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Fresh beef, containing .5% dextrose, was inoculated with Streptococcus lactis , incubated for 3, 5 and 7 d at 7±1°C, and then used to manufacture frankfurters. The frankfurters were vacuum-packaged and stored for 1, 2, 4 and 6 weeks at 3±1°C. Microbial quality was evaluated by examining frankfurters for aerobic and psychrotrophic counts at the end of each storage period. The level of nisin, an antibiotic substance produced by this strain of S. lactis , was also monitored in fresh meat and processed products. During the incubation period, no appreciable reduction in pH occurred in fresh meat. Growth of psychrotrophs was slower in treated than untreated fresh meat, but whether this was due to presence of nisin or competition resulting from the heavy inoculation with S. lactis is unclear. Before and after processing, nisin levels in the 5- and 7-d incubation-treated groups were higher than in the 3-d group, and all treated groups had higher nisin levels than untreated groups. Little difference in aerobic and psychrotrophic growth was noted with storage time between treated and untreated groups for the 3-d incubation period, but well defined differences were noted in the 5- and 7-d incubation groups, with aerobic growth in treated groups being significantly slowed through the 6-week storage period, and psychrotrophs being 1 to 1.5 log cycles lower at the 6-week evaluation. The growth patterns suggest that nisin played a role in the reduced growth rate of bacteria in treated groups.

A series of 2 H ,4 H -2-[3,5-dimethyl-4-(substituted) phenyl azo pyrazol-1-yl] carbonyl methyl-3-oxo-1,4-benzothiazine derivatives have been synthesized by the reaction of 2 H ,4 H -2-hydrazino carbonyl methyl-3-oxo-1,4-benzothiazine with acetyl acetone derivatives using ultrasound in lesser time with higher yields. All the synthesized compounds were investigated for their antibacterial activities. The result indicated that the compounds show convincing activities against Gram-positive bacteria ( Bacillus subtilis and Streptococcus lactis ) when compared with standard drug (ampicillin trihydrate). These compounds were also synthesized by conventional method and their structures have been elucidated on the basis of spectral analyses and chemical reactions.

This study investigated the inhibitory effects of Lactobacillus casei AST18 as a biocontrol agent on fungi spoilage in yogurt. In a yogurt preservation period experiment and mould-proof accelerated testing at 4 °C, the addition of 2% (v/v) L. casei AST18 in yogurt completely inhibited mould growth; mould growth was significantly delayed and spore germination was inhibited at 30 °C. The addition of L. casei AST18 improved the quantity of Lactobacillus , but the number of Streptococcus lactis in 2% AST18-added yogurt decreased by 1.0 Log (cfu/mL) compared with that in the blank group. L. casei AST18 exerted no significant influence on viscosity, water holding capacity, sensory quality, pH and titratable acidity. The post-acidification effect of L. casei AST18 was low. L. casei AST18 conspicuously inhibited the growth of filamentous fungi. Therefore, L. casei AST18 can be used as a bioprotective culture in yogurt to prevent fungal growth and extend shelf life.

Three mozzarella cheeses were prepared from three different milk sources i.e. cow (C), buffalo (B) and their mix (A) milks using DL (Streptococcus lactis and cremoris; S. Diacetalactic, Leuconostoc cremoris) and yoghurt cultures. Milk was standardized to C/F: 0.90/0.92 and process optimized for using all milk sources. The samples were analyzed for physico-chemical and functional properties, sensory attributes and protein and fat losses. Except ash and yield at 50% moisture; fat, protein, actual yield, pH, acidity and moisture were significantly influenced by milk sources. From sensory result, cow and mix cheeses were significantly superior, whereas functional properties were superior for cow cheese but buffalo and mix cheeses had higher nutritive value. From overall comparison, cow mozzarella cheese was ranked most suitable for pizza topping. J. Food Sci. Technol. Nepal, Vol. 6 (94-101), 2010 DOI: http://dx.doi.org/10.3126/jfstn.v6i0.8268

1. It has been shown on serological and biochemical grounds thatStr. faecalisfalls into Lancefield's group D.Str. liquefaciensis considered to be a variety ofStr. faecalis, andStr. glycerinaceus(Orla Jensen) has insufficient claim to a separate name.2.Str. lactishas, on biochemical and serological grounds, been clearly differentiated fromStr. faecalisand the other members of group D.3. Reasons are given for assigningStr. lactisto a new serological group ‘N’ into whichStr. cremorisalso falls.

By a series of quantitative assays of concentrates prepared in a variety of ways, it has been shown that the previously described growth factor now called "strepogenin," required by certain hemolytic streptococci, is very similar to and possibly identical with the recently described growth factors for Lactobacillus casei and Smith's Streptococcus lactis. An improved procedure for assay of strepogenin with the aid of Lactobacillus casei has been described, and additional properties of the growth factor have been presented.

Des progres significatifs ont ete realises au cours des dernieres annees pour elucider la regulation du metabolisme des sucres chez Lactococcus lactis et d'autres bacteries lactiques. Comme la connaissance de ces mecanismes constitue un outil precieux pour l'ingenierie metabolique, une vue d'ensemble des differents systemes de controle globaux des bacteries Gram positives est presentee, avec une attention particuliere portee a l'exclusion par l'inducteur, l'expulsion par l'inducteur et la repression catabolique. L'exclusion par l'inducteur correspond au phenomene ou l'addition d'un sucre rapidement metabolisable comme le glucose dans le milieu resulte en une moindre consommation des autres sucres. Dans les memes circonstances, un autre systeme, appele expulsion par l'inducteur est actif et reduit la concentration intracellulaire des sucres phosphoryles en les dephophorylant et en otant les sucres de la cellule. Les deux mecanismes sont dependants de l'etat de phosphorylation du residu serine-46 du phosphotransporteur HPr qui depend d'une kinase activee par un metabolite. Outre un controle allosterique, le metabolisme des sucres peut aussi etre regule au niveau transcriptionnel. Un exemple de regulation transcriptionnelle est la repression catabolique ou la presence d'un sucre rapidement metabolisable dans le milieu reduit la transcription des genes requis pour l'utilisation des autres sucres. Les bacteries Gram positives expriment leur repression catabolique via un regulateur transcriptionnel, CcpA. Pour analyser le role de HPr et CcpA dans le metabolisme de Lactococcus lactis, les genes codants pour HPr et CcpA, ptsH et ccpA respectivement, ont ete clones et analyses.

ii ÖZET Doktora Tezi STREPTOCOCCUS LACTİS1 DE FAJ DİRENÇLİLİ?ÎNİN PLAZMİDLERLE OLAN ÎLİŞKÎSÎ ve FAJ DİRENÇLİ LİK PLAZMİDLERİNİN FAJA DUYARLI SUSLARA AKTARIM OLANAKLARI ÜZERİNDE ARAŞTIRMALAR Mustafa AKÇELİK Ankara üniversitesi Fen Bilimleri Enstitüsü Gıda Bilimi ve Teknolojisi Anabil im Dalı Danışman: Prof. Dr. Nezihe TUNAİL 1991, Sayfa: 163 Jüri: Prof. Dr. Nezihe TUNAİL : Prof. Dr. M. Hilmi PAMİR : Prof. Dr. Gürdal ALAEDDİNO?LU Bu çalışmada, starter kullanılmaksızın üretilen Türk beyaz peynirlerinin peyniraltı sularından izole edilmiş olan streptococcus lactis suşlarında faj direnç mekanizmaları. araştırılmıştır. Virulent laktik fajlar, Türk çiğ sütlerin den ve değişik ülkelerin endüstriyel fermentasyon süreçlerinde çeşitli ürün ve ortamlardan izole edilmiştir. 28 i streptococcus lactissusu, direnç özelliklerinin belirlenme si amacı ile 11 laktik faja karşı denenmiş ve bu suşların altısının tüm fajlara karşı tamamen dirençli olduğu saptanmıştır. Bu altı susta faj direnç sistemlerinin 32 Kb. veIll 26.1 Kb. (Kilobaz) büyüklükte iki farklı plazmidin varlığına bağlı olduğu ve 32 Kb. lık plazmidin adsorbsiyonun engellenmesi, 26.1 Kb. lık plazmidin ise ısı duyarlı, faj direnç sistemlerini kodladığı belirlenmiştir. 32 Kb. lık plazmidin ayrıca proteolitik aktivite, laktoz fermentasyonu ve yüksek -2 -4 sıklıkta konjugasyon (verici hücre başına 1.2x10 - 4.8x10 konjugant) özelliklerini de kontrol ettiği örneklenmiştir. 26.1 Kb. lık plazmidin ise Streptococcus lactis suşlarında ısı duyarlı faj direnç dışında başka hiçbir özelliği kodlamadığı saptanmıştır. Her iki direnç plazmidi de protoplast transformasyonu yöntemiyle, plazmid içermeyen streptococcus lactis suşlarına aktarılarak, plazmid DNA'nm ug.mmda 4 5 1.4x10 - 2x10 oranında transformant oluşumu sağlanmıştır. Ayrıca elektronmikroskopik incelemeler sonucu Türk faj izolatlarının morfolojik özellikleri belirlenmiştir. ANAHTAR KELİMELER : Streptococcus lactis, plazmid, faj di renç, transformasyon, konjugasyon IV ABSTRACT Ph.D. Thesis RESEARCHES ON THE RELATIONSHIP BETWEEN PLASMIDS AND PHAGE INSENSITIVITY IN STREPTOCOCCUS LACTIS AND TRANSFER POSSIBILITIES OF THE PHAGE RESISTANCE PLASMIDS TO PHAGE SENSITIVE STRAINS Mustafa AKÇELİK Ankara University Graduate School of Natural and Applied Sciences Department of Food Science and Technology Supervisor: Prof. Dr. Nezihe TUNAİL 1991, Page: 163 ; Jury: Prof.Dr. Nezihe TUNAİL : Prof. Dr. M. Hilmi PAMİR : Prof. Dr. Gür dal ALAEDDİNO?LU In this study, the phage resistance mechanisms of Streptococcus lactis strains, isolated from whey of Turkish white pickled cheese which produced using without industrial starter, were investigated. Virulent lactic phages were isolated from Turkish raw milk and various products and media of industrial fermentation processes of different countries. Twenty eight streptococcus lactis strains were / tested for resistance to eleven different lactic phages and six of these strains were found to be completely resistantaganist all the phages. The phage resistance systems in the six strains were found to be determined by two different plasmids, having molecular sizes of 32 kilobase and 26.1 kilobase, and they were shown to encode interference of phage adsorption and heat sensitive phage resistance systems, respectively,. Furthermore, the 32 kilobase plasmid was also demostrated to be responsable for proteolytic activity, _2 lactose fermentation and high frequency conjugation (1.2x10 -4 4.8x10 conjugant per donor cell) functions. The 26.1 kilobase plasmid was found to carry none of the characters in the streptococcus lactis strains except the heat sensitive phage resistance system. Both of the resistant plasmids could be transformed into plasmid free streptococcus lactis strains by protoplast transformation method with the frequency rate 4 5 of 1.4x10 - 2x10 transformant per ug of plasmid DNA. In addition, the morphogical characters of the Turkish phage isolates were determined electronmicroscopically. KEY WORDS: Streptococcus lactis, plasmid, phage resistance, transformation, conjugation 163

Characterization of psychrotrophic bacteria from cultured milk has shown that most (72%) of the psychrotrophs which were isolated originated from the starter culture. Streptococcus lactis subsp. diacetilactis was the most abundant organism and dominated both Streptococcus cremoris and Leuconostoc cremoris , which was the least abundant. The doubling time of S. lactis subsp. diacetilactis at 7 C was 20 h and 54 h in Trypticase Soy Broth at pH 7.0 and 5.0, respectively. This indicates a growth potential which could be significant to the quality of the product over a long storage period. Psychrotrophic coliforms which had optimal growth temperatures within the range of 15 to 30 C could also be found in small numbers. Their survival in the product was limited and it was concluded that they have little effect on shelf-life.

Raw whole milk was processed under vacuum at 60, 68.3, or 76.6 C and distillate (5 ml/100 ml of milk) was collected in cold traps. Unprocessed and processed samples of milk were autoclaved, steamed, or heated in a waterbath at 60, 68.3, or 76.6 C. Each heated processed milk was subdivided into two portions; distillate (5:95) was added to one portion and distilled (in glass) demineralized water (5:95) was added to the other. All samples were inoculated with Streptococcus lactis 4175 and incubated at 22 C. Growth and activity of S. lactis were monitored by determining the number of organisms, pH, and titratable acidity after 0, 4, 7, 10, and 13 h of incubation. Distillate collected at 60 C slightly retarded growth of S. lactis in steamed milk. Both growth and activity of this organism were reduced when distillate collected at 68.3 C was added to autoclaved and waterbath-treated (68.3 C) milk; this was not true for steamed milk. When milk was processed at 76.6 C, the resultant distillate had only a very slight effect on behavior of S. lactis in autoclaved milk. In steamed milk, although its generation time was unaffected, acid production by S. lactis was enhanced when the distillate was added. When processed milk was heated at 76.6 C, distillate collected at 76.6 C caused a slight increase in the generation time of S. lactis.

Streptococcus lactis 7962, which ferments lactose slowly, has a lactose phosphoenolpyruvate-dependent phosphotransferase system and low phospho-beta-galactosidase activity, in addition to high beta-galactosidase activity. Lactose 6'-phosphate accumulated to a high concentration (greater than 100 mM) in cells growing on lactose. In contrast, lactic streptococci, which ferment lactose rapidly and use only the lactose-phosphotransferase system for uptake, contained high phospho-beta-galactosidase activity and low concentrations (0.9 to 1.6 mM) of lactose 6'-phosphate. It is concluded that rate-limiting phospho-beta-galactosidase activity is primarily responsible for defective lactose metabolism in S. lactis 7962.

Nutrient broth inoculated with Salmonella typhimurium was dispensed into epoxy-lined aerosol cans. Twenty-five volatile compounds were then individually added to the cans to yield or non-volatile compounds were then individually added to the cans to yield final concentrations of 1, 10, 100, and 1000 ppm of each compound. Compounds tested included fatty acids (formic, acetic, butyric, hexanoic, octanoic and decanoic), aldehydes (formaldehyde, acetaldehyde, propionaldehyde, and glyoxal), ketones (acetone, 2-butanone, and diacetyl), amines (propyl and hexylamine), alcohols (furfurol and methanol), sulfur compounds (methylsulfide, methylsulfone, methanethiol, and ethanethiol), acetonitrile, chloroform, ether, and ethylenedichloride. Bacteria were enumerated at intervals during incubation at 37 C. Shorter chain fatty acids generally inhibited S. typhimurium more than did longer chain acids. At 10 ppm formic acid was most effective of those tested and at 1 ppm fatty acids were generally not inhibitory. Formaldehyde and glyoxal were more inhibitory than acetaldehyde and propionaldehyde. Diacetyl was most effective of the three ketones tested. Low concentrations of acetone or 2-butaoone sometimes enhanced growth of S. typhimurium. Acetonitrile at all concentrations tested significantly inhibited S. typhimurium during the terminal stages of incubation. Ether (10 ppm), chloroform (10 ppm), ethylenedichloride (100 ppm), and methylsulfone (100 ppm) generally caused significant reduction in growth of S. typhimurium. Ethanethiol was more detrimental to growth of S. typhimurium than were methylsulfide or methanethiol; amines were more inhibitory than alcohols.

Generation times and acid production after 6 and 24 h by 20 strains of Streptococcus lactis of dairy origin were determined in heat treated (80 C for 30 min) and sterilized cow's and ewe's milk. Ewe's milk enhanced growth of the streptococci, with significantly (P &A;0.001) shorter generation times and higher acid production after 6 h incubation than cow's milk, probably due to its higher vitamin content. The stronger buffer capacity of ewe's milk allowed a higher (P &A;0.001) acid production after 24 h than cow's milk. A stimulatory effect of sterilization on generation time and acid production after 24 h was observed in cow's milk. However, the heat treated ewe's milk was shown to be a better substrate than sterilized ewe's milk for Str. lactis. We thank C. Garc ía-Aser and M. A. Rodríguez-Marín for N determinations and C. Burbano for Ca and Mg analysis. © 1983, Proprietors of Journal of Dairy Research. All rights reserved.

Summary Physiological properties, properties of their lactate dehydrogenases and DNA /DNA relatedness have been determined for strains of streptococci which grow at 10°C but not at 45 °C; these include strains of Streptococcus lactis, Streptococcus cremoris and Streptococcus raffinolactis. The results show that S. lactis and S. cremoris are variants of a single species unrelated to S. raffinolactis and also to a group of strains which cannot be separated from S. lactis on easily determined physiological properties. Several strains could not be classified but one strain received as Lactobacillus hordniae proved to be a variant of S. lactis by DNA/DNA homology and the properties of its lactate dehydrogenase, although its physiological properties would not indicate that it belongs to that species.

SummaryClones ofStreptococcus lactisand ofStr. cremorisresistant to some bacteriophages that could virtually annihilate the parent strains were selected by plating very large numbers of the bacteria in the presence of the phage. Some of the resistant strains were shown to form acid rapidly in milk and to be satisfactory for cheese-making. They were not lysogenic. Adsorption of the phage to which the parent culture had been sensitive still occurred with 3 strains but not with 4 others. Rates of mutation from phage sensitivity to phage resistance were determined for 2 original strains ofStr. lactisand were found to be of the same order as the rates quoted in the literature for other mutations. The mutation rates of phages to forms which could attack the resistant strains were too low to be measured.

A self-transmissible (Tra+) plasmid encoding determinants for restriction and modification activities (R+/M+) from Streptococcus lactis ME2 was isolated and characterized. The 28-kilobase (kb) plasmid (pTN20) was detected in lactose-fermenting (Lac+) transconjugants generated from matings between S. lactis N1, and ME2 variant, and a plasmid-free recipient, S. lactis LM2301. The plaquing efficiencies of prolate- and small isometric-headed phages were reduced on transconjugants containing either pTN20 (R+/M+ Tra+) or 100-kb plasmids encoding Lac+, R+/M+, and Tra+. Lac+ transconjugants which harbored pTR1040 (Lac+) and pTN20 (R+/M+) were phenotypically R-/M- and transferred Lac+ at low frequency in subsequent matings to give rise to 100-kb R+/M+ plasmids. R+/M+ activities and high-frequency conjugal transfer ability were detected in Lac+ transconjugants that contained pTR1041 (Lac+) and pTN20 (R+/M+). No 100-kb R+/M+ plasmids were recovered after these matings, suggesting that pTR1041 was mobilized by pTN20 through a process that resembled plasmid donation. pTR1041 was identical to pTR1040 but contained an additional 3.3-kb DNA fragment. These data suggested that phenotypic expression of R+/M+ and Tra+ is affected by coresident Lac+ plasmids. Restriction enzyme analysis and hybridization reactions demonstrated that the 100-kb R+/M+ plasmid was formed by a cointegration event between pTR1040 (Lac+) and pTN20 (R+/M+ Tra+) during conjugal transfer via a conductive-type process. This is the first report that defines self-transmissible restriction and modification plasmids in the lactic streptococci.

The effects of rennet level, culture composition and strain, and microbial contamination were evaluated as potential causes for bitterness in commercial quarg. Rennet level was the critical factor in the development of bitter flavor. When rennet was decreased from 3876 to 388 units/1000kg of milk, bitterness was reduced and an acceptable product with an extended shelf life was obtained. The yield of laboratory produced quargs ranged from 28.42% for 3876 units/1000kg rennet to 18.05% with no rennet addition. Rennet of 388 units/1000kg resulted in laboratory and commercial yields of 21.90 and 22.50%, respectively. The microbiological results indicated low psychrotrophs, coliforms, and yeast and molds counts during the 6-wk storage trial, suggesting that bitterness was not the outcome of microbial contaminants. The nature of the starters tested (mixed cultures of Streptococcus lactis and Streptococcus cremoris with and without S. diacetilactis , or single strains of S. cremoris 134 and 584) did not play an important role in bitterness development during storage.

The properties of the lactate dehydrogenases, percent guanine plus cytosine in the deoxyribonucleic acid (DNA), and DNA/DNA hybridization studies have shown that three strains of group N streptococci do not belong to either Streptococcus lactis or Streptococcus cremoris. The biochemical properties of the three strains were published about 25 years ago, and at that time the strains were not assigned to any species. The three strains are here identified as members of Streptococcus raffinolactis Orla-Jensen and Hansen.

In a mixed culture of Saccharomycopsis fibuliger Y76 and Streptococcus lactis 65.1 on starch the main interactions are commensalism and competition. Oxygen-limited batch and continuous cultures of S. fibuliger showed accumulation of sugars. When oxygen was used as an external regulatory parameter in the mixed culture lactic acid, acetic acid and formic acid were formed. Ethanol produced by S. lactis was most likely assimilated by S. fibuliger. Continuous mixed cultures were stable under conditions of oxygen limitation at the dilution rates tested (0.10 h−1 and 0.15 h−1). Conversion yields of 35 to 40% were obtained but may be improved.

Streptococcal plasmid pAMβ1 was conjugally transferred from Streptococcus lactis KB953 (a transformant of pAMβ1) into Lactobacillus casei 239. A unique transconjugant, L. casei C2, was found to contain a small (11.1 kilobase pair) plasmid, pLY201, which was derived by a deletion event from pAMβ1. Restriction analysis revealed that pLY201 was missing approximately 58% of the original pAMβ1 genome, and contained 5 single restriction sites for Ava I, Eco RI, Pvu II, Hpa I and Kpn I. Physical analyses revealed that the stability and copy number of pLY201 were elevated compared with those of pAMβ1 in L. casei . In addition, pLP201 was no longer tranferable by conjugation.

Summary The presence of plasmid DNA was demonstrated for all of 51 investigated strains of Streptococcus lactis, S. lactis subsp. diacetylactis , and S. cremoris . The plasmid patterns of representative strains analysed by agarose gel electrophoresis of the DNA are documented. This pattern does not appear to provide a basis for species differentiating. Lactosenegative derivatives having lost some of their plasmids were obtained by curing with nalidixic acid.