Bioprocessed Brewers' Spent Grain Improves Nutritional and Antioxidant Properties of Pasta.

Brewers' spent grain (BSG), the by-product of brewing, was subjected to a xylanase treatment followed by fermentation with Lactiplantibacillus plantarum PU1. Bioprocessed BSG has been used as ingredient to obtain a fortified semolina pasta which can be labeled as "high fiber" and "source of protein" according to the European Community Regulation No. 1924/2006. Compared to native BSG, the use of bioprocessed BSG led to higher protein digestibility and quality indices (essential amino acid index, biological value, protein efficiency ratio, nutritional index), as well as lower predicted glycemic index. Bioprocessing also improved the technological properties of fortified pasta. Indeed, brightfield and confocal laser scanning microscopy revealed the formation of a more homogeneous protein network, resulting from the degradation of the arabinoxylan structure of BSG, and the release of the components entrapped into the cellular compartments. The extensive cell wall disruption contributed to the release of phenols, and conferred enhanced antioxidant activity to the fortified pasta. The persistence of the activity was demonstrated after in vitro-mimicked digestion, evaluating the protective effects of the digested pasta towards induced oxidative stress in Caco-2 cells cultures. The fortified pasta showed a peculiar sensory profile, markedly improved by the pre-treatment, thus confirming the great potential of bioprocessed BSG as health-promoting food ingredient.

Fermentation Biotechnology Applied to Cereal Industry By-Products: Nutritional and Functional Insights.

Cereals are one of the major food sources in human diet and a large quantity of by-products is generated throughout their processing chain. These by-products mostly consist of the germ and outer layers (bran), deriving from dry and wet milling of grains, brewers' spent grain originating from brewing industry, or others originating during bread-making and starch production. Cereal industry by-products are rich in nutrients, but still they end up as feed, fuel, substrates for biorefinery, or waste. The above uses, however, only provide a partial recycle. Although cereal processing industry side streams can potentially provide essential compounds for the diet, their use in food production is limited by their challenging technological properties. For this reason, the development of innovative biotechnologies is essential to upgrade these by-products, potentially leading to the design of novel and commercially competitive functional foods. Fermentation has been proven as a very feasible option to enhance the technological, sensory, and especially nutritional and functional features of the cereal industry by-products. Through the increase of minerals, phenolics and vitamins bioavailability, proteins digestibility, and the degradation of antinutritional compounds as phytic acid, fermentation can lead to improved nutritional quality of the matrix. In some cases, more compelling benefits have been discovered, such as the synthesis of bioactive compounds acting as antimicrobial, antitumoral, antioxidant agents. When used for baked-goods manufacturing, fermented cereal by-products have enhanced their nutritional profile. The key factor of a successful use of cereal by-products in food applications is the use of a proper bioprocessing technology, including fermentation with selected starters. In the journey toward a more efficient food chain, biotechnological approaches for the valorization of agricultural side streams can be considered a very valuable help.

Use of Selected Lactic Acid Bacteria and Quinoa Flour for Manufacturing Novel Yogurt-Like Beverages.

This study aimed at investigating the suitability of quinoa for making yogurt-like beverages. After the selection of the adequate technological parameters, the fermentation was carried out by using different lactic acid bacteria strains: a probiotic (Lactobacillus rhamnosus SP1), an exopolysaccharides (EPS)-producing (Weissella confusa DSM 20194), and one isolated from quinoa (Lactobacillus plantarum T6B10). During the 20 h of fermentation, W. confusa caused the highest viscosity increase. All the strains had improved concentration of free amino acids and γ-Aminobutyric acid (GABA), polyphenols availability, antioxidant activity (up to 54%), and protein digestibility. The nutritional index (NI) was the highest when L. rhamnosus SP1 was used. The starch hydrolysis index in vitro ranged from 52 to 60. During storage at 4 °C, viscosity and water holding capacity decreased with the exception of the beverage fermented with W. confusa, while all the nutritional characteristics remained stable or slightly increased. Sensory analyses showed that beverages had good textural and organoleptic profiles. Besides the well-known positive properties of the raw matrix, fermentation allowed the obtainment of beverages with different features. Due to the nutritional and functional characteristics conferred to the quinoa beverages, the use of the probiotic and EPS-producing strains showed adequate potential for the industrial application.

Italian legumes: effect of sourdough fermentation on lunasin-like polypeptides.

BACKGROUND: There is an increasing interest toward the use of legumes in food industry, mainly due to the quality of their protein fraction. Many legumes are cultivated and consumed around the world, but few data is available regarding the chemical or technological characteristics, and especially on their suitability to be fermented. Nevertheless, sourdough fermentation with selected lactic acid bacteria has been recognized as the most efficient tool to improve some nutritional and functional properties. This study investigated the presence of lunasin-like polypeptides in nineteen traditional Italian legumes, exploiting the potential of the fermentation with selected lactic acid bacteria to increase the native concentration. An integrated approach based on chemical, immunological and ex vivo (human adenocarcinoma Caco-2 cell cultures) analyses was used to show the physiological potential of the lunasin-like polypeptides. RESULTS: Italian legume varieties, belonging to Phaseulus vulgaris, Cicer arietinum, Lathyrus sativus, Lens culinaris and Pisum sativum species, were milled and flours were chemically characterized and subjected to sourdough fermentation with selected Lactobacillus plantarum C48 and Lactobacillus brevis AM7, expressing different peptidase activities. Extracts from legume doughs (unfermented) and sourdoughs were subjected to western blot analysis, using an anti-lunasin primary antibody. Despite the absence of lunasin, different immunoreactive polypeptide bands were found. The number and the intensity of lunasin-like polypeptides increased during sourdough fermentation, as the consequence of the proteolysis of the native proteins carried out by the selected lactic acid bacteria. A marked inhibitory effect on the proliferation of human adenocarcinoma Caco-2 cells was observed using extracts from legume sourdoughs. In particular, sourdoughs from Fagiolo di Lamon, Cece dell'Alta Valle di Misa, and Pisello riccio di Sannicola flours were the most active, showing a decrease of Caco-2 cells viability up to 70 %. The over-expression of Caco-2 filaggrin and involucrin genes was also induced. Nine lunasin-like polypeptides, having similarity to lunasin, were identified. CONCLUSIONS: The features of the sourdough fermented legume flours suggested the use for the manufacture of novel functional foods and/or pharmaceuticals preparations.

Synthesis of 2-methoxy benzoquinone and 2,6-dimethoxybenzoquinone by selected lactic acid bacteria during sourdough fermentation of wheat germ.

BACKGROUND: In the last decade, several studies described the promising cytotoxic activity of fermented wheat germ towards cancer cell lines and during in vivo clinical trials. Recent data suggested that the antiproliferative, antimetastatic and immunological effects of this preparation are mainly attributed to quinones. This study aimed at exploiting the potential of sourdough lactic acid bacteria fermentation to release 2-methoxy benzoquinone, and 2,6-dimethoxybenzoquinone, which are naturally present in wheat germ as glycosylated and non-physiologically active form. RESULTS: Preliminarily, forty strains of lactic acid bacteria, previously isolated from wheat germ, were in vitro screened based on ß-glucosidase activity. Lactobacillus plantarum LB1 and Lactobacillus rossiae LB5 were selected based on the highest enzyme activity and on technology features. These strains were used in combination to ferment wheat germ. Raw wheat germ, without bacterial inoculum, was subjected to the same incubation and used as the control. The sourdough fermented wheat germ was characterized based on microbiological, physico-chemical and biochemical features. During incubation, the release of the non-glycosylated and physiologically active 2-methoxy benzoquinone, and 2,6-dimethoxybenzoquinone was almost completed during 24 h. Compared to the control, the concentration of the above bioactive compounds increased almost 4 and 6-folds. Both raw wheat germ (control) and sourdough fermented wheat germ were ex vivo assayed for the anti-proliferative activity towards various cell lines of germ cell tumor, colon carcinoma and ovarian carcinoma. While no effect was found for the raw wheat germ, the sourdough fermented preparation markedly and variously affected the human tumor cell lines. The values of IC50 ranged from 0.105 ± 0.005 to 0.556 ± 0.071 mg/ml, with a median value of IC50 of 0.302 mg/ml. CONCLUSIONS: These results are comparable to those found for other well-known pharmaceutical preparations, and may disclose the use of the sourdough fermented wheat germ as an ingredient, nutritional supplement and/or anticancer drug.

Lactic acid fermentation as a tool to enhance the functional features of Echinacea spp.

BACKGROUND: Extracts and products (roots and/or aerial parts) from Echinacea ssp. represent a profitable market sector for herbal medicines thanks to different functional features. Alkamides and polyacetylenes, phenols like caffeic acid and its derivatives, polysaccharides and glycoproteins are the main bioactive compounds of Echinacea spp. This study aimed at investigating the capacity of selected lactic acid bacteria to enhance the antimicrobial, antioxidant and immune-modulatory features of E. purpurea with the prospect of its application as functional food, dietary supplement or pharmaceutical preparation. RESULTS: Echinacea purpurea suspension (5%, wt/vol) in distilled water, containing 0.4% (wt/vol) yeast extract, was fermented with Lactobacillus plantarum POM1, 1MR20 or C2, previously selected from plant materials. Chemically acidified suspension, without bacterial inoculum, was used as the control to investigate functional features. Echinacea suspension fermented with Lb. plantarum C2 exhibited a marked antimicrobial activity towards Gram-positive and -negative bacteria. Compared to control, the water-soluble extract from Echinacea suspension fermented with Lactobacillus plantarum 1MR20 showed twice time higher radical scavenging activity on DPPH. Almost the same was found for the inhibition of oleic acid peroxidation. The methanol extract from Echinacea suspension had inherent antioxidant features but the activity of extract from the sample fermented with strain 1MR20 was the highest. The antioxidant activities were confirmed on Balb 3T3 mouse fibroblasts. Lactobacillus plantarum C2 and 1MR20 were used in association to ferment Echinacea suspension, and the water-soluble extract was subjected to ultra-filtration and purification through RP-FPLC. The antioxidant activity was distributed in a large number of fractions and proportional to the peptide concentration. The antimicrobial activity was detected only in one fraction, further subjected to nano-LC-ESI-MS/MS. A mixture of eight peptides was identified, corresponding to fragments of plantaricins PlnH or PlnG. Treatments with fermented Echinacea suspension exerted immune-modulatory effects on Caco-2 cells. The fermentation with Lb. plantarum 1MR20 or with the association between strains C2 and 1MR20 had the highest effect on the expression of TNF-α gene. CONCLUSIONS: E. purpurea subjected to lactic acid fermentation could be suitable for novel applications as functional food dietary supplements or pharmaceutical preparations.

Yogurt-like beverages made of a mixture of cereals, soy and grape must: microbiology, texture, nutritional and sensory properties.

Cereal (rice, barley, emmer and oat) and soy flours and concentrated red grape must were used for making vegetable yogurt-like beverages (VYLB). Two selected strains of Lactobacillus plantarum were used for lactic acid fermentation, according to a process which included the flour gelatinization. All VLYB had values of pH lower than 4.0 and both selected starters remained viable at ca. 8.4 log cfu/g throughout storage. All VLYB showed high values of apparent viscosity and water holding capacity. During fermentation, lactic acid bacteria consumed glucose, fructose, and malic acid, which was supplied with grape must. Compared to control vegetable yogurt-like beverages (CVYLB), without bacterial inoculum, an increase of total free amino acids (FAA) was found during fermentation and storage. Also the concentration of polyphenolic compounds and ascorbic acid (ASC) was higher in VLYB compared to CVYLB. This was reflected on the antioxidant activity. As determined by Solid Phase Micro-Extraction/Gas-Chromatography/Mass-Spectrometry analysis, several volatile compounds were identified. Beverages made with the mixture of rice and barley or emmer flours seemed to possess the best combination textural, nutritional and sensory properties.

Selected lactic acid bacteria synthesize antioxidant peptides during sourdough fermentation of cereal flours.

A pool of selected lactic acid bacteria was used for the sourdough fermentation of various cereal flours with the aim of synthesizing antioxidant peptides. The radical-scavenging activity of water/salt-soluble extracts (WSE) from sourdoughs was significantly (P < 0.05) higher than that of chemically acidified doughs. The highest activity was found for whole wheat, spelt, rye, and kamut sourdoughs. Almost the same results were found for the inhibition of linoleic acid autoxidation. WSE were subjected to reverse-phase fast protein liquid chromatography. Thirty-seven fractions were collected and assayed in vitro. The most active fractions were resistant to further hydrolysis by digestive enzymes. Twenty-five peptides of 8 to 57 amino acid residues were identified by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry. Almost all of the sequences shared compositional features which are typical of antioxidant peptides. All of the purified fractions showed ex vivo antioxidant activity on mouse fibroblasts artificially subjected to oxidative stress. This study demonstrates the capacity of sourdough lactic acid bacteria to release peptides with antioxidant activity through the proteolysis of native cereal proteins.

Synthesis of the cancer preventive peptide lunasin by lactic acid bacteria during sourdough fermentation.

This study aimed to exploit the potential of sourdough lactic acid bacteria to release lunasin during fermentation of cereal and nonconventional flours. The peptidase activities of a large number of sourdough lactic acid bacteria were screened using synthetic substrates. Selected lactic acid bacteria were used as sourdough starters to ferment wholemeal wheat, soybean, barley, amaranth, and rye flours. Proteinase activity during fermentation was characterized by SDS-PAGE analysis of the water-soluble extracts. Albumins having molecular masses of 18 to 22 kDa, which included the size of lunasin precursors, were markedly affected by proteolysis of lactic acid bacteria. After fermentation, lunasin from the water-soluble extracts was quantified, purified, and identified through RP-HPLC and nano-LC-ESI-MS analyses. Compared to control doughs, the concentration of lunasin increased up to 2-4 times during fermentation. Lactobacillus curvatus SAL33 and Lactobacillus brevis AM7 synthesized the highest concentrations of lunasin in all the flours. Besides the presence of the entire lunasin sequence, fragments containing the immunoreactive epitope RGDDDDDDDDD were also found. This study shows that fermentation by lactic acid bacteria increased the concentration of lunasin to levels that would suggest new possibilities for the biological synthesis and for the formulation of functional foods.

Antifungal activity of Wickerhamomyces anomalus and Lactobacillus plantarum during sourdough fermentation: identification of novel compounds and long-term effect during storage of wheat bread.

This study aimed at investigating the antifungal activity of Wickerhamomyces anomalus and sourdough lactic acid bacteria to extend the shelf life of wheat flour bread. The antifungal activity was assayed by agar diffusion, growth rate inhibition, and conidial germination assays, using Penicillium roqueforti DPPMAF1 as the indicator fungus. Sourdough fermented by Lactobacillus plantarum 1A7 (S1A7) and dough fermented by W. anomalus LCF1695 (D1695) were selected and characterized. The water/salt-soluble extract of S1A7 was partially purified, and several novel antifungal peptides, encrypted into sequences of Oryza sativa proteins, were identified. The water/salt-soluble extract of D1695 contained ethanol and, especially, ethyl acetate as inhibitory compounds. As shown by growth inhibition assays, both water/salt-soluble extracts had a large inhibitory spectrum, with some differences, toward the most common fungi isolated from bakeries. Bread making at a pilot plant was carried out with S1A7, D1695, or a sourdough started with a combination of both strains (S1A7-1695). Slices of the bread manufactured with S1A7-1695 did not show contamination by fungi until 28 days of storage in polyethylene bags at room temperature, a level of protection comparable to that afforded by 0.3% (wt/wt) calcium propionate. The effect of sourdough fermentation with W. anomalus LCF1695 was also assessed based on rheology and sensory properties.

Molecular adaptation of sourdough Lactobacillus plantarum DC400 under co-cultivation with other lactobacilli.

This work was aimed at investigating the molecular mechanisms of Quorum Sensing (QS) in Lactobacillus plantarum DC400 when co-cultured with other sourdough lactobacilli. The growth and survival of L. plantarum DC400 was not affected when co-cultivated with Lactobacillus sanfranciscensis DPPMA174 or Lactobacillus rossiae A7. Nevertheless, 2-DE analysis showed that the level of protein expression of L. plantarum DC400 increased under co-culture conditions. Although several proteins were commonly induced in both co-cultures, the highest induction was found in co-culture with L. rossiae A7. Overexpressed proteins, related to QS and stress response mechanisms, were identified: DnaK, GroEL, 30S ribosomal protein S1 and S6, ATP synthase subunit beta, adenosylmethionine synthetase (MetK), phosphopyruvate hydratase, phosphoglycerate kinase, elongation factor Tu, putative manganese-dependent inorganic pyrophosphatase, d-lactate dehydrogenase, triosephosphate isomerase, fructose-bisphosphate aldolase and nucleoside-diphosphate kinase. As shown by real-time PCR, expression of the luxS gene of L. plantarum DC400 was also affected during co-cultivation. According to overexpression of MetK and luxS during co-cultivation, synthesis of AI-2-like substances was also influenced by the type of microbial co-cultures. This study showed that expression of some genes/proteins, also QS-related, in L. plantarum was influenced by co-cultivation of other sourdough lactobacilli.