Safety Evaluation of Goat Milk Added with the Prebiotic Inulin Fermented with the Potentially Probiotic Native Culture Limosilactobacillus mucosae CNPC007 in Co-culture with Streptococcus thermophilus QGE: Analysis of Acute and Repeated Dose Oral Toxicity.

Despite functional goat milk products having emerged due to their importance for human nutrition and health, few studies have assessed the safety of consumption of goat dairy products containing potentially probiotic autochthonous lactic acid bacteria supplemented with prebiotic carbohydrates. Aiming this field, this study evaluated the safety of goat's milk fermented with Streptococcus thermophilus QGE, the autochthonous Limosilactobacillus mucosae CNPC007 culture, and the prebiotic inulin, through single- and repeated-dose oral toxicity tests (SDT and RDT, respectively) in animals. Ten female Swiss Webster mice were used for SDT evaluation - 2 groups, SDTc (20 mL/kg of filtered water) and SDTt (20 mL/kg of fermented milk) - and 40 Wistar rats for RDT - RDT3, RDT6, and RDT12 (treated with fermented milk at doses of 3 mL/kg, 6 mL/kg, and 12 mL/kg, respectively) and also RDTc (12 mL/kg of filtered water). For SDT, no signs of mortality or toxicity were observed, and the animals maintained the expected weight gain and feed intake. The RDT trials did not show mortality or signs of toxicity, as well as no change in body weight and organs, in the hematological and biochemical parameters, and also in relation to morphology and histology. Since the fermented milk did not cause any toxic effect in the conditions evaluated, it can be said that its no-adverse effect level (NOAEL) was considered to be higher than 20 mL/kg/day. Thus, the fermented milk with L. mucosae CNPC007 and inulin was considered to be of low toxicity, safe for use in rodents, and allowed for use in further studies.

Autochthonous adjunct culture of Limosilactobacillus mucosae CNPC007 improved the techno-functional, physicochemical, and sensory properties of goat milk Greek-style yogurt.

We evaluated the performance of Limosilactobacillus mucosae CNPC007 as an autochthonous adjunct culture in the production of goat milk Greek-style yogurt. The techno-functional, physicochemical, and sensory characteristics of the control yogurt (containing only starter culture, CY) and the probiotic yogurt (with the probiotic strain added, PY) were assessed during 28 d of refrigerated storage. Furthermore, we determined the survival of the strain throughout the gastrointestinal tract under simulated conditions. The PY yogurt had a lower extent of proteolysis index and a higher depth of proteolysis index. These results indicate that the proteolytic enzymes of L. mucosae may have a possible action in PY. The PY formulation exhibited viscosity almost 1.5 times as high as CY over the refrigeration period, probably due to higher production of exopolysaccharides by the probiotic strain, which directly interferes with the microstructure, texture, and viscosity of the product. The PY formulation received higher scores for color, flavor, and global acceptance at 1 d of storage and higher texture scores at 28 d. The counts of L. mucosae remained high (>7 log cfu/g and >8.5 log cfu/g) throughout mouth-ileum digestion and storage, respectively, in PY. The autochthonous adjunct culture of L. mucosae CNPC007 can be used for production of a novel potentially probiotic goat yogurt without negatively affecting the general characteristics of the product quality, adding value associated with maintaining its functional potential.

Functional Properties of Lactobacillus mucosae Strains Isolated from Brazilian Goat Milk.

The search for probiotic candidates among lactic acid bacteria (LAB) isolated from food may uncover new strains with promising health and technological properties. Lactobacillus mucosae strains attracted recent research attention due to their ability to adhere to intestinal mucus and to inhibit pathogens in the gastrointestinal tract, both related to a probiotic potential. Properties of interest and safety aspects of three Lb. mucosae strains (CNPC006, CNPC007, and CNPC009) isolated from goat milk were investigated employing in vitro tests. The presence of genetic factors related to bile salt hydrolase production (bsh), intestinal adhesion properties (msa, map, mub, and ef-tu), virulence, and biogenic amine production were also verified. All strains exhibited the target map, mub, and ef-tu sequences; the msa gene was detected in CNPC006 and CNPC007 strains. Some of the searched sequences for virulence factors were detected, especially in the CNPC009 strain; all strains carried the hyl gene, related to the production of hyaluronidase. Lb. mucosae CNPC007 exhibited a high survival rate in simulated gastric and enteric conditions. Besides, all strains exhibited the bsh sequence, and CNPC006 and CNPC007 were able to deconjugate salts of glycodeoxycholic acid (GDC). Regarding technological properties for dairy product applications, a relatively higher milk acidification and clotting capacity, diacetyl production, and proteolytic activity were registered for CNPC007 in comparison to the other strains. Collectively, the results aim at Lb. mucosae CNPC007 as a promising probiotic candidate for application in dairy products, deserving further studies to confirm and explore its potential.

Lectin, hemolysin and protease inhibitors in seed fractions with ovicidal activity against Haemonchus contortus / Lectina, hemolisina e inibidores de protease em frações de sementes com atividade ovicida contra Haemonchus contortus

Bioactive molecules of plant species are promising alternatives for the chemical control of gastrointestinal nematodes in ruminants. Extracts of native and exotic seed species from Brazil's semi-arid region were tested in vitro in an egg hatch assay and the bioactivity of their proteins was investigated. Each seed species was subjected to three extractions with three types of solvents. All the seeds showed ovicidal activity, which varied according to the solvents. Higher ovicidal activity was found in the molecule fractions of low molecular weight (<12 kDa) for Albizia lebbeck, Ipomoea asarifolia, Jatropha curcas, Libidibia ferrea, Moringa oleifera and Ricinus communis (P<0.05, Bonferroni test). The two fractions of Crotalaria spectabilis showed the same ovicidal activity (P>0.05, Bonferroni test). Hemagglutinating activity was detected in the fractions of C. spectabilis and M. oleifera fractions, hemolysin activity in the A. lebbeck and M. oleifera fractions, serine protease inhibitory activity in the A. lebbeck, I. asarifolia, J. curcas, M. oleifera and R. communis fractions, cysteine protease inhibitor activity in the M. oleifera fraction, and no protein activity in the L. ferrea fraction. The results of this work reveal new plant species with a potential for use in controlling nematode parasites in goats, thus opening a new field of research involving plant protein molecules with ovicidal properties.

Moléculas bioativas de espécies vegetais são alternativas promissoras ao controle químico dos nematoides gastrintestinais em ruminantes. Extratos de sementes de espécies nativas e exóticas do Semiárido Brasileiro foram testados in vitro em ensaio de eclosão de ovos e investigada a natureza proteica da bioatividade. Três extrações com três solventes foram feitas para cada semente estudada. Todas as sementes apresentaram atividade ovicida, variando com o solvente utilizado. Maior taxa de inibição da eclosão concentrou-se nas frações de moléculas de baixa massa molecular (<12 kDa) para Albizia lebbeck, Ipomoea asarifolia, Jatropha curcas, Libidibia ferrea, Moringa oleifera e Ricinus communis (P<0,05, teste de Bonferroni). Crotalaria spectabilis mostrou atividade nas duas frações, sem diferença entre elas (P>0,05, teste de Bonferroni). Observou-se atividade hemaglutinante nas frações de C. spectabilis e M. oleifera, de hemolisina em A. lebbeck e M. oleifera, de atividade inibidora de protease da serina em A. lebbeck, I. asarifolia, J. curcas, M. oleifera e R. communis, de atividade inibidora de protease da cisteína em M. oleifera e nenhuma atividade proteica na fração de L. ferrea. Os resultados revelaram novas espécies botânicas com potencial de controle de nematoides em caprinos e um novo campo de pesquisa, o estudo de moléculas de origem proteica com atividade ovicida.