Bactérias láticas produtoras de folato e riboflavina: Isolamento e avaliação do seu potencial de aplicação na produção de produtos lácteos caprinos com alto teor de vitaminas / Folate and riboflavin producing lactic acid bacteria: isolation and evaluation of their potential application for production of goat dairy products with higher vitamin content

O valor nutricional e as características físico-químicas fazem com que os produtos lácteos caprinos sejam muito apreciados e tenham um alto valor agregado. Muitas bactérias láticas (BAL) possuem a capacidade de produzir compostos com propriedades benéficas à saúde, inclusive vitaminas do grupo B, como a riboflavina (B2) e o folato (B9), que participam de importantes funções metabólicas. O presente estudo objetivou isolar e identificar BAL capazes de produzir folato e riboflavina, utilizando leite de cabra cru e de queijos de cabra como fonte, para em seguida, avaliar a produção destas vitaminas em leite de cabra UHT, para estimar uma possível aplicação na produção de derivados lácteos de cabra com teores mais altos destas vitaminas. Um total de 179 isolados de BAL, sendo 87 provenientes de leite e 92 de queijos, foram obtidas e analisados quanto à produção de folato e de riboflavina extracelular (EC) e intracelular (IC), empregando-se ensaios microbiológicos apropriados. A produção de folato em meio de cultura a 37ºC foi observada em 151 isolados (84,4%) e de riboflavina em 15 isolados (8,4%), sendo que 14 produziram as duas vitaminas concomitantemente. A média da produção folato total (EC + IC) foi 138,8 µg/L, sendo que em 77 isolados (51%) a produção estava acima da média. Houve diferença significativa entre as médias de produção de folato total pelos isolados de leite e de queijo. A média da produção de riboflavina total (EC + IC) foi 363,7 µg/L, sendo que em 9 isolados (60%) a produção foi acima da média. Com base no RAPD-PCR e sequenciamento da porção 16S do rDNA, foram obtidos 19 perfis genéticos diferentes e identificadas 7 espécies, com predominância de Streptococcus thermophilus (7 isolados), Weissella paramensenteroides (6 isolados) e Lactococcus lactis subsp. lactis (4 isolados). Oito isolados produtores de folato acima da média foram selecionados e submetidos à avaliação da produção de folato em leite de cabra UHT a 37°C, sendo 7 positivas, com média da concentração de folato total de 120,55 µg/L, variando de 12,97 a 261,91 µg/L. Os melhores produtores de folato em leite de cabra UHT foram Lc. lactis subsp. lactis FP368 e St. thermophilus FP34v, FP170v e FP268v. A concentração de folato produzido por estes isolados foi acima da média, evidenciando seu interessante potencial de aplicação na produção de novos derivados lácteos caprinos com teores aumentados desta vitamina. Por outro lado, não foi possível detectar a produção de riboflavina em leite de cabra por nenhum dos isolados testados

Due to the high nutritional value and physicochemical characteristics, goat milk and cheeses are highly appreciated. Many strains of lactic acid bacteria (LAB) are able to produce vitamins from the B complex, such as riboflavin (B2) and folate (B9). These vitamins have important metabolic roles. The present study aimed to isolate riboflavin- and folate-producing LAB strains from goat milk and cheeses, and evaluate the potential application of those strains in the production of goat dairy products with higher content of these vitamins. A total of 179 LAB isolates were obtained from milk (87) and cheese (92) samples. The isolates were evaluated for production of extra (EC) and intracellular (IC) riboflavin and folate, applying appropriate microbiological methods. Among these isolates, 151 (84.4%) were able to produce folate, while 15 (8.4%) displayed the ability to produce riboflavin, and 14 produced both vitamins. The average production of total folate (EC + IC) was 138.8 µg/L, and the amount of folates produced by 77 isolates (51%) were above this average. The differences observed for the average production of total folate from milk and cheese isolates were statistically significant. For total riboflavin (EC+ IC), the average rate was 363.7 µg/L. Nine isolates (60%) presented production rates above the average. No significant difference was observed between the average production of total riboflafin from milk or cheese isolates (319.3 µg/L and 379.8 µg/L, respectively). Based on RAPD-PCR and 16S rDNA sequencing, 19 different genetic profiles were obtained and 7 species were identified, with predominance of Streptococcus thermophilus (7 isolates), Weissella paramensenteroides (6 isolates), and Lactococcus lactis subsp. lactis (4 isolates). Eight isolates that produced folate above the average were selected and tested for vitamins production in UHT goat milk at 37°C. Seven isolates produced an average of 120.55 µg/L of folate in the milk and concentration varied from 12.97 to 261,91 µg/L. The best folate producers were Lactococcus lactis subsp. lactis FP368, Streptococcus thermophilus FP34v, Streptococcus thermophilus FP170v and Streptococcus thermophilus FP268v. The amount of folate produced by these isolates surpassed the average, and was above the amounts described in other studies, evidencing their potential application in the production of goat dairy products with higher content of folate. None of the tested isolates was able to produce riboflavin in UHT goat milk

Active immunization against riboflavin carrier protein results in peri-implantation embryonic loss leading to pregnancy termination in rats: use of alternate adjuvants.

To investigate the mechanism of pregnancy termination following immuno-neutralization of riboflavin carrier protein (RCP) and to use acceptable adjuvants, we actively immunized female rats with reduced and carboxymethylated RCP (RCM-RCP) using various adjuvants (during primary immunization) such as sodium phthalylated lipopolysaccharide (SPLPS), purified S. typhi outer membrane proteins (porins) and a combination of them. Rats (5-14 per group) were immunized with alugel adsorbed RCM-RCP (100 microg/dose) either alone or with SPLPS or porins or SPLPS+porins. Control animals received RCM-RCP emulsified with Fruend's completelincomplete adjuvants (FCA/FIA). All animals received five boosters at intervals of 21 days. The lowest (4 X 10(-3)) and the highest (> 70 X 10(-3)) anti-RCM-RCP antibody titers were observed in alugel adsorbed-RCM-RCP group and control groups, respectively. Immunized animals showed reduced fertility following 3rd, 4th and 5th boosters. Reduction in fertility was 30-60% in alugel adsorbed RCM-RCP group, 90-100% in FCA-RCM-RCP group and 80-90% in SPLPS+porins group. Fertility reduction was not strictly correlatable with the serum antibody titers. RCP-specific IgG could be localized in the uterine endometrial glands and luminal epithelial cells in the immunized animals. Animals in the FCA/FIA group showed abnormal implantation/resorption sites and their histological sections showed degenerated embryos. But, day 5 preimplantation embryos were normal. These results show that (a) SPLPS+porins can be used as adjuvants in place of FCA/FIA for active immunization against RCM-RCP and (b) early termination of pregnancy in the immunized animals is due largely to the failure of normal embryo implantation.

Immunoreactive riboflavin carrier protein concentration during ovulatory cycle in the common marmoset (Callithrix jachhus): role of estrogen.

Common marmoset, a non-human primate, is increasingly being used as a model system in reproductive endocrinology. Marmosets do not menstruate but have normal cycle length of 28 +/- 2 days. The presence of protein immunologically and functionally similar to the well characterized chicken riboflavin carrier protein (cRCP) has been demonstrated in circulation in pregnant marmosets. Marmoset RCP (mRCP) has been partially purified and the molecular weight of the immunoreactive protein is similar to cRCP. The source of RCP in rodents appears to be maternal liver, suggesting thereby that RCP levels could be modulated by changing hormonal pattern that occurs during the cycle. To study the hormonal control of marmoset RCP, immunoreactive RCP levels were monitored during normal cycle. Plasma progesterone was estimated by RIA and mRCP was measured using a heterologous cRCP RIA. When mRCP and progesterone were analysed for a period of 40 days (n = 5) in adult females, very low amount of mRCP was measured during the luteal phase and a single sharp peak was observed during the follicular phase of the cycle. These data suggested that the induction of mRCP was regulated by estrogen. Direct evidence for the role of estrogen in the elaboration of RCP, was the observation that exogenous administration of estradiol 17 B to immature marmosets resulted in a time and dose dependent increase in plasma RCP levels. As early as 24 hr following hormonal administration, a measurable increase in mRCP with a maximum increase, 5-fold over the zero period was seen 3 days following a single administration of estradiol 17 B (5 mg/kg body wt). RCP levels declined thereafter and was indistinguishable by day 7. These studies indicate that the marmoset RCP levels are regulated by estrogen.

Análise de vitaminas do complexo B em carnes preparadas em serviço de alimentaçäo / Complex B vitamins analyse in meats prepared in alimentation service

Apesar da evoluçäo tecnológica, estudos sobre a estabilidade de nutrientes em alimentos preparados em Serviços de Alimentaçäo têm recebido pouca atençäo. No entanto, é cada vez maior o número de pessoas que fazem suas refeiçöes nestes estabelecimentos. Uma vez que o conteúdo em vitaminas é um dos fatores mais importantes na determinaçäo da qualidade dos alimentos processados, o objetivo deste estudo foi definir métodos de análise quantitativa para tiamina, riboflavina e ácido nicotínico por CLAE em carnes, para, entäo, avaliar a influência da preparaçäo em Serviço de Alimentaçäo sobre o conteúdo destas vitaminas em diferentes cortes de carne bovina, suína e de frango. Objetivou-se também avaliar a estabilidade das vitaminas citadas após métodos de preparaçäo tradicionais comparados com métodos de preparaçäo em forno combinado (um dos equipamentos de cocçäo mais modernos utilizados em Serviço de Alimentaçäo)...

Revisión bibliográfica actualizada sobre cambios dentofaciales y malformaciones congéticas inducidas por deficiencia aguda de riboflavina / A current review of the literature on dentofacial changes and congenital malformations induced by acute riboflavin deficiency

La riboflavina no es sintetizada por animales superiores en cantidad suficiente como para cubrir las necesidades nutricionales, por lo cual debe ingerirse en los alimentos. La deficiencia aguda de riboflavina en edad embrionaria puede ocasionar malformaciones esqueléticas y alteraciones bucofaciales, siendo el esqueleto membranoso el primero en manifestar los signos de este sindrome. Entre las diferentes anomalías tenemos: braquignacia, fusión de las costillas superiores, ausencia de tibia, atresia del esófago, excenfalia, protrusión de la lengua, incisivos inferiores ausentes, paladar hendido, atrofia de los músculos milhoideo y masetero, sindactilia, abducción de las manos. Se acepta que el agentes causante de estas alteraciones es la hiporriboflavinosis, la cual afecta la multiplicación de células mesenquimatosas durante la vida intrauterina. Si bien estas anomalías no son exclusivas de la deficiencia de riboflavina, se puede afirmar que ciertas deficiencias nutricionales en madres durante el período de gestación pueden causar defectos congénitos en los descendientes.

Hormonal modulation of secretion of immunoreactive riboflavin carrier protein by adult rat Leydig cells in vitro.

Adult rat Leydig cells in culture synthesize and secrete riboflavin carrier protein (RCP) as demonstrated by [35S]-methionine incorporation into newly synthesized proteins followed by immunoprecipitation as well as specific radioimmunoassay. LH stimulates the secretion of RCP 4-fold which could be inhibited upto 75% by an aromatase inhibitor. 8-bromo-cyclic AMP and cholera toxin could mimic the LH stimulated secretion of the carrier protein. The extent of stimulation of RCP secretion brought about by exogenous estradiol-17 beta is comparable to that of LH. The antiestrogen tamoxifen, when added along with either LH or estrogen, inhibited the stimulated levels significantly. These results show that the estrogen-inducible riboflavin carrier is secreted by Leydig cells under positive regulation of LH.

Immunohistochemical localization of riboflavin carrier protein in testicular cells of mammals.

Using specific polyclonal antibodies against chicken riboflavin carrier protein (cRCP), immunocytochemical localization of riboflavin carrier protein was carried out in testicular sections and isolated cells of mammals. A positive reaction was observed in the developing germ cells of rat testis, especially in meiotic and post-meiotic germ cells such as pachytene spermatocytes, round spermatids and spermatozoa. In addition both the somatic cells of the testis, viz. Leydig and Sertoli cells with vital function in germ cell proliferation and differentiation, displayed a moderate to strong staining reaction. This was further confirmed using in utero X-irradiated rat testis devoid of germ cells. Different types of cells isolated from testis when subjected to immunostaining showed similar patterns of reaction as in the intact tissue. Mature spermatozoa from different mammals (rat, bull and monkey) exhibited strong staining reaction in their head regions localized mainly in acrosomal caps. It is suggested that the testicular riboflavin carrier protein has a role in cell to cell communication and may be crucial during development of germ cells especially at the meiotic and post-meiotic stages.

Improved presentation of antigenic sites in enzyme-linked immunosorbent assay: antigenicity of reduced and carboxymethylated riboflavin carrier protein.

Disulphide reduced and carboxymethylated riboflavin carrier protein (RCM-RCP), an unfolded derivative of chicken RCP, does not bind riboflavin and there is a drastic reduction in its ability to interact with antiserum to cRCP. Antibodies to RCM-RCP are directed against sequential epitopes(s). On radioiodination of RCM-RCP, a maximum of 30-50% binding at dilution of 1:500 was obtained with rabbit antibodies RCM-RCP [n = 5]. However, high titer antibodies was obtained when radioiodinated RCM-RCP was immobilized on ELISA microtiter plates suggesting that immobilization of RCM-RCP leads to either preservation of antigenic sites or improved presentation of the antigenic determinants. An avidin-biotin system was utilized to develop an ELISA.

Termination of pregnancy in mice following antiserum administration to modified chicken riboflavin carrier protein.

Rabbit antibodies to native riboflavin carrier protein (RCP), are to a large extent directed towards conformational epitopes and antibodies to disulphide bond reduced carboxymethylated RCP (RCM-RCP) are towards sequential epitopes. The major cyanogen bromide (CNBr) fragments and tryptic fragments of RCM-RCP interact with both antiserum to RCM-RCP and RCP. Passive immunization of pregnant mice with antibodies to RCM-RCP results in bioneutralization, leading to termination of pregnancy. Recently, a major tryptic fragment of RCM-RCP (24 +/- 2 kd) which could assume conformation at the antibody combining site of native RCP, obtained following mild trypsinization has been identified [Natraj et al. J. Biosci, 15 (1990) 341]. Rabbit antibodies to RCM-RCP treated with trypsin generated antibodies of low titer which interacted with RCM-RCP as well as RCP. The interaction of this antibody with RCP was of high affinity and could be displaced with RCP. The bioneutralizing ability of the antibody was demonstrated by its ability to cause termination of pregnancy in mice.

Enzyme-linked immunosorbent assay for riboflavin carrier protein and modified protein: application for epitope analysis.

Rabbit antibodies to native riboflavin carrier protein (RCP), are to a large extent directed towards the conformational epitopes and antibodies to disulphide bond reduced carboxymethylated riboflavin carrier protein (RCM-RCP) to the sequential epitopes. Taking advantage of this premise and in order to map the epitopes of RCP recognized by the antibodies, enzyme-linked immunosorbent assays were validated for RCP and RCM-RCP using the Avidin-Biotin system. The usefulness of these assays were illustrated when antigenicity of peptides derived from RCM-RCP following trypsinization were examined. Two major (T1,T2) and one minor peptide (T3) fractions were obtained when the tryptic peptides were fractionated on DEAE-cellulose. RCP has a blocked N-terminal. Tryptic peptides (T1 and T2) on microsequencing revealed the absence of an N-terminal amino acid, indicating that these fragments emanate from the N-terminal region of RCP. In support of this observation is the finding that antipeptide antibody to cRCP (10-24) of cRCP interacted with T1 as well as T2 indicating the presence of the sequential epitope (10-24) of cRCP in these fragments. In RCP-ELISA, only T2 displaced RCP and peptides T1 and T2 displaced RCM-RCP in RCM-RCP ELISA. Differences in the ability of these fragments (T1 and T2) to displace RCP and RCM-RCP reflect the subtle changes in the spatial structures of these epitopes in RCP and RCM-RCP.

Ultraviolet radiation-induced photodegradation and 1O2, O2-. production by riboflavin, lumichrome and lumiflavin.

Although UVA (320-400 nm) is considered less harmful to skin as compared to UVB (290-320 nm) and UVC (200-290 nm) radiation, certain endogenous chromophores may enhance UVA-induced cutaneous reactions by largely O2-dependent photodynamic reactions. Photodegradation pattern and singlet oxygen (1O2), superoxide anion radical (O2-.) producing capacity of riboflavin (RF), lumiflavin (LF) and lumichrome (LC) were examined to assess their phototoxic potential under UVA. Photolysis of RF upon exposure to UVA, UVB or UVC revealed considerable degradation to LF and LC with a near identical spectral pattern of photodegradation between 250-500 nm. Both LF and LC were stable to UVA (3 J/cm2) and UVB (400 mJ/cm2), whereas RF was photodegraded by 30 and 20%, respectively, under similar irradiation conditions. UVA-sensitized LF and LC respectively, produced nearly 15% higher and 60% lower yield of 1O2 in comparison to RF, whereas, O2-. was generated predominently by RF. Both RF and LF thus appeared to be potential chromophores for evoking deleterious effects of UVA in normal human skin.

Riboflavin and Thiamine Absorption

For the purpose of estimating the maximum absorption of riboflavin and thiamine from the intestine, large test doses of riboflavin and thiamine were administered orally to apparently normal male subjects, receiving normal Korean diets, and their maximum absorption were estimated by determining the excretion in urine. The maximum absorption of riboflavin and thiamine were estimated 10.700 and 3.800 mg. respectively.