semi-liquid matrices, e.g. with a paddle mixer, for a time of about 15-20 min, at a temperature of 10-25°C, at 100-150 rpm.

Any ingredients added in step f.9 may be added to the fermented matrix after appropriate sanitisation, e.g. pasteurisation of the fruit, or other suitable treatment, if required, as known in the field.

The vegetable beverage based on maize germ described herein is also an object of the present invention and has the following advantageous distinguishing features, shown for example in Example 6:

- like conventional yoghurt it has: i) high viscosity, it is “spoonable”; ii) high cell density, in fact the lactic ferments used as starter in the finished product and for the entire shelf-life survive at cell density equal to or greater than 10 ⁸ cfu/ml;

- is produced exclusively with vegetable ingredients and can therefore be labelled as vegetarian" or "vegan”;

- contains no milk or milk derivatives, resulting for this reason "lactose-free";

- lends itself to numerous possibilities of differentiation, through the addition of further ingredients to the basic formulation, allowing it to respond to market needs;

- has a naturally high dietary fibre content, in particular higher than the average content of a conventional yoghurt and averaging about 2.5-3.5/100g of product;

- contains exclusively vegetable proteins;

- has plenty of essential fatty acids and a lower fat content than a yoghurt made from whole milk. Vegetable cheese

A process for preparing a vegetable cheese on the basis of maize germ according to the invention is illustratively shown in Figure 4.

The process for producing vegetable cheese on the basis of maize germ comprises the process steps described above followed by a step of mixing the roasted and fermented maize germ with roasted maize germ and any additional ingredients and further steps of heat processing and shaping as described below.

In an exemplary embodiment, the process involves: g.1 separating the maize germ from the other maize fractions; g.2 roasting the maize germ and subsequent grinding and dehydration; g.3 fermenting the maize germ by means of an admixture of lactic bacteria comprising at least one strain selected from Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413. The fermentation is carried out at a temperature between 20 and 35°C, for example at 30°C, for a time between 8 and 24 hours until reaching a pH between 3.8 and 4.5, for example in the range 4.0-4.5, and a final cell density of the microorganisms in the range from 1 to 7 x 10 ⁹ cfu/ml, or from 1 to 8 x 10 ⁹ cfu/ml; g.4 mixing the germ obtained at the end of step g.3, also referred to herein as roasted and fermented maize germ or fG _T , with germ obtained at the end of step g.2, also referred to herein as roasted maize germ or G _t , in a ratio G _T :fG _T between 1:1 and 10:1, and with flour and/or cereal starch and water at the following percentages:

- admixture of roasted maize germ and roasted, fermented and dehydrated maize germ at from 60 to 70% of the final weight of the formulation, preferably 65%;

- flour and/or cereal starch at from 15 to 30% of the final weight of the formulation, preferably 8%;

- water at from 10 to 20% of the final weight of the formulation, preferably 15%; g.5 thermal processing by means of heating at a temperature between 75 and 90°C, for example 85°C, for a time between 10 and 30 minutes, for example 15 minutes. The processing is useful to ensure a reduction of the microbial load and to facilitate the formation of a cohesive structure; g.6 optional addition of acidifying agents; g.7 shaping and pressing using bundles or containers to give the admixture its final shape; g.8 optional pasteurization; g.9 refrigeration at from 0 to 8°C, e.g. at 4°C, and subsequent packaging in package for food use and distribution in the refrigerated chain.

The flour or starch used in step g.4 may for example be from the following cereals: maize, wheat, oat, rice.

The acidifying agents used in step g.6 may be lactic acid, acetic acid and/or citric acid, preferably citric acid is used.

The preferred size of the cheese varies from 100 to 2000g.

In step g.7 mechanical pressing is preferred as it facilitates draining and the elimination of superfluous water.

In step g.8, pasteurisation is useful to ensure a longer shelf-life.

The vegetable cheese thus obtained typically has a shelf-life of 30-90 days, depending on the final pH and water activity values.

The vegetable cheese on the basis of maize germ described herein is also an object of the present invention and has the following advantageous distinguishing features, shown for example in Example 7: - the product is made exclusively with vegetable ingredients and can therefore be labelled as "vegetarian" or "vegan";

- contains no milk or milk derivatives, resulting for this reason "lactose-free";

- has a naturally high dietary fibre content, in particular higher than that of a conventional cheese;

- has a high protein content, all of which of vegetable nature;

- has an abundance of essential fatty acids.

The present invention will now be illustrated by means of examples.

EXAMPLES

Example 1

Nutritional and microbiological characterisation of roasted and unroasted maize germ

1.1 Chemical and microbiological analyses

Protein (total nitrogen x 5.7), lipids, humidity, total dietary fibre and ash from roasted (G _T ) and unroasted (G) maize germ were determined according to the methods approved by the American Association of Cereal Chemists (AACC, 2010) and identified by the following codes 46-11A, 30- 10.01 , 44-15A, 32-05.01 and 08-01.01. The available carbohydrates were calculated as the difference [100 - (protein + lipids + ash + total dietary fibre)]. Protein, lipids, carbohydrates, total dietary fibre and ash were expressed as % of dry matter (d.m.).

Microbiological characterisation was carried out by homogenising 10 g of sample with 90 ml of peptonised water (0.1% peptone and 0.85% NaCI). The total aerobic mesophilic bacterial load was assessed by counting on Plate Count Agar medium (PCA, Oxoid, Basingstoke, Hampshire, UK) at 30°C for 48 hours while lactic bacteria were enumerated using modified de Man, Rogosa and Sharpe (mMRS supplemented with 1% maltose and 5% fresh yeast extract, pH 5.6). Enterobacteriaceae were enumerated on Violet Red Bile Glucose Agar (VRBGA, Oxoid) at 37 °C for 24 hours. The yeasts and moulds were enumerated on Soboroud Dextrose Agar (SDA, Oxoid) and Potato Dextrose Agar (PDA, Oxoid), respectively, at 25°C for 48 hours.

The chemical composition and microbiological characterisation of roasted maize germ (G _T ) and unroasted maize germ (G) is shown in Table 1. Thermal processing resulted in a G _T with humidity four times lower than G.

As expected, G and G _T contained high levels of fat (up to about 33% DM).

Probably, due to the thermal processing, none of the investigable microbial groups were detectable in 1 g G _T . In contrast, with the exception of yeasts, G showed cell densities of about 2 to 5 Iog10 cfu/g of the microbial groups determined (Table 1).

Table 1. Chemical and microbiological characterisation of roasted (G _T ) and unroasted (G) maize germ.

Data are the result of three independent experiments ± standard deviation (n=3); ^* Data are expressed on dry weight (d.m.); ^# Data are expressed as Iog10 cfu/g.

Example 2

Fermentation of maize germ and characterisation of fermented maize germ (sourdough based on maize germ)

2.1 Fermentation of the maize germ

The fermentation process was carried out on doughs consisting of maize germ and water. Specifically, 62.5 g of roasted maize germ (i-G _T ) and unroasted maize germ (i-G) were mixed with 37.5 ml of drinking water containing the cell suspension of each lactic bacteria ( Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413 in a 1 :1 ratio). The DY (dough yield, dough weight x 100/flour weight) was 160 and the initial cell density of each lactic bacteria was about 7.0 -7.8 log 10 cfu/g (1-7 x 10 ⁷ cfu/g). Mixing was carried out manually for 5 minutes and fermentation was carried out at 30°C for 24 hours. After fermentation (i-fG _T and i-fG), the samples were stored at 4°C and analysed within 2 hours. Such doughs correspond to what is referred to in the present invention as "sourdough based on maize germ". Non-inoculated doughs were used as controls (i-G _T and i-G).

2.2 Monitoring of maize germ fermentation

The pH and total titratable acidity (TTA) values and the cell density of the lactic bacteria in the doughs before (i-G _T and i-G) and after fermentation (i-fG _T and i-fG) were determined as follows.

The pH values were measured by means of a pH meter (Model 507, Crison, Milan, Italy) provided with a solid food probe. TTA was measured on 10 g of bread homogenised with 90 ml of distilled water for 3 min in a Bag Mixer 400P (Interscience, St Norn, France). The TTA was expressed as the volume (ml) of 0.1 N NaOH reguired to achieve a pH value of 8.3. The cell density of the lactic bacteria was determined as shown in Example 1.

2.3 Characterisation of the fermented maize germ

Water/salt-soluble extracts (WSE) of fermented (i-fG _T and i-fG) and non-fermented (i-G _T and i-G) doughs, prepared as described by Weiss et al. (Weiss et al., 1993. Electrophoresis 14:805-16) were used to determine the concentration of lactic and acetic acid, peptides and total free amino acids (TFAA). The determination of the organic acids in WSE was performed by means of High Performance Liguid Chromatography (HPLC) using HPLC AKTA Purifier™ system (GE Healthcare Bio-Sciences, Uppsala, Sweden), with refractive index detector (Perkin Elmer Corp., Waltham, MA). The fermentation guotient (QF) was determined as the molar ratio between lactic and acetic acid. The peptides were analysed by reversed-phase fast performance liguid chromatography (RP- FPLC) while TFFA were analysed by ion exchange chromatography with post-column derivatisation with ninhydrin (Biochrom 30, Biochrom Ltd., Cambridge Science Park, England).

2.4 Determination of phytic acid of fermented maize germ

The effect of fermentation on the concentration of anti-nutritional factors (ANF) was determined. In particular, the concentration of phytic acid was measured using Megazyme K-PHYT 05/07 kit (Megazyme International Ireland Limited, Bray, Ireland). 2.5 Determination of total polyphenol concentration and radical scavenqinq activity

Methanolic extracts (EM) were used for the spectrophotometric determination of total phenolic compounds using the Folin-Cicolteau reagent. The antioxidant activity of the EMs was determined on the synthetic DPPH radical by measuring its radical scavenging activity spectrophotometrically (Rizzello et al., 2010). The antioxidant activity was also determined on aqueous extracts (WSE).

2.6 Determination of lipase activity

Tributyrin was used as a substrate to determine the lipase activity of the extract of the doughs i-G _T , i-G, i-fG _T and i-fG by agar diffusion assay (Lawrence et al., 1967). Nature 213:1264-65). The agar plates contained 1% (w/v) triglycerides, 0.02% (w/v) sodium azide and 50 mM phosphate buffer, pH 8.0. As reported by Lin et al. (Lin et al., 1983. Plant Pathology 73:460-63) this pH value is optimal for endogenous maize germ lipase activity. The activity was expressed as the minimum dilution of the enzyme preparation that failed to provide a detectable zone of hydrolysis after 24 hours of incubation at 30°C.

2.7 Statistical analysis

All data were obtained from triplicate analysis. The data were subjected to one-way ANOVA, usinq

Statistica 12.0 software (StatSoft Inc., USA). The siqnificance is expressed at P<0.05.

Biochemical and nutritional characteristics of maize qerm douqhs

The biochemical and nutritional characteristics of the maize germ doughs before fermentation (i-G and roasted germ i-G _T ) and after fermentation (i-fG and roasted germ i-fG _T )are shown in Table 2. i-G and i-G _T had similar pH and TTA values, being about 6.35 and 8.7 NaOH 0.1 M ml, respectively (Table 2). However, the lactic acid concentration was significantly higher in i-G probably due to the acidifying activity of the endogenous microbiota. The acetic acid was not detectable in any of the samples before fermentation. Significant differences were also found for TFAA and peptide concentrations, being higher in i-G (Table 2).

In general, i-G _T was characterised by lower concentrations of lactic acid, TFFA, peptides and phytic acid than i-G. The partial denaturation of endogenous proteases and the significant reduction of the resident microbiota due to thermal processing could explain the lower values of TFFA and peptide concentrations. Similarly, the reduction of lactic bacteria, and more generally, microbial density led to a reduction in the concentration of lactic acid in i-G _T .

After 24 hours of fermentation with L. plantarum DSM 33412 and L. brevis DSM 33413, the pH values of i-fG and i-fG _T were lower than the corresponding unfermented doughs (i-G and i-G _T , respectively), with a lower value in i-fG _T . In contrast, TTA values increased during fermentation, being significantly higher in i-fG _T than in i-fG. The cell density of the inoculated starters increases in both i-fG and i-fG _T by about two logarithmic cycles.

The concentration of lactic acid in l _F G and l _F G _T was about 10 to 100 times higher than i-G and i-G _T , respectively (Table 2). A similar trend was found for the acetic acid. The QF was only determined in the fermented samples, being about 4.7. Fermented samples (i-fG and i-fG _T ) had significantly higher concentrations of TFAA (up to 80%) and peptides (up to 35%) than i-G and i-G _T . In addition, the presence of unroasted germ (i-G and i-fG) resulted in higher values than samples containing thermally processed germ (i-G _T and i-fG _T ).

The increase in amino acid concentrations during fermentation may be due to the proteolytic activity of the lactic bacteria and endogenous proteases that have been activated under the acidic conditions of fermentation (Thiele et al., 2002. Cereal Chemistry 79:45-5). The higher concentrations of TFAA and peptides in i-fG compared to i-fG _T can be explained by the contribution of the endogenous germline protease activity (G) and the endogenous microbial proteolytic activity. Furthermore, the lower lactic acid concentration found in i-fG could be explained by the reduced acidification efficiency of the inoculated lactic bacteria, influenced by the competition with the endogenous microbiota.

In terms of nutritional properties, i-G contained higher concentrations of phytic acid than i-G _T , but no significant differences were found in terms of total phenols. Furthermore, the radical scavenging activity in WSE was lower in i-G. Fermentation resulted in a decrease and increase in phytic acid concentration (up to 50%) and radical scavenging activity in WSE (up to 30-fold), respectively (Table 2). A slight increase in the concentration of total phenols was also found (Table 2).

l-Gwas the only sample to show lipase activity (35.4 ± 1.7 μg/ml). Both roasting and fermentation appear to have completely inhibited the enzyme activity (Table 2).

Fermentation with selected lactic bacteria helped to create the optimal environment for the endogenous phytase (myo-inositol-hexakisphosphate phosphohydrolase, EC 3.1.3.8) of the maize germ (Poutanen et al., 2009. Food Microbiology 26:693-699) which significantly reduced the concentration of phytic acid. The optimal pH of a phytase purified from maize seedlings was 4.8 (Laboure et al., 1993. Biochemical Journal 295:413-419). Proteolysis by endogenous proteases and microbial peptidases during fermentation may have led to the release of peptides with antioxidant activity, thus explaining the increased radical scavenging activity in WSE in i-fG and i- fG _T .

Example 3

Fermentation and characterisation of the maize germ fermented with Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413 individually and in a 10:1 and 1:10 ratio

3.1 Fermentation of the maize germ

The fermentation process was carried out on doughs consisting of maize germ (unroasted, G, and roasted, G _T ) and water. Like in Example 2, 62.5 g of maize germ was mixed with 37.5 ml of drinking water containing a cell suspension of the starter lactic bacteria in the following proportions and manner:

-Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413 inoculated in a 1 :1 ratio, i.e. at a density of 5 x 10 ⁷ cfu/g each (like in Example 2);

- Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413 inoculated in a 10:1 ratio, i.e. at a density of 5 x 10 ⁷ cfu/g for the former and 5 x 10 ⁶ cfu/g for the latter;

- Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413 inoculated in a 1:10 ratio, i.e. at a density of 5 x 10 ⁶ cfu/g for the former and 5 x 10 ⁷ cfu/g for the latter;

- Lactobacillus plantarum DSM 33412 at a density of 5 x 10 ⁷ cfu/g;

- Lactobacillus brevis DSM 33413 at a density of 5 x 10 ⁷ cfu/g;

Therefore, 5 different doughs were prepared with unroasted maize germ, G, and 5 doughs with roasted maize germ, G _T , differing in the type and/or proportions of the starter lactic bacteria.

Mixing was carried out manually for 5 minutes and fermentation was carried out at 30°C for 24 hours. After fermentation, the samples were stored at 4°C and analysed within 2 hours. Such doughs correspond to what is referred to in the present invention as "sourdough based on maize germ".

The pH values, total titratable acidity and cell density of the lactic bacteria of the doughs were determined like in Example 2.

Water/salt-soluble extracts (WSE) of the fermented doughs were used to determine the concentration of lactic and acetic acid, peptides and total free amino acids (TFAA), as described in Example 2. The fermentation quotient (QF) was determined as the molar ratio between lactic and acetic acid.

The phytic acid concentration was measured using Megazyme K-PHYT 05/07 kit (Megazyme International Ireland Limited, Bray, Ireland).

The spectrophotometric determination of total phenolic compounds using the Folin-Ciocalteau reagent and the antioxidant activity was carried out as described in Example 2.

The lipase activity of the dough extract was determined on tributyrin, as described in Example 2.

Results and comments

In all examined cases, the growth of the strains during incubation corresponds to 2 logarithmic cycles and therefore results in a final cell density in the fermented doughs in the range 1-8 x 10 ⁹ cfu/g.

The data shown in Tables 3 and 4 demonstrate that both with the use of a single strain Lactobacillus plantarum DSM 33412 or Lactobacillus brevis DSM 33413 and with different ratios between the two strains Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413 1:1 , 1 :10 and 10:1 results in terms of acidification, titratable acidity, lactic and acetic acid synthesis, fermentation quotient, release of free amino acids (TFAA) and peptides, as well as the impact on total polyphenol concentration and radical scavenging activity (on both methanolic and aqueous extracts), phytic acid degradation and lipase activity reduction were comparable and showed no significant differences (P>0.05).

The same applies to the use of the single strains: also in this case, the results obtained on both unroasted (G) and roasted (G _T ) maize germ matrix are to be considered as not significantly different (P>0.05) from the results obtained with both bacterial strains.

Therefore, in all examples, the advantages of the invention are obtained.

It can also be noted that the doughs obtained with both strains Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413 have improved performance compared to doughs obtained with a single strain: the average data show a better general trend in acidification and pro- technological performance.

Inoculums with cell densities below 10 ⁶ cfu/g result, with the same fermentation times, in fermentation performances and therefore product characteristics not comparable to those described in Examples 2 and 3 (data not shown). Although the extension of the fermentation times in these cases may lead to comparable results, it is not considered to be cost-effective from a process management point of view; such an extension of fermentation times would also expose the matrix to undesirable microbial contamination.

Inoculations above 10 ⁷ cfu/g lead to results that are not significantly different from those demonstrated in Example 3, and therefore not economically convenient (costs for obtaining the cells of the starter microorganisms not justified).

Example 4

Use of the sourdough based on maize germ for the production of oven-baked leavened products (bread)

4.1 Preparation of a wheat bread fortified with fermented toasted maize germ

A bread (DY, 180) made from wheat ( Triticum aestivum, cv Appulo) fortified with roasted and fermented maize germ (i-fG _T ) was produced by employing the two-step protocol commonly used for sourdough bread production (Rizzello et al., 2016. Food Microbiology 56: 1-13).

I-G _T produced as per Example 2 was fermented with L. plantarum DSM 33412 e L. brevis DSM 33413 at 30° C for 24 hours (i-fG _T , step I, corresponding to "sourdough based on maize germ")', then ,I _F G _T was mixed with wheat flour, water and brewer's yeast at a speed of 60 xg for 5 minutes with a high-speed mixer IM 58 (Mecnosud, Flumeri, Italy) and allowed to rest for 1 .5 hours at 30° C (step II). The chemical composition of the wheat flour used was as follows: humidity, 14.2%; protein (N x 5.70), 11.5% (dry matter); fat, 1.6%; ash, 0.6% and total soluble carbohydrates, 86.3%. In detail, i-fG _T was used at 25% (weight/weight). At the end of the resting step (step II), the risen dough (500 g) was baked at 220° C for 50 minutes (Combo 3, Zucchelli, Verona, Italy), resulting in a wheat bread enriched with roasted and fermented maize germ (p-i-fG _T ). A wheat bread (p-F, DY 180) obtained with only brewer's yeast and without the addition of l _F G _T was used as a control. The brewer's yeast was added at a rate of 1.5% (w/w) (which corresponds to a final Saccharomyces cerevisiae cell density of about 9 log 10 cfu/g) in all the doughs for step II only. Salt was not used. All breads were cooled to room temperature before analysis.

4.2 Characterisation of a wheat bread fortified with roasted and fermented maize germ

The values of the biochemical (pH and TTA, organic acid concentration) and nutritional characteristics (protein, fat, carbohydrate, total fibre, TFAA, total phenols and phytic acid and radical scavenging activity) were determined as reported above. The specific volume of the breads was measured according to the approved method AACC 10-05.01 (American Association for Clinical Chemistry, 2010).

The in-vitro protein digestibility ( IVPD ) of the breads (p-l _F G _T e p-F) was determined using the method proposed by Akeson and Stahmann (Akeson and Stahmann. 1964. Journal of Nutrition 83:257-261) with some modifications (Rizzello et al., 2014. Food Microbiology 37:59-68). The samples were subjected to a seguential enzymatic processing mimicking in-vivo digestion in the gastrointestinal tract and IVPD was expressed as the percentage of total protein that was solubilised after enzymatic hydrolysis. The protein concentration in the digested and undigested fractions was determined by the Bradford method (Bradford, 1976. Analitical Biochemistry 72: 248- 254). Starch hydrolysis analysis was carried out on the breads. The procedure mimicked the in- vivo digestion of starch (De Angelis et al., 2009. European Food Research and Technology 229: 593-601 ). Aliguots of bread, containing 1 g starch, were subjected to an enzymatic process and the glucose content released was measured using the D-Fructose/D-Glucose Assay Kit (Megazyme Inti., Ireland). The degree of starch digestion was expressed as the percentage of potentially available and hydrolysed starch after 180 min. The p-F bread was used as a control to estimate the hydrolysis index (HI = 100). The predicted glycaemic index (pGI) was calculated using the eguation: pGI = 0.549 x HI + 39.71 (Capriles and Areas, 2013. Food & Function 4:04-10).

4.3 Biochemical and nutritional characteristics of bread containing roasted and fermented maize germ

The biochemical and nutritional characteristics of the breads are summarised in Tables 3 and 4. The values of pH, TTA and concentrations of lactic and acetic acids were respectively lower and higher in the breads enriched with roasted and fermented maize germ than in the control bread (p- F).

Furthermore, the fermentation quotient value was higher in fortified bread, reaching values considered optimal for a good sensory profile (Hammes and Ganzle, 1998. Microbiology of fermented foods. 199-216).

The use of roasted and fermented maize germ as an ingredient in baking resulted in high fibre (up to about 9.9 % d.m.) and protein (about 12.9 % d.m.) content compared to p-F. Significantly higher concentrations (up to about 2-fold) of TFAA were found in p-i-fG _T compared to p-F.

The higher concentration of TFAA, which can be considered as an indication of the degree of proteolysis operated by the lactic bacteria during the fermentation process, in p-i-fG _T was reflected in the IVPD, which was up to 70% higher than p-F. In contrast, a significant reduction in HI (about 30%) was found in p-i-fG _T compared to p-F.

The high fibre content (EC Regulation no. 1924/2006) of bread fortified with roasted and fermented maize grains is not the only nutritional benefit of fortification. In fact, fermentation of the roasted maize germ with LAB positively influenced other nutritional characteristics such as IVPD and HI, resulting in a bread with a high nutritional profile. High TFAA content and increased protein digestibility were achieved when i-fG _T was used to fortify bread.

These data were probably mainly due to the intense proteolysis operated by endogenous and microbial enzymes (Pontonio et al., 2017. Journal of Cereal Science 77:235-242). Furthermore, biological acidification operated by the lactic bacteria positively affected starch hydrolysis (HI) De Angelis et al., 2009. European Food Research and Technology 229: 593-601 ).

A significant reduction in phytic acid concentration (about 40 %), in accordance with what was found in i-fG _T , was found in p-i-fG _T compared to p-F.

Table 5. Biochemical properties of wheat bread fortified with maize germ sourdough (obtained from roasted maize germ). The data relating to a bread obtained with the sole use of brewer's yeast and therefore not containing i-fG _T (p-F) have been reported. p-F was used as a control.

Table 6. Nutritional characteristics of wheat bread fortified with roasted and fermented maize germ. The data relating to a bread obtained with the sole use of brewer's yeast (p-F) and therefore not containing maize germ sourdough i-fG _T have been reported. p-F was used as a control.

Example 5 Production of the spreadable cream

5.1 Formulation and production of the spreadable cream based on maize germ

The production of the spreadable cream involved the preliminary preparation of roasted and fermented maize germ. As described above, the preparatory operations involved separation of the maize germ from the other maize fractions; roasting of the maize germ and fermentation of the maize germ by inoculation with Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413. Fermentation was carried out at a temperature of 30°C for 24 hours. The stabilisation of the roasted and fermented maize germ was carried out by means of dehydration.

The formulation was prepared by mixing the following ingredients:

- roasted and fermented maize germ, 25% with respect to the total weight of the preparation;

- roasted maize germ, 25% with respect to the total weight of the preparation;

- seed oil, 15% with respect to the total weight of preparation;

- Cacao, 15% with respect to the total weight of preparation;

- cocoa mass, 10% with respect to the total weight of preparation; - “cane” sugar, 10% with respect to the total weight of preparation;

Mixing was carried out in a ball mill for 40 min at 25°C.

The production process is schematised in Figure 2.

- Characterisation of the spreadable cream

Protein (total nitrogen x 5.7), lipids, humidity, total dietary fibre and ash were determined according to the methods approved by the American Association of Cereal Chemists (AACC, 2010) and identified by the following codes 46-11 A, 30-10.01 , 44-15A, 32-05.01 and 08-01.01. The available carbohydrates were calculated as the difference [100 - (protein + lipids + ash + total dietary fibre)]. Protein, lipids, carbohydrates, total dietary fibre and ash were expressed as % of dry matter (d.m.). The results obtained are shown in the following Table 7. Table 7. Nutrition label for the spreadable cream ^* The data are the result of three independent production and analysis ± standard deviation (n=3).

Example 6

Production of the yoghurt-style drink

6.1 Formulation and production of a yoghurt-style drink based on roasted maize germs

The production of the yoghurt-style drink involved the preliminary preparation of roasted and fermented maize germ. As described above, the preparatory operations involved separating the maize germ from the other maize fractions and roasting of the maize germ.

The formulation was then prepared by mixing (with a paddle mixer, for a time of 15 min, at a temperature of 25°C) the following ingredients:

- roasted maize germ 8% of the final weight of the formulation;

- maize flour 8% of the final weight of the formulation;

- water: 84% of the final weight of the formulation.

The admixture was then subjected to the following steps:

- thermal processing: 85°C for 10 minutes;

- cooling to 8°C in 5 minutes;

- heating to 30°C, and inoculation of the selected starters Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413 (cell pellets collected by centrifugation from late-phase culture), at a cell density of 5 x 10 ⁷ cfu/g;

- fermentation at 30°C for 24 hours,

-refrigeration at 4°C for 30 days

The maize flour used as an ingredient had the following composition: humidity, 12.4%; protein, 8.6%; lipids, 2.7%; carbohydrates, 75.8% (of which starch 65.4% and fibre 3.1%); ash, 0.5%.

6.2 Chemical and microbiological characterisation

Protein (total nitrogen x 5.7), lipids, humidity, total dietary fibre and ash were determined according to the methods approved by the American Association of Cereal Chemists (AACC, 2010) and identified by the following codes 46-11 A, 30-10.01 , 44-15A, 32-05.01 and 08-01.01. The available carbohydrates were calculated as the difference [100 - (protein + lipids + ash + total dietary fibre)]. Protein, lipids, carbohydrates, total dietary fibre and ash were expressed as % of dry matter (d.m.). The results of the characterisation are shown in the following table.

The microbiological characterisation was carried out by homogenising 10 g of sample with 90 ml of peptonated water (0.1% peptone and 0.85% NaCI) at 0, 15 and 30 days of refrigerated storage. The total aerobic mesophilic bacterial load was assessed by counting on Plate Count Agar medium (PCA, Oxoid, Basingstoke, Hampshire, UK) at 30°C for 48 hours while lactic bacteria were enumerated using modified de Man, Rogosa and Sharpe (mMRS supplemented with 1% maltose and 5% fresh yeast extract, pH 5.6). Enterobacteriaceae were enumerated on Violet Red Bile Glucose Agar (VRBGA, Oxoid) at 37 °C for 24 hours. The yeasts and moulds were enumerated on Soboroud Dextrose Agar (SDA, Oxoid) and Potato Dextrose Agar (PDA, Oxoid), respectively, at 25°C for 48 hours.

At the end of the fermentation process, the pH of the drink was egual to 4.3 and the cell density of lactic bacteria was 3 x 10 ⁹ cfu/g. The nutrition label for the yoghurt-like drink is shown in table 8. If compared to a yoghurt made from whole milk, the vegetable drink containing maize flour and maize germ (8% each) has about 40 and 30% lower protein and fat content, respectively; and higher available carbohydrate content (6.3 vs. 4.3 g/100g yoghurt). However, while yoghurt contains no dietary fibre, the vegetable drink provides 2.8 g/100 g of fibre.

The microbiological analyses carried out at 15 and 30 days of refrigerated storage confirm the survival at high cell density (never less than 10 ⁸ cfu/ml) of the lactic bacteria used as starters and the hygienic safety of the product (Table 9).

Table 8. Nutrition label for the yoghurt-style drink based on roasted maize germ.

Table 9. Microbiological analysis of the yoghurt-style drink based on roasted maize germ described in Example 6, 15 and 30 days of refrigerated storage

Example 7 Vegetable cheese production

7.1 Formulation and production of a vegetable cheese on the basis of roasted maize germs

The production of the vegetable cheese involved the preliminary preparation of roasted and fermented maize germ. As described above, the preparatory operations involved separation of the maize germ from the other maize fractions; roasting of the maize germ and fermentation of the maize germ by inoculation with Lactobacillus plantarum DSM 33412 and Lactobacillus brevis DSM 33413. Fermentation was carried out at a temperature of 30°C for 24 hours. The stabilisation of the roasted and fermented maize germ was carried out by means of dehydration.

The production of the vegetable cheese on the basis of maize germ then involved the following steps: - mixing of the following ingredients:

- roasted maize germ 35% of the final weight of the formulation;

- roasted and fermented maize germ (dehydrated), 30% of the final weight of the formulation;

- maize flour 20%; - water: 15% of the final weight of the formulation, - thermal processing, 85°C for 15 minutes.

- shaping in pieces of 500g and soft pressing.

- storage under refrigerated conditions (4° C)

7.2 Characterisation Protein (total nitrogen x 5.7), lipids, humidity, total dietary fibre and ash were determined according to the methods approved by the American Association of Cereal Chemists (AACC, 2010) and identified by the following codes 46-11 A, 30-10.01 , 44-15A, 32-05.01 and 08-01.01. The available carbohydrates were calculated as the difference [100 - (protein + lipids + ash + total dietary fibre)]. Protein, lipids, carbohydrates, total dietary fibre and ash were expressed as % of dry matter (d.m.). The results obtained are shown in the following Table 10.

Table 10. Nutrition label for vegetable cheese on the basis of roasted maize germ