The process of acetaldehyde formation by the yogurt bacterium Streptococcus thermophilus is described in this paper. Attention was focused on one specific reaction for acetaldehyde formation catalyzed by serine hydroxymethyltransferase (SHMT), encoded by the glyA gene. In S. thermophilus, SHMT also possesses threonine aldolase (TA) activity, the interconversion of threonine into glycine and acetaldehyde. In this work, several wild-type S. thermophilus strains were screened for acetaldehyde production in the presence and absence of L-threonine. Supplementation of the growth medium with L-threonine led to an increase in acetaldehyde production. Furthermore, acetaldehyde formation during fermentation could be correlated to the TA activity of SHMT. To study the physiological role of SHMT, a glyA mutant was constructed by gene disruption. Inactivation of glyA resulted in a severe reduction in TA activity and complete loss of acetaldehyde formation during fermentation. Subsequently, an S. thermophilus strain was constructed in which the glyA gene was cloned under the control of a strong promoter (P LacA ). When this strain was used for fermentation, an increase in TA activity and in acetaldehyde and folic acid production was observed. These results show that, in S. thermophilus, SHMT, displaying TA activity, constitutes the main pathway for acetaldehyde formation under our experimental conditions. These findings can be used to control and improve acetaldehyde production in fermented (dairy) products with S. thermophilus as starter culture.Yogurt is a product obtained through milk fermentation with a specific yogurt starter culture consisting of a mixture of two species of lactic acid bacteria (LAB), Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus (23). The main roles of this mixed starter in the production of yogurt are (i) acidification through the conversion of lactose into lactic acid, (ii) creation of the viscous texture by the production of exopolysaccharides, and (iii) development of the typical yogurt flavor (29).The typical yogurt flavor is caused by lactic acid, which imparts an acidic and refreshing taste, and a mixture of various carbonyl compounds like acetone, diacetyl, and acetaldehyde, the latter of which is considered the major flavor component (10,11,21,29). The relatively high concentration of acetaldehyde (in the range of 5 to 21 mg/liter) found in yogurt must be due to a low utilization rate of this metabolite since the yogurt bacteria lack the main enzyme for acetaldehyde conversion into ethanol, alcohol dehydrogenase (12).The production of acetaldehyde by LAB seems to be strain dependent. L. delbrueckii subsp. bulgaricus has been reported by some authors to be a greater acetaldehyde producer than S. thermophilus, whereas other authors have reported the contrary (21, 27). Although it is presently unclear what the major pathway for acetaldehyde production by LAB is, several metabolic pathways have been shown to lead to its formation (5, 24) and it is possible that more th...

Bacteriocins produced by fifteen strains of Lactococcus lactis (14 L. lactis subsp. lactis and one L. lactis subsp. cremoris) were heat resistant, sensitive to several proteolytic enzymes and active over a wide range of pH. Their resistance to the heating was greatly influenced by the pH. Only the strain L. lactis subsp. lactis ITAL 383 produced a bacteriocin with a wide activity spectrum, similar to nisin of L. lactis subsp. lactis ATCC 11454. This bacteriocin inhibited closely related species and other Gram-positive microorganisms including Listeria monocytogenes and Staphylococcus aureus, but it was not active against the Gram-negative bacteria tested. The identification of partially purified antimicrobial compounds by SDS-PAGE showed that bacteriocin produced by strain ITAL 383 had the same molecular weight of nisin produced by L. lactis subsp. lactis ATCC 11454.

International audienceUF-cheeses have been successfully developed in many countries. However, proteolysis and the general extent of ripening were shown to be far slower compared to traditional varieties. The absence of starter lysis has been cited as a plausible explanation. In this work, propionibacteria and lactococci cells were disrupted using a new pilot homogenizer. Crude broken suspension (CBS), or cell-free extract (CFE), obtained after centrifugation, were added to UF-St Paulin retentate (concentration factor 6) made with a usual lactic starter. RO water was added to the control. Proteolysis was estimated by NCN, NPN and free amino acids. Neutral volatile compounds were determined by GC-MS. A low extent of ripening was noted in the control and the absence of starter lysis was effectively proved using immunodetection of lactococci cytoplasmic proteins. The addition of lactococci CBS or CFE increased free amino acid content (1.5 to 3 times) whereas propionibacteria CBS or CFE exhibited no significant increase, even when cheeses were ripened at 20 °C instead of 12 °C. By contrast, addition of propionibacteria CBS generated a significant increase in several volatile compounds like alcohols and ketones, whereas CFE did not, showing that the presence of live cells was required to form these compounds. CBS or CFE of lactococci did not significantly change the volatile compound profile. In conclusion, it was possible to influence the ripening of UF-cheese by the addition of crude broken bacterial cell suspensions. Other strains and species should now be investigated.Impact d'extraits cellulaires de lactocoques et de propionibactéries sur l'affinage de fromages UF : étendue de la protéolyse et profils CG-MS. Les fromages UF se sont développés avec succès dans de nombreux pays. Cependant, sans action technologique complémentaire, la protéolyse et par conséquent l'affinage sont nettement plus lents que dans les produits obtenus par les procédés traditionnels. L'absence de lyse du levain pourrait constituer une des causes possibles. Dans ce travail, des cellules de P. freudenreichii et de Lactococcus sp. ont été "cassées " à l'aide d'un nouvel homogénéisateur pilote. Les suspensions cassées brutes (CBS) ou les extraits intracellulaires (CFE) obtenus après centrifugation, ont été ajoutés à des rétentats UF de type St Paulin (concentration $\times$6) acidifiés à l'aide d'un levain lactique mésophile commercial. De l'eau osmosée était ajoutée au témoin. La protéolyse a été estimée par la mesure des teneurs en NCN, NPN et en acides aminés libres ; les composés volatils neutres par CG-MS. Dans le témoin, cette protéolyse était très limitée et l'absence de lyse du levain commercial (L. lactis) a été effectivement démontrée. L'addition de CBS ou de CFE de Lactococcus augmentait la teneur en acides aminés libres (1,5 à 3 fois). Cet accroissement n'était pas observé lors de l'addition de CBS ou de CFE de P. freudenreichii, même lorsque la température d'affinage était élevée à 20 °C au lieu de 12 °C. Par contre, l'addition de ...

One hundred sixty seven strains of Lactococcus lactis were screened for bacteriocin production by well diffusion assay of GM17 agar. Fourteen (8.4%) produced antimicrobial activity other than organic acids, bacteriophages or hydrogen peroxide. The frequency of bacteriocin production ranged from 2% in L. lactis subsp. cremoris up to 12% in L. lactis subsp. lactis. Antimicrobial activities were not observed in any strain of L. lactis subsp. lactis var. diacetylactis. Among thirteen bacteriocin-producing strains and two nisin-producing strains (L. lactis subsp. lactis ATCC 11454 and L. lactis subsp. lactis CNRZ 150), eight (53%) were characterized as lactose-positive (Lac + ) and proteinase-negative (Prt -). The bacteriocin-producing cultures were also characterized on the basis of plasmid content. All strains had 2 to 7 plasmids with molecular weights varying from 0.5 to 28.1 Mdal. Four strains (ITAL 435, ITAL 436, ITAL 437 and ITAL 438) showed identical profiles and the other were quite distinct.

83 ResumoAtualmente, são lançados no mercado diversos novos produtos, sendo que na área de laticínios a ênfase é para os funcionais. Esta pesquisa propõe o desenvolvimento de uma bebida láctea fermentada, denominada buttermilk, que seja probiótica, apresente opções de sabor, que tenha versões dietéticas e que atenda à legislação brasileira. A primeira etapa do trabalho consistiu na determinação do fluxograma de preparo do produto para determinar os melhores momentos para adição de sacarose, sucralose e da cultura probiótica. Na etapa seguinte, foi avaliada a qualidade microbiológica de corantes e aromatizantes para empregá-los, sem tratamento térmico, no produto fermentado. Posteriormente, foram analisadas as amostras de buttermilk de diversos sabores, durante o armazenamento, para verificar se a adição de sacarose, edulcorante, aromatizantes e corantes interferiria nas contagens microbiológicas. Os resultados indicaram que a adição da cultura probiótica deve ser feita pré-fermentação e que corantes e aromatizantes podem ser adicionados no produto já fermentado. Observou-se que as amostras de buttermilk com sacarose adicionadas de agentes de cor e aromatizantes tenderam a apresentar menores contagens de bifidobactérias após a estocagem. Porém, todos os sabores de buttermilk se mantiveram adequados à legislação quanto aos aspectos de higiene e de número de bifidobactérias. B. animalis subsp. lactis apresentou excelente viabilidade durante o armazenamento do produto (média de 1,3.10 8 UFC.mL -1 ). As amostras de buttermilk podem, assim, ser consideradas seguras para consumo, além de potencialmente funcionais. Palavras-chave: buttermilk; probiótico; viabilidade; aspectos higiênico-sanitários. AbstractNew food products are launched on the market nearly every day and the main focus of the dairy industry is on functional products. The aim of this research project is to develop a fermented probiotic dairy product -buttermilk -, in a variety of flavors -including diet versions -in compliance with Brazilian regulations. As a first step, the manufacturing process of buttermilk was tested and evaluated to determine the best method and process step to add sucrose, sucralose and the probiotic culture. The next step consisted of evaluating the microbiological quality of coloring agents and flavor compounds intended to be added -without prior heat treatment -to the fermented product. In the next stage of the project, buttermilk samples of different flavors were analyzed during cold storage to observe whether sugar, sweetener, flavor compounds and coloring agents would interfere with microbiological counts. The results obtained at the end of the storage period indicated that the probiotic culture should be added prior to fermentation, while flavor compounds and coloring agents could be added directly to the fermented product after completing the culturing process. It was observed that the buttermilk samples containing sucrose and added with flavor compounds and coloring agents tended to show lower bifidobacterial counts after s...

One of the key focuses of today's dairy industry worldwide is the continued development of new products, especially probiotic-based products. Buttermilk is originally a by-product of butter making fermented by Mesophilic Aromatic Cultures (MAC). It can also be made by fermentation of pasteurized whole milk or skimmed milk. This product is not marketed in Brazil. The objectives of this work were: (1) to develop a selective medium for Bifidobacterium animalis subsp. lactis enumeration and (2) to determine the viability of this microorganism during the shelf life of the buttermilk. Skim milk added with 10% sucrose or 0.03% sucralose was pasteurized and inoculated with a composite starter culture consisting of 1% MAC (containing Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis biovar. diacetylactis and Leuconostoc mesenteroides subsp. cremoris) and 2% Bifidobacterium animalis subsp. lactis. To attain selective counts of Bif. animalis subsp. lactis the MRS agar supplemented with 0.5% L-cysteine hydrochloride at 10%, 1% lithium chloride at 10%, 0.01% aniline blue and 0.5% dicloxacillin at 0.1% was modified by increasing the antibiotic concentration, addition of NaCl, adjusting pH to 4.8 or increasing the incubation temperature (from 37 to 45ºC). Raising the incubation temperature to 45ºC was found to be efficient in inhibiting the MAC cultures, even in media not added with dicloxacillin. Bif. animalis subsp. lactis exhibited high viability in the product. The buttermilk product prepared with sucrose and sweetener contained in excess of 10 8 cfu.ml -1 bifidobacteria throughout the shelf life of the product (28 days).

Probiotic buttermilk‐like fermented milk in different flavors in versions with sucrose and sucralose were processed in a pilot plant. The Mesophilic Aromatic Culture CHN‐22 and Bifidobacterium animalis Bb12 were the cultures employed for fermentation. Physicochemical composition, apparent viscosity, diacetyl content, viability of cultures during storage, and acceptance of product via sensorial analysis were assessment factors. The high viability of the probiotic culture was observed (average 8.08 log cfu/ml) during refrigerated storage (28 days) of buttermilk and good acceptance of the product via sensory test. However, the probiotic culture might have been responsible for the loss of diacetyl, a volatile compound that is a characteristic feature of the product.

The typical yogurt flavor is caused by acetaldehyde produced through many different pathways by the yogurt starter bacteria L. bulgaricus and S. thermophilus. The attention was focused on one specific reaction for acetaldehyde and folic acid formation catalyzed by serine hydroxymethyltransferase (SHMT), encoded by the glyA gene. In S. thermophilus, this enzyme SHMT also plays the typical role of the enzyme threonine aldolase (TA) that is the interconvertion of threonine into glycine and acetaldehyde. The behavior of engineered S. thermophilus strains in milk fermentation is described, folic acid and acetaldehyde production were measured and pH and counts were followed. The engineered S. thermophilus strains StA2305 and StB2305, have the glyA gene (encoding the enzyme serine hydroxymethyltransferase) overexpressed. These engineered strains showed normal growth in milk when it was supplemented with Casitione. When they were used in milk fermentation it was observed an increase in folic acid and in acetaldehyde production by StA2305 and for StB2305 it was noticed a significative increase in folic acid formation.