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.

Goat Cheese Produced with Sunflower (Helianthus annuus L.) Seed Extract and a Native Culture of Limosilactobacillus mucosae: Characterization and Probiotic Survival.

The microbiological and biochemical properties of a goat cheese produced using Helianthus annuus (sunflower) seed extract as a coagulant and the potentially probiotic autochthonous culture Limosilactobacillus mucosae CNPC007 were examined in comparison to a control cheese devoid of the autochthonous culture. Throughout a 60-day storage period at 6 ± 1 °C, lactobacilli maintained a count of above 8 log CFU/g. Additionally, its viability in cheeses subjected to the in vitro gastrointestinal conditions demonstrated improvement over this period. Specifically, the recovery of lactobacilli above 6 log CFU/g was observed in 16.66% of the samples in the first day, increasing to 66.66% at both 30 and 60 days. While total coliforms were detected in both cheese trials, this sanitary parameter exhibited a decline in L. mucosae cheeses during storage, falling below the method threshold (<3 MPN/g) at 60 days. This observation suggests a potential biopreservative effect exerted by this microorganism, likely attributed to the higher acidity of L. mucosae cheeses at that point (1.80 g/100 g), which was twice that of the control trial (0.97 g/100 g). Furthermore, distinct relative proportions of >30 kDa, 30-20 kDa, and <20 kDa proteins during storage was verified for L. mucosae and control cheeses. Consequently, either the H. annuus seed extract or the L. mucosae CNPC007 autochthonous culture influenced the biochemical properties of the cheese, particularly in terms of proteolysis. Moreover, L. mucosae CNPC007 acidification property resulted in a biopreservative effect throughout the storage period, indicating the potential as a promising source of probiotics for this product.

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.

ß-Glucosidase activity and antimicrobial properties of potentially probiotic autochthonous lactic cultures.

Background: The demand for lactic acid bacteria products, especially probiotics, has increased. Bacteria that increase polyphenol bioavailability and act as bio preservatives are sought after. This study aims to identify autochthonous lactic acid cultures from EMBRAPA that demonstrate ß-glucosidase activity and inhibitory effect on microbial sanitary indicators. Methods: Cell-free extracts were obtained by sonicating every 5 s for 40 min. The extracts were mixed with cellobiose and incubated at 50 °C. The reaction was stopped by immersing the tubes in boiling water. The GOD-POD reagent was added for spectrophotometer readings. Antimicrobial activity was tested against reference strains using the agar well diffusion method. Lactic cultures in MRS broth were added to 0.9 cm wells and incubated. The diameter of the inhibition zones was measured to determine the extension of inhibition. Results: Only L. rhamnosus EM1107 displayed extracellular ß-glucosidase activity, while all autochthonous strains except L. plantarum CNPC020 demonstrated intracellular activity for this enzyme. L. plantarum CNPC003 had the highest values. On the other hand, L. plantarum CNPC020, similarly to L. mucosae CNPC007, exhibited notable inhibition against sanitary indicators. These two strains significantly differed from the other five autochthonous cultures regarding S. enterica serovar Typhimurium ATCC 14028 inhibition (P < 0.05). However, they did not differ from at least one positive control in terms of inhibition against S. aureus ATCC 25923 and E. coli ATCC 25922 (P > 0.05). Therefore, it is advisable to consider these cultures separately for different technological purposes, such as phenolics metabolism or bio preservative activity. This will facilitate appropriate selection based on each specific property required for the intended product development.

Mo-HLPs: New flocculating agents identified from Moringa oleifera seeds belong to the hevein-like peptide family.

Cationic peptides found in Moringa oleifera seeds belong to different protein families and are described as the main flocculating agents of the species. In this study we report the identification and isolation of four new flocculant peptides, called Mo-HLPs 1-4, belonging to the family of hevein-like peptides, previously only known for their members' antimicrobial activity. Purification of the peptides followed two sequential membrane ultrafiltration steps and separation by reverse-phase liquid chromatography. Proteomic analyses showed that Mo-HLPs are extremely basic (pI >10) cysteine-rich molecules with molecular masses between 4.5 and 4.8 kDa and with a highly conserved chitin-binding domain. Searches in BLAST revealed high similarity of Mo-HLPs with hevein and other hevein-like peptides and 90% identity with morintides, which are members of the 8C-hevein-like subfamily found in M. oleifera leaves. Mo-HLPs microflocculation assays showed distinct coagulation/flocculation efficiencies, promoting turbidity reduction levels between 67 and 89% in synthetic turbid water. Activity variations were attributed to the substitution of some amino acids among the isoforms, which may have altered the final net charge of the molecules. The identification of Mo-HLPs represents the discovery of a new group of cationic peptides involved in the flocculation properties of M. oleifera seeds. SIGNIFICANCE: The study reveals the presence of hevein-like peptides in Moringa oleifera seeds. It is reported for the first time that members of this family have properties to act as flocculating agents of importance for water treatment processes. The identification of these peptides as well as new functional assignment broadens the horizon for speculation on new species which could act as sources of green coagulants for sustainable water treatment, and contributes to the knowledge about occurrence, distribution, molecular and active diversity of peptides belonging to the hevein-like family.

Inactivation of lectins from castor cake by alternative chemical compounds.

Enabling the use of castor cake in animal feeding is an excellent alternative strategy to reduce feed costs. The cake is a by-product derived from the extraction of the castor oil by the biodiesel industry whose chemical composition is satisfactory despite the presence of antinutritional factors like toxic lectins, which require detoxification before it can be used as a dietary ingredient. The aim of the present study was to evaluate alternative chemical sources in the degradation and inactivation of ricin and Ricinus communis agglutinin (RCA), two lectins from castor cake. Ten chemical compounds were evaluated: sodium hydroxide, monodicalcium phosphate, dicalcium phosphate, calcium oxide, calcium hydroxide, calcitic limestone, magnesian limestone, urea, potassium chloride, and sodium chloride. Gel electrophoresis indicated 100% lectin degradation only in the cakes treated with 90 g sodium hydroxide and 2500 mL water per kg of cake. The hemagglutination assay was crucial to providing innocuousness to the treated cakes, with total absence of hemagglutinating activity observed in the castor cakes treated with 60 or 90 g sodium hydroxide in water volumes equal to or higher than 1500 mL/kg of castor cake and in the cakes treated with 90 g calcium oxide with 2500 or 3000 mL water/kg castor cake. Thus, though depending on the concentration of the chemical compound and on the volume of water per kilogram of treated cake, sodium hydroxide and calcium oxide showed to be promising chemical products for degradation and complete inactivation of the lectins present in castor cake to allow its use as an ingredient in animal diets.

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.

Efeito da oligofrutose (FOS) sobre a capacidade de acidificação da cultura nativa Lactobacillus plantarum CNPC 003 isolada ou em co-cultura com Streptococcus thermophilus em leite de cabra / Influence of oligofrutose (FOS) on the acidification capacity of the indigenous culture Lactobacillus plantarum CNPC 003 solely or in coculture with Streptococcus thermophilus in goat milk

Objetivou-se analisar o comportamento fermentativo da cultura Lactobacillus plantarum CNPC 003 em leite de cabra em quatro tratamentos por 6 horas: T1 – L. plantarum CNPC 003; T2 – L. plantarum + oliogofrutose (FOS); T3 – L. plantarum + Streptococcus thermophilus; T4 – com L. plantarum + S. thermophilus + FOS. Os tratamentos estudados diferiram significativamente entre si em relação à acidez e ao pH após 6h de fermentação (p<0,05), tendo sido verificada uma influência positiva do uso de S. thermophilus e da adição de FOS sobre esses parâmetros. Do mesmo modo, a presença de FOS melhorou a viabilidade dos microrganismos estudados após 6h de fermentação. A utilização de S. thermophilus e de FOS é uma alternativa viável para o uso em leites fermentados contendo a cepa nativa L. plantarum CNPC 003.

Lectin, hemolysin and protease inhibitors in seed fractions with ovicidal activity against 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.

Biodegradação do bagaço de cana-de-açúcar por microrganismos ruminais de caprinos e ovinos / Biodegradation of sugarcane bagasse by ruminal microorganisms from sheep or goats

No presente estudo objetivou-se avaliar a degradação do bagaço de cana-de-açúcar (BCA) integral (BIN) ou hidrolisado (BH) pela microbiota ruminal de caprinos e ovinos de raças naturalizadas do Nordeste brasileiro e o potencial desses animais como fontes de microrganismos e/ou enzimas celulolíticas para degradação da fibra do BCA. Para hidrólise do BCA foi utilizada uma solução de NaOH a 50%, 30% na matéria seca (MS). Foram determinadas as concentrações de MS, proteína bruta (PB), cinzas (CZ), fibra detergente neutro (FDN), fibra detergente ácido (FDA), celulose (Cel), hemicelulose (Hcel) e lignina (Lig). A degradação in situ da FDN do BH foi determinada pela incubação ruminal em sacos de náilon nos tempos: 0, 6, 24 e 96 horas. A técnica de duas etapas preconizada por Tilley e Terry foi utilizada para determinar a digestibilidade in vitro da MS (DIVMS). Foi coletado conteúdo ruminal dos animais quatro horas após a infusão de 100g de BIN via fístula ruminal para a separação de microrganismos associados às fases líquida e sólida, utilizando filtragem e extração com tampão fosfato de sódio 50 mM e pH 6,9, respectivamente. A fração sólida foi submetida ao cultivo in vitro com o substrato BIN, por 96 horas, para determinação da taxa de degradação da MS, FDN e atividade celulolítica. O pH foi determinado nos tempos de cultivo de 24, 48 e 96 horas. O pré-tratamento com NaOH aumentou a DIVMS (P<0,05). Não houve efeito de espécie sobre a DIVMS (P>0,05). A inclusão de NaOH aumentou a degradação in situ da FDN, apresentando os ovinos menor tempo de colonização (TC). Houve solubilização da Hcel, da Cel e da Lig no BCA pré-tratado com NaOH. A atividade celulolítica se concentrou na fração sólida independente da espécie doadora do inóculo, sendo observado crescimento microbiano no cultivo in vitro do BIN a partir dessa fração. O pH aumentou com o tempo de cultivo in vitro. Microrganismos ruminais de caprinos e ovinos naturalizados do Nordeste brasileiro colonizaram e degradaram o BIN e BH. O NaOH pode ser utilizado no pré-tratamento alcalino do BCA.

The purpose of this study was to evaluate the biodegradation of sugar cane bagasse (SCB), whole (WB) or hydrolyzed (HB) by ruminal microorganisms from Brazilian naturalized goat and sheep breeds, and the animals' potential as a source of microorganisms and/or cellulolytic enzymes for the biodegradation of SCB fiber. SCB hydrolysis was performed using 50% sodium hydroxide solution on 30% of dry matter (DM). The concentrations of DM, crude protein (CP), ether extract (EE), ash, neutral detergent fiber (NDF), acid detergent fiber (ADF), cellulose (Cel), hemicellulose (Hcel) and lignin (Lig) were determined in the WB and HB. For the in situ biodegradation of NDF, the HB was placed in nylon bags and incubated in rumen for 0, 6, 24 and 96 hours. The two-step technique recommended by Tilley and Terry was used to determine the in vitro digestibility of DM (IVDDM). Four hours after infusion of WB (100 g) through the ruminal fistula, the rumen content was collected from the animals and the microorganisms associated with the liquid and solid phases were separated, the former by filtration and the latter by extraction with 50 mM sodium phosphate buffer, pH 6.9. The solid fraction was subjected to in vitro culture with WB substrate for 96 hours to determine the biodegradation rate of DM, NDF and cellulolytic activity. The pH was measured after culture times of 24, 48 and 96 hours. Pretreatment with NaOH increased the IVDDM (P <0.05). No species effect on the IVDDM was detected (P> 0.05). The addition of NaOH increased the in situ biodegradation rate of NDF, with sheep showing a lower lag time (LT). There was a solubilization of Hcel, Cel and Lig in SCB pretreated with NaOH. Cellulolytic activity was higher in the solid phase, irrespective of the inoculum source, and microbial growth was observed in in vitro cultures using microorganisms from the solid phase and WB as substrate. The pH of the in vitro culture increased over time. Ruminal microorganisms from Brazilian naturalized goat and sheep breeds successfully colonized and biodegraded both WB and HB. Sodium hydroxide proved useful as an alkaline pretreatment for SCB.

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.