Quantitative determination of active Bowman-Birk isoinhibitors, IBB1 and IBBD2, in commercial soymilks

Naturally-occurring serine protease inhibitors of the Bowman-Birk family exert their potential chemopreventive and/or therapeutic properties via protease inhibition. In this study, we have quantified the amounts of active BBI isoinhibitors, IBB1 and IBBD2, in six commercial soymilks. By using cation exchange chromatography, the BBI isoinhibitors were isolated and their specific trypsin inhibitory activity was used to estimate their amounts in soymilk samples. IBB1 and IBBD2 concentrations ranged from 0.44 to 5.20 and 0.27 to 4.60 mg/100 ml of soymilk, respectively; total BBI, considered as the sum of both isoinhibitors, ranged from 0.60 to 9.07 mg/100 ml of soymilk. These data show that physiologically relevant amounts of active BBI are present in commercial soymilk and may exert potential health-promoting effects.A.C. acknowledges support by ERDF-co-financed Grant from the Spanish CICYT (AGL2011-26353). A.C. is involved in COST Action FA1005 INFOGEST on Food Digestion. L.C.B.R. acknowledges support from CAPES (Programa Institucional de Doutorado Sanduíche no Exterior–PDSE process number 9004-11-4), Brazil. We are grateful to the proteomics Facility of University of Cordoba (Spain) for carrying out peptide mass fingerprinting. B.H.-L. thanks Ministry of Economy and Competitiveness for her “Ramón y Cajal” contract.Peer Reviewe

Prebiotic properties of non-fructosylated α-galactooligosaccharides from PEA (Pisum sativum L.) using infant fecal slurries

The interest for naturally-occurring oligosaccharides from plant origin having prebiotic properties is growing, with special focus being paid to supplemented products for infants. Currently, non-fructosylated α-galactooligosaccharides (α-GOS) from peas have peaked interest as a result of their prebiotic activity in adults and their mitigated side-effects on gas production from colonic bacterial fermentation. In this study, commercially available non-fructosylated α-GOS from peas and β-galactooligosaccharides (β-GOS) derived from lactose were fermented using fecal slurries from children aged 11 to 24 months old during 6 and 24 h. The modulatory effect of both GOS on different bacterial groups and bifidobacteria species was assessed; non-fructosylated α-GOS consumption was monitored throughout the fermentation process and the amounts of lactic acid and short-chain fatty acids (SCFA) generated were analyzed. Non-fructosylated α-GOS, composed mainly of manninotriose and verbascotetraose and small amounts of melibiose, were fully metabolized and presented remarkable bifidogenic activity, similar to that obtained with β-GOS. Furthermore, non-fructosylated α-GOS selectively caused an increase on the population of Bifidobacterium longum subsp. longum and Bifidobacterium catenulatum/pseudo-catenulatum. In conclusion, non-fructosylated α-GOS could be used as potential ingredient in infant formula supplemented with prebiotic oligosaccharides.This research was funded by the Spanish Ministry of Economy, Industry and Competitiveness, grant numbers AGL2017-83772-R and AGL2017-84614-C2-1-R (AEI/FEDER,UE); the Spanish Ministry of Science, Innovation and Universities grant number RTI2018-101273-J-I00 (JIN Program) and AGR2011-7626 from Junta de Andalucía

The cytotoxic effect of Bowman-Birk isoinhibitors, IBB1 and IBBD2, from soybean (Glycine max) on HT29 human colorectal cancer cells is related to their intrinsic ability to inhibit serine proteases

Bowman-Birk inhibitors (BBI) from soybean and related proteins are naturally occurring protease inhibitors with potential health-promoting properties within the gastrointestinal tract. In this work, we have investigated the effects of soybean BBI proteins on HT29 colon adenocarcinoma cells, compared with non-malignant colonic fibroblast CCD-18Co cells. Two major soybean isoinhibitors, IBB1 and IBBD2, showing considerable amino acid sequence divergence within their inhibitory domains, were purified in order to examine their functional properties, including their individual effects on the proliferation of HT29 colon cancer cells. IBB1 inhibited both trypsin and chymotrypsin whereas IBBD2 inhibited trypsin only. Despite showing significant differences in their enzyme inhibitory properties, the median inhibitory concentration values determined for IBB1 and IBBD2 on HT29 cell growth were not significantly different (39.9+/-2.3 and 48.3+/-3.5 μM, respectively). The cell cycle distribution pattern of HT29 colon cancer cells was affected by BBI treatment in a dose-dependent manner, with cells becoming blocked in the G0-G1 phase. Chemically inactive soybean BBI had a weak but non-significant effect on the proliferation of HT29 cells. The anti-proliferative properties of BBI isoinhibitors from soybean reveal that both trypsin-and chymotrypsin-like proteases involved in carcinogenesis should be considered as potential targets of BBI-like proteins.This work was carried out with the financial support of grants AGR 2006-00706 and AGL 2007-60007/ALI from Junta de Andalucía and Spanish CICYT, respectively. Alfonso Clemente acknowledges receipt of a Ramon and Cajal contract.Peer Reviewe

Quantitative determination of active Bowman-Birk isoinhibitors, IBB1 and IBBD2, in commercial soymilks

Resumen del póster presentado al 2nd International Conference on Food Digestion celebrado en Madrid (España) del 6 al 8 de marzo de 2013.Peer Reviewe

Estudio de la digestibilidad ileal y actividad prebiótica in vivo de galacto-oligosacáridos sintetizados a partir de lactosa y lactulosa

Resumen del trabajo presentado al III Workshop Probióticos, Prebióticos y Salud. Evidencia Científica, celebrado en Castelldefels (Barcelona) del 15 al 16 de diciembre de 2011.Los galacto-oligosacáridos (GOS) son mezclas de oligosacáridos basados en unidades de galactosas, producidos mediante transgalactosilación de la lactosa (GOS-La), usando como catalizador β-galactosidasas. Recientemente, se han sintetizado y caracterizado nuevos GOS a partir de lactulosa (GOS-Lu), observándose que dichos carbohidratos poseen un efecto bifidogénico in vitro. Para confirmar su potencial prebiótico, el objetivo del presente trabajo fue estudiar la digestibilidad de GOS sintetizados a partir de lactosa y lactulosa, evaluando su actividad prebiótica in vivo y la diversidad poblacional de bifidobacterias en el intestino grueso (ciego y colon). Para ello, se alimentaron ratas Wistar durante 14 días, con dietas al 1% (p/p) de GOS-Lu, GOS-La y lactulosa, respectivamente, incluyendo como marcador indigestible Cr2O3. La presencia de estos carbohidratos en el íleon se detectó y cuantificó mediante GC-MS, determinándose su grado de digestibilidad. El efecto prebiótico in vivo así como la modulación de las poblaciones de bifidobacterias del contenido intestinal se determinaron mediante qPCR y DGGE, respectivamente. Los trisacáridos de GOS-Lu demostraron ser mucho menos digeribles en el íleon (12.5%) que los trisacáridos presentes en GOS-La (53.2 %), mientras que la lactulosa y los disacáridos presentes en GOS-Lu no se digirieron. Estos resultados indican que los GOS-Lu alcanzan el intestino grueso en mayor proporción que los GOS-La. Mediante qPCR, se observó una mayor actividad bifidogénica tras ingesta de GOS-Lu, comparado con GOS-La, presentando las muestras de contenido intestinal una mayor diversidad poblacional de bifidobacterias.En conclusión, los resultados obtenidos con GOS-Lu ponen de manifiesto su capacidad para alcanzar el intestino grueso en concentraciones fisiológicamente significativas debido a su baja digestibilidad ileal, además de ejercer un efecto positivo en la modulación de la microbiota intestinal y en la diversidad poblacional de bifidobacterias, en comparación con prebióticos comercialmente disponibles (GOS-La).Peer Reviewe

An Updated Review of Soy-Derived Beverages: Nutrition, Processing, and Bioactivity

The global market for plant-based drinks is experiencing rapid growth driven by consumer demand for more sustainable diets, including vegetarian and vegan options. Soy beverages in particular are gaining popularity among individuals with lactose intolerance and milk protein allergies. They are considered an excellent source of high-quality protein, vitamin B, unsaturated fatty acids, and beneficial phytochemicals such as phytosterols, soy lecithins, and isoflavones. This review presents a comprehensive market survey of fifty-two soy beverages available in Spain and other European countries. The predominant category among those evaluated was calcium and vitamin-fortified drinks, accounting for 60% of the market. This reflects the need to address the nutritional gap compared to cow’s milk and meet essential dietary requirements. The review covers the technological aspects of industrial soy milk production, including both traditional methods and innovative processing techniques. Additionally, it analyzes multiple studies and meta-analyses, presenting compelling evidence for the positive effects of soy beverages on various aspects of health. The review specifically examines the contributions of different components found in soy beverages, such as isoflavones, proteins, fiber, and oligosaccharides. Moreover, it explores controversial aspects of soy consumption, including its potential implications for growth, puberty, fertility, feminization, and the thyroid gland

In vitro fermentation of novel enzymatically synthesized fruit-oligosaccharides by infant fecal microbiota

Resumen de la comunicación oral presentada en el XII Workshop Sociedad Española de Microbiota, Probióticos y Prebióticos y I Congreso Sociedad Iberoamericana de Microbiota, Probióticos y Prebióticos. 15-18 de septiembre (Madrid, 2021

The anti-proliferative properties of Bowman-Birk proteins from legumes on colon cancer cells are related to their ability to inhibit trypsin- and chymotrypsin-like proteases

Resumen del trabajo presentado al BIT's Inaugural Symposium on Enzymes & Biocatalysis celbrado en Shanghai (China) del 22 al 24 de abril de 2010.Bowman-Birk inhibitors (BBI) from legumes, such as soybean, pea, lentil and chickpea, are naturally-occurring protease inhibitors with potential health-promoting properties within the gastrointestinal tract. Recently, we have demonstrated that BBI proteins are structurally and functionally resistant to the harsh environment of the gastrointestinal tract in vivo. BBI proteins from chickpea seeds can resist both acidic conditions and the action of proteolytic enzymes, and transit through the pig stomach and small intestine without major degradation, permitting significant amounts to reach the large intestine in active form. Further studies have demonstrated that the protease (trypsin- and chymotrypsin-like) inhibitory activities of soybean BBI are unaffected by the metabolic/proteolytic activity of faecal microbiota. We demonstrated that soybean BBI, consisting of multiple isoinhibitors, inhibited the in vitro cell growth of HT29 human colorectal adenocarcinoma cells as a consequence of their intrinsic ability to inhibit the proteolytic activities of serine proteases, where chemically inactive BBI showed no such effect. In contrast, the growth of non-malignant colonic fibroblast CCD-18Co cells was unaffected by soybean BBI. In our studies, sequence variation within the inhibitory domains suggested to be of biological relevance. Two major soybean isoinhibitors, IBB1 and IBBD2, showing considerable amino acid sequence divergence within and between their inhibitory domains, were purified in order to evaluate their individual effects on the proliferation of HT29 colon cancer cells. The anti-proliferative properties of individual isoinhibitors from soybean reveal that both trypsin- and chymotrypsin-like proteases involved in carcinogenesis should be considered as potential targets of BBI-like proteins.Peer Reviewe

The bifidogenic effect of galacto-oligosaccharides in rats is dependent of their structural characteristics

Resumen del trabajo presentado al IV Workshop Probióticos, Prebióticos y Salud: Evidencia Científica celebrado en Madrid (España) del 31 de enero al 1 de febrero de 2013.A comparative study to evaluate the resistance to gut digestion, fermentability and modulatory effect of gut microbiota in the large intestine of growing rats (5 wk old) fed novel galacto-oligosaccharides (GOS) derived from lactulose (GOS-Lu) or commercially available derived from lactose (GOS-La) was performed. Rats were fed either a control (AIN-93G) or experimental diet containing 1% (wt:wt) of GOS-Lu or GOS-La for 14 d. Quantitative analysis of oligomers from dietary and ileal samples demonstrated that the trisaccharide fraction of GOS-Lu was significantly more resistant to gut digestion than that from GOS-La, whereas the disaccharide fraction of GOS-Lu was fully resistant to the extreme environment of the upper digestive tract. The low ileal digestibility rates of GOS-Lu was attributed to the great resistance of galactosyl-fructoses to mammalian digestive enzymes, highlighting the key role played by monomer composition and linkage type involved in the oligosaccharide chain. Di- and trisaccharides were completely fermented within the large intestine, enabling both GOS to have a potential prebiotic function. Indeed, the novel GOS-Lu exerted a stronger bifidogenic effect in large intestine than GOS-La, with the former having a significant and selective increase of Bifidobacterium animalis. In addition, both GOS types showed a significant increase in Eubacterium rectale/Clostridium coccoides population relative to control. These data support a direct relationship between patterns of resistance to digestion and bifidogenic properties of galacto-oligosaccharides.Peer Reviewe