The effect of yoghurt on some components of the gut microflora and on the metabolism of lactose in the rat

Feeding yoghurt or base milk (from which the yoghurt was prepared by fermentation) to rats increased the counts of coliforms in the gut whereas the counts of lactobacilli were reduced by yoghurt but not by the base milk. Lactobacillus bulgaricus survived in the guts of gnotobiotic and conventional rats when yoghurt was fed continuously. Streptococcus thermophilus also survived in gnotobiotic rats but its ability to survive in conventional rats could not be examined. Both organisms failed to colonise the gut when a small inoculum of yoghurt was administered orally to germfree rats maintained on the stock diet. Streptococcus thermophilus but not Lact. bulgaricus grew in the rat diet when tested in vitro. Two enzyme systems (beta-galactosidase and lactase) were studied using, respectively, o-nitrophenyl-beta-D-galactopyranoside (ONPG) and lactose as the test substrates. Enzyme levels estimated with both substrates increased in the gut contents when rats were fed yoghurt but an increase was only found with ONPG in the intestinal mucosa fraction. The bacterial origin of all this increased activity is discussed. The other lactose-containing diets did not affect enzyme activity to the same degree. Feeding yoghurt changed the lactobacillus flora from one which was predominantly heterofermentative (Lact. reuteri ) to one which was predominantly homofermentative (Lact. salivarius).     

Fermentation of milk by Streptococcus salivarius subspecies salivarius and Streptococcus salivarius subspecies thermophilus and their use to the yoghurt manufacturer

Unlike Streptococcus salivarius subspecies thermophilus, Streptococcus salivarius subspecies salivarius fails to grow symbiotically in milk in the presence of Lacto-bacillus bulgaricus , does not produce large quantities of the flavour volatiles, acetal-dehyde or diacetyl and is unable to stimulate growth of Lact. bulgaricus by producing formate. Although Strep, salivarius subspecies salivarius and thermophilus have similar DNA base composition and belong in the same DNA homology group, the former is unsuitable for milk fermentations such as yoghurt because fermentation of milk using this organism results in products with poor flavour, aroma and texture.     

Production of yoghurt with mild taste by a Lactobacillus delbrueckii subsp. bulgaricus mutant with altered proteolytic properties

In this communication, we describe the isolation of a Lactobacillus delbrueckii subsp. bulgaricus 92063 mutant strain named pH-P11, which differed from the parent strain by low proteolytic activity and altered regulation of expression of lacZ in the presence of glucose or lactose. In the presence of lactose, beta-galactosidase activity was approximately twice as high in pH-P11 than in the wild type. pH-P11 exhibited protosymbiosis together with Streptococcus thermophilus. Yoghurt produced with pH-P11 was characterized by low acidity and little post-acidification during storage. The organoleptic properties (absence of bitterness and other off-flavors, weak sourness, and clear yoghurt taste) were those of a typical "yoghurt mild". This mild flavor was achieved at rather high cell counts of lactobacilli even at the end of shelf-life. High cell counts in conjunction with high beta-galactosidase activity make pH-P11 an interesting strain for application in yoghurt especially designed for consumers with lactose malabsorption. In contrast to "yoghurt mild", which is predominantly produced with Lactobacillus acidophilus together with Streptococcus thermophilus, the product obtained by fermentation with pH-P11 and Streptococcus thermophilus concurs with international standards for yoghurt. During frequent sub-culturing, strain pH-P11, which is supposed to differ from the wild type by one or a few so-far-not-characterized mutations, showed sufficient stability for application in industrial production.     

Production of D-tagatose, a low caloric sweetener during milk fermentation using L-arabinose isomerase

Lactobacillusdelbrueckii subsp. bulgaricus and Streptococcus thermophilus are used for the biotransformation of milk in yoghurt. During milk fermentation, these lactic acid bacteria (LAB) hydrolyze lactose producing a glucose moiety that is further metabolized and a galactose moiety that they are enable to metabolize. We investigated the ability of L. bulgaricus and S. thermophilus strains expressing a heterologous L-arabinose isomerase to convert residual D-galactose to D-tagatose. The Bacillus stearothermophilus US100l-arabinose isomerase (US100l-AI) was expressed in both LAB, using a new shuttle vector where the araA US100 gene is under the control of the strong and constitutive promoter of the L. bulgaricus ATCC 11842 hlbA gene. The production of L-AI by these LAB allowed the bioconversion of D-galactose to D-tagatose during fermentation in laboratory media and milk. We also established that the addition of L-AI to milk also allowed the conversion of D-galactose into D-tagatose during the fermentation process.     

Prediction of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus populations in yoghurt by Curie point pyrolysis-mass spectrometry.

We evaluated the potential of pyrolysis-mass spectrometry (PyMS) for quantifying the binary mixed population of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in yoghurt. For this purpose, a new analytical approach was developed. The yoghurt was transparised and its total bacterial population was recovered by centrifugation and estimated by turbidimetric measurement. The quantity of each population (L. bulgaricus, S. thermophilus) was then estimated in the pellet by PyMS, and the data were analysed by artificial neural networks (ANNs). In parallel, streptococci and lactobacilli were numerated on SYL agar and these data were used as reference values to predict the bacterial counts of each population by PyMS. A close correlation was established between the streptococci and the lactobacilli counts on SYL agar and PyMS measurements (r(2)=0.98 for S. thermophilus and r(2)=0.96 for L. bulgaricus). Combined turbidimetric measurement and PyMS/ANNs seemed to be a powerful method for obtaining rapid counts of binary mixtures of bacteria in yoghurt.     

Induction of 2'-5' A synthetase activity and interferon in humans by bacteria used in dairy products.

The production of interferon by fasted human subjects in response to lactic bacteria Lactobacillus bulgaricus and Streptococcus thermophilus was evaluated in vivo and in vitro. The 2'-5' A synthetase activity of blood mononuclear cells was used to estimate interferon production following a single ingestion of 10(11) bacteria in yoghurt or sterile milk (controls). The level of the 2'-5' A synthetase of the yoghurt fed subjects was 83% (p = 0.002) higher than that of the milk fed controls 24 hours after ingestion. The baseline value remained unchanged in the control group. Blood mononuclear cells from a second group of subjects, were cultured with lactic bacteria for 48 hours, their cell-free supernatants contained gamma interferon. These results suggest that a transient production of interferon can be induced in healthy subjects by the lactic bacteria used in food processing.     

Detection of extracellular bound proteinase in EPS-producing lactic acid bacteria cultures on skim milk agar

AIMS: Skim milk agar was developed to investigate extracellular cell-bound proteinase in yogurt cultures, Streptococcus thermophilus and Lactobacillus bulgaricus. METHODS AND RESULTS: The Lact. bulgaricus cultures produced more extracellular cell-bound proteinase than did Strep. thermophilus cultures. Strong positive correlations between the size of the exopolysaccharide (EPS) layer and extracellular cell-bound proteinase were found for both Streptococcus and Lactobacillus cultures. CONCLUSION: Strong positive linear relationships existed between the EPS size and colony size and the diameter of clear zone and colony size for Streptococcus cultures, whereas weak positive linear relationships were observed for Lactobacillus cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: These data are useful to validate the relationship between extracellular proteinase and the EPS size of LAB. Also, a convenient medium to detect the presence of extracellular cell-bound proteinase of LAB is valuable for dairy industries.     

Note: evaluation of selective media for the enumeration of Bifidobacterium sp. in milk

Pure cultures of three species of bifidobacteria (Bifidobacterium longum, Bif. adolescentis and Bif. bifidum), Lactobacillus acidophilus and a mixed culture of Lact. delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus were each enumerated on two differential media and six selective media for the enumeration of bifidobacteria. The appearance of the colonies on the differential media was as expected but when mixed cultures were present, it proved extremely difficult to distinguish one species from another. Of the selective media, AMC, RMS, NPNL and BL-OG performed well in that they gave good recoveries of bifidobacteria and were inhibitory to the growth of Lact. delbrueckii subsp. bulgaricus, Strep. salivarius subsp. thermophilus and Lact. acidophilus. However, of these four media, AMC was most convenient as it is based on a commercially available medium, whereas the others must be made up from individual constituents. The AMC agar is thus a good choice for the routine enumeration of bifidobacteria from mixed cultures.     

郑州部分市售酸奶中乳酸菌的分离鉴定与益生特性的研究

目的调查郑州部分市售的酸奶中嗜热乳酸链球菌与保加利亚乳酸杆菌存在情况与益生特性。方法无菌采取样品进行乳酸菌分离,并对分离菌进行形态学、生化特性、产酸性、耐酸性、耐胆汁性、高温耐受性和抑菌性等生物学特性研究。结果从8种品牌中分离到4株保加利亚乳酸杆菌、7株嗜热链球菌,且分离菌株均能产酸、对高温具有一定的耐受性,在pH 3.0以上均能生长良好,而且分离菌的抑菌物质具有一定的热稳定性,保加利亚乳酸杆菌经70、100℃热处理抑菌活性基本不变。嗜热链球菌经70℃热处理后保留90%以上的抑菌活性,但经100℃高温处理后抑菌活性有所降低。分离菌产生的抑菌物质对胰蛋白酶敏感,保加利亚乳酸杆菌在偏中性条件下抑菌活性较高,而嗜热链球菌在偏中性条件下丧失抑菌活性。结论本研究为河南市售酸奶质量以及为消费者提供有利信息奠定基础。     

Anaerobic fermentation of Lactobacillus bulgaricus and streptococcus thermophilus on 3% nonfat dry milk with pure and mixed culture

Two different strains of Lactobacillus bulgaricus (ATCC-11842 and Microlife) and two strains of Streptococcus thermophilus (ATCC-19258 and Microlife) were grown anaerobically on 3% nonfat dry milk in pure and mixed culture using batch followed by fed batch culture. Samples were collected every 30 min. Concentrations of lactose, galactose, lactic acid, and other products present were measured using high pressure liquid chromatography. A spectrophotometric method from the literature was modified and used to determine microbial biomass concentrations. Relative cell numbers of the two organisms were measured microscopically in mixed culture. The results are presented in tabular form. S. thermophilus (Microlife) showed different growth characteristics compared to the other cultures. This culture utilized most of the galactose that was formed and produced greater amounts of lactic acid and biomass.     

Evidence for the conjugal transfer of the broad host range plasmid pIP501 into strains of Lactobacillus helveticus

The conjugative broad host range plasmid pIP501 was transferred from Streptococcus faecalis to a series of strains of lactic streptococci used commercially as dairy starter cultures. With these transconjugants as donors the plasmid was exconjugated to two strains of Lactobacillus helveticus and a commercially used strain of Strep. thermophilus. There was evidence that the plasmid could transfer between isogenic derivatives of one of the strains of Lact. helveticus. Transfer from Lact. helveticus to Strep. faecalis was also detected but at a low frequency. There was no evidence for the conjugal transfer of plasmid pIP501 into a strain of Lact. bulgaricus by exconjugation from either lactic streptococci or Lactobacillus sp.     

Evidence for the conjugal transfer of the broad host range plasmid pIP501 into strains of Lactobacillus helveticus

The conjugative broad host range plasmid pIP501 was transferred from Streptococcus faecalis to a series of strains of lactic streptococci used commercially as dairy starter cultures. With these transconjugants as donors the plasmid was exconjugated to two strains of Lactobacillus helveticus and a commercially used strain of Strep, thermophilus. There was evidence that the plasmid could transfer between isogenic derivatives of one of the strains of Lact. helveticus. Transfer from Lact. helveticus to Strep. faecalis was also detected but at a low frequency. There was no evidence for the conjugal transfer of plasmid pIP501 into a strain of Lact. bulgaricus by exconjugation from either lactic streptococci or Lactobacillus sp.     

Utilization of spline functions for smoothing fermentation data and for estimation of specific rates

A response surface method of smoothing fermentation data with spline functions is presented. The available electron balance is used to optimally select the values of the smoothing parameters associated with the spline functions. The method is applied to six sets of anaerobic fermentation data in which pure and mixed cultures are grown in batch followed by fed batch culture. Lactobacillus bulgaricus and Streptococcus thermophilus are cultured on 3% dry milk. Measured concentrations of biomass, lactose, galactose, lactic acid, and other acid products are smoothed using spline functions. Values of specific growth rate, specific lactose consumption rate, specific galactose formation rate, and specific acid product formation rate are estimated and the consistency of the results is examined using the available electron balance. The results show that the method works reasonably well, but that an upper bound should be used for the smoothing parameters to obtain accurate estimates of the derivative quantities.     

An Agar Medium for the Differential Enumeration of Streptococcus thermophilus and Lactobacillus bulgaricus in Yoghurt

A differential medium has been developed for the simultaneous enumeration of Lactobacillus bulgaricus and Streptococcus thermophilus from yoghurt. After incubating in a 'candle jar' at 42°C, a first count can usually be made at 24 h.     

Characterization of dominant microbiota of a Ghanaian fermented milk product, nyarmie, by culture- and nonculture-based methods

AIMS: To characterize the predominant micro-organisms in a Ghanaian traditional fermented dairy product, nyarmie, made from cows' milk, using both culture- and nonculture-based methods. METHODS AND RESULTS: Samples of nyarmie were analysed from three production sites in Accra, by determining the counts on selective culture media. The microbial diversity occurring in nyarmie was also evaluated by 16S/18S ribosomal DNA PCR amplification and denaturing gradient gel electrophoresis. Results showed that nyarmie contained lactococci and lactobacilli in the range of 10(8) and 10(10) CFU ml(-1), respectively, and yeasts at around 10(7) CFU ml(-1). The pH ranged between 3.49 and 4.25. The predominant lactic acid bacteria (LAB) in nyarmie were Leuconostocmesenteroides ssp. mesenteroides, Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lact.helveticus, Lact. delbrueckii ssp. lactis and Lactococcus lactis, while Saccharomyces cerevisiae was the predominant yeast species. Lactobacillus delbrueckii ssp. delbrueckii was not detected by cultivation but its predominance was revealed by PCR-DGGE analysis. CONCLUSIONS: The flora in products from different producers varied in the LAB composition present and may result in variations in product quality. SIGNIFICANCE AND IMPACT OF THE STUDY: Development and use of starter cultures for nyarmie may be beneficial in improving the consistency of product quality.     

Carbohydrate metabolism is essential for the colonization of Streptococcus thermophilus in the digestive tract of gnotobiotic rats

Streptococcus thermophilus is the archetype of lactose-adapted bacterium and so far, its sugar metabolism has been mainly investigated in vitro. The objective of this work was to study the impact of lactose and lactose permease on S. thermophilus physiology in the gastrointestinal tract (GIT) of gnotobiotic rats. We used rats mono-associated with LMD-9 strain and receiving 4.5% lactose. This model allowed the analysis of colonization curves of LMD-9, its metabolic profile, its production of lactate and its interaction with the colon epithelium. Lactose induced a rapid and high level of S. thermophilus in the GIT, where its activity led to 49 mM of intra-luminal L-lactate that was related to the induction of mono-carboxylic transporter mRNAs (SLC16A1 and SLC5A8) and p27(Kip1) cell cycle arrest protein in epithelial cells. In the presence of a continuous lactose supply, S. thermophilus recruited proteins involved in glycolysis and induced the metabolism of alternative sugars as sucrose, galactose, and glycogen. Moreover, inactivation of the lactose transporter, LacS, delayed S. thermophilus colonization. Our results show i/that lactose constitutes a limiting factor for colonization of S. thermophilus, ii/that activation of enzymes involved in carbohydrate metabolism constitutes the metabolic signature of S. thermophilus in the GIT, iii/that the production of lactate settles the dialogue with colon epithelium. We propose a metabolic model of management of carbohydrate resources by S. thermophilus in the GIT. Our results are in accord with the rationale that nutritional allegation via consumption of yogurt alleviates the symptoms of lactose intolerance.     

Preliminary characterization of microflora of Comté cheese

The evolution of the microflora of three Comté cheeses made in duplicate with raw milk from three different sources was followed during ripening. The same starter was used with each type of milk. The comparison of the cheeses did not reveal any significant difference in the development of the microflora. Starter lactic acid bacteria ( Streptococcus thermophilus and Lactobacillus helveticus) , which are added at the beginning of manufacture, decreased quickly in the first stages of ripening supporting the hypothesis of cell autolysis. Other micro-organisms, i.e. homofermentative and heterofermentative lactobacilli ( Lact. delbrueckii ssp. lactis , Lact. paracasei ssp. paracasei , Lact. rhamnosus and Lact. fermentum ), pediococci, enterococci and propionibacteria grew in cheese from small numbers in fresh curd. The characterization of Strep. thermophilus by pulsed-field gel electrophoresis showed that wild strains were also able to grow in the curd. The values for the genome size of 11 Strep. thermophilus strains determined in this investigation were in the range of 1·8–2·3 Mbp. The potential role of starter and raw milk microflora in cheese flavour development was considered.     

Yoghurt fermentation at elevated temperatures by strains of Streptococcus thermophilus expressing a small heat-shock protein: application of a two-plasmid system for constructing food-grade strains of Streptococcus thermophilus

Streptococcus thermophilus S4 expressing a small heat-shock protein from the plasmid pSt04-encoded copy of shsp, is able to carry out fermentation at elevated temperature, i.e., at 50 degrees C. In yoghurt culture together with Lactobacillus delbrueckii subsp. bulgaricus, fermentation at elevated temperature results in a mild yoghurt with low post-acidification and improved stability of the starter bacteria during storage at 4 degrees C. To transfer pSt04 into commercial S. thermophilus yoghurt starter strains, a two-plasmid system was constructed. A helper plasmid providing a selectable antibiotic marker, but relying on the repA gene of pSt04, was transformed together with pSt04. After isolation of transformants, the helper plasmid was readily lost upon incubation of transformants in antibiotic-free medium, thus yielding food-grade strains carrying pSt04 only. Successful application of the system was demonstrated.