Hilde Marit Østlie

GROWTH AND METABOLISM OF SELECTED STRAINS OF PROBIOTIC BACTERIA IN MILK

Abstract Growth and metabolism of five probiotic strains with well-documented health effects were studied in ultra-high temperature (UHT) treated milk, supplemented with 0.5% (w/v) tryptone or 0.75% (w/v) fructose. The probiotic strains were Lactobacillus acidophilus La5, Lb. acidophilus 1748, Lactobacillus rhamnosus GG, Lactobacillus reuteri SD 2112 and Bifidobacterium animalis BB12. Fermentation was followed for 72 h at 37 °C and the samples were analysed for pH, log cfu ml−1, volatile compounds, organic acids and carbon dioxide. The strains reduced pH from 6.7 to between 3.9 and 4.4 after 24 h of incubation. All strains attained viable cell counts above 8.7–9.18 log cfu ml−1 after 6–16 h of incubation. The two Lb. acidophilus strains showed a stable level of viable cells during 12–72 h of incubation but the three other strains showed a reduction of 0.4–1.1 log cfu ml−1 from 24 to 72 h of incubation. However, all strains showed cell levels between 7.8 and 8.7 log cfu ml−1 after 72 h of incubation. After 48 h of incubation, the amount of lactic acid produced varied according to strain from 6949 to 14,000 mg kg−1 and acetic acid produced varied from 0 to 6901 mg kg−1. Three of the strains metabolised citrate but only low amounts of diacetyl and acetoin were detected within strains, 0.2–0.8 and 6.5–10 mg kg−1, respectively. Carbon dioxide produced varied from 221 to 3942 mg kg−1 and was connected to the citrate-fermenting ability of the strain used and their carbohydrate fermentation pathway. Three of the strains produced detectable levels of acetaldehyde and the concentration varied from 9.4 to 12.6 mg kg−1 after 24 h of incubation. All five probiotic strains showed very different profiles of metabolites during fermentation; however, the two Lb. acidophilus strains were the most alike. Our findings show the importance of controlling the fermentation time since the probiotic strains produced different amounts of metabolic products according to fermentation time.

Sensory evaluation and consumer acceptance of naturally and lactic acid bacteria-fermented pastes of soybeans and soybean–maize blends.

Fermented pastes of soybeans and soybean–maize blends were evaluated to determine sensory properties driving consumer liking. Pastes composed of 100% soybeans, 90% soybeans and 10% maize, and 75% soybeans and 25% maize were naturally fermented (NFP), and lactic acid bacteria fermented (LFP). Lactic acid bacteria fermentation was achieved through backslopping using a fermented cereal gruel, thobwa. Ten trained panelists evaluated intensities of 34 descriptors, of which 27 were significantly different (P < 0.05). The LFP were strong in brown color, sourness, umami, roasted soybean-and maize-associated aromas, and sogginess while NFP had high intensities of yellow color, pH, raw soybean, and rancid odors, fried egg, and fermented aromas and softness. Although there was consumer (n = 150) heterogeneity in preference, external preference mapping showed that most consumers preferred NFP. Drivers of liking of NFP samples were softness, pH, fermented aroma, sweetness, fried egg aroma, fried egg-like appearance, raw soybean, and rancid odors. Optimization of the desirable properties of the pastes would increase utilization and acceptance of fermented soybeans.

Genomic Characterization of Dairy Associated Leuconostoc Species and Diversity of Leuconostocs in Undefined Mixed Mesophilic Starter Cultures

Undefined mesophilic mixed (DL-type) starter cultures are composed of predominantly Lactococcus lactis subspecies and 1–10% Leuconostoc spp. The composition of the Leuconostoc population in the starter culture ultimately affects the characteristics and the quality of the final product. The scientific basis for the taxonomy of dairy relevant leuconostocs can be traced back 50 years, and no documentation on the genomic diversity of leuconostocs in starter cultures exists. We present data on the Leuconostoc population in five DL-type starter cultures commonly used by the dairy industry. The analyses were performed using traditional cultivation methods, and further augmented by next-generation DNA sequencing methods. Bacterial counts for starter cultures cultivated on two different media, MRS and MPCA, revealed large differences in the relative abundance of leuconostocs. Most of the leuconostocs in two of the starter cultures were unable to grow on MRS, emphasizing the limitations of culture-based methods and the importance of careful media selection or use of culture independent methods. Pan-genomic analysis of 59 Leuconostoc genomes enabled differentiation into twelve robust lineages. The genomic analyses show that the dairy-associated leuconostocs are highly adapted to their environment, characterized by the acquisition of genotype traits, such as the ability to metabolize citrate. In particular, Leuconostoc mesenteroides subsp. cremoris display telltale signs of a degenerative evolution, likely resulting from a long period of growth in milk in association with lactococci. Great differences in the metabolic potential between Leuconostoc species and subspecies were revealed. Using targeted amplicon sequencing, the composition of the Leuconostoc population in the five commercial starter cultures was shown to be significantly different. Three of the cultures were dominated by Ln. mesenteroides subspecies cremoris. Leuconostoc pseudomesenteroides dominated in two of the cultures while Leuconostoc lactis, reported to be a major constituent in fermented dairy products, was only present in low amounts in one of the cultures. This is the first in-depth study of Leuconostoc genomics and diversity in dairy starter cultures. The results and the techniques presented may be of great value for the dairy industry.

Survival of lactic acid and propionibacteria in low- and full-fat Dutch-type cheese during human digestion ex vivo.

The survival of selected bacteria in semi‐hard experimental cheeses was studied after exposure to human gastric and duodenal juices in an ex vivo model. Experimental cheeses (10 and 28% fat) were supplemented with different strains of Lactobacillus sp. and Propionibacterium sp. and ripened for 7 and 70 weeks. After digestion, greater numbers of the adjunct bacteria we rerecorded in the 7‐week‐old cheeses compared to the well‐matured cheeses (70 weeks). The bacterial survival was strain dependent, and influenced by the fat content of the cheese. Lactobacilli showed better survival, especially when in low‐fat cheeses. The strains of propionibacteria also survived well during the digestion of the low‐fat cheeses. The results confirmed that cheese can potentially be a good carrier matrix for bacteria to the intestine. In addition, it has been shown that different strains present in cheese have different abilities to survive the conditions of the gastrointestinal tract. Younger cheese was indicated to be a better carrier, possibly because the bacteria present in those cheeses have had shorter exposure to the stress conditions occurring in cheese during prolonged maturation.

Identification and Characterization of Lactic Acid Bacteria Involved in Natural and Lactic Acid Bacterial Fermentations of Pastes of Soybeans and Soybean-Maize Blends Using Culture-Dependent Techniques and Denaturing Gradient Gel Electrophoresis

Pastes of soybeans and soybean-maize blends were fermented with inoculum through back-slopping using lactic acid bacteria (LAB) fermented cereal gruel, thobwa and without inoculum (naturally). LAB involved in the fermentations were characterized using culture-dependent and culture-independent analyses. Decreases in pH from 6.4 to 3.9–4.2 and from 6.9 to 5.4–5.8 after 72 h were observed in LAB fermented pastes (LFP) and in naturally fermented pastes (NFP), respectively. LAB increased from 5.0 to 8.7–9.6 log10 cfu/g in NFP and from 8.1 to 9.3 log10 cfu/g in LFP. LAB in both fermentations were heterofermentative lactobacilli (82.4%) and homofermentative cocci (17.6%), of which 44.7% and 42.9% were exopolysaccharide producers, respectively. Principal component analysis based on carbohydrate fermentation, CO2 production and arginine hydrolysis showed four clusters dominated by Lactobacillus fermentum, Weissella confusa, Lactobacillus brevis 1 and Pediococcus pentosaceus, respectively. Sequencing of 16S rDNA gene confirmed Lb. fermentum, W. confusa/W. cibaria, and P. pentosaceus as identities of species in three clusters. Denaturing gradient gel electrophoresis (DGGE) confirmed these species as the dominant microbiota. DGGE showed higher similarity in microbial profiles of LFP throughout fermentation and low similarity in NFP during early and late stages of fermentation.

Metabolite changes during natural and lactic acid bacteria fermentations in pastes of soybeans and soybean–maize blends

The effect of natural and lactic acid bacteria (LAB) fermentation processes on metabolite changes in pastes of soybeans and soybean–maize blends was studied. Pastes composed of 100% soybeans, 90% soybeans and 10% maize, and 75% soybeans and 25% maize were naturally fermented (NFP), and were fermented by lactic acid bacteria (LFP). LAB fermentation processes were facilitated through back-slopping using a traditional fermented gruel, thobwa as an inoculum. Naturally fermented pastes were designated 100S, 90S, and 75S, while LFP were designated 100SBS, 90SBS, and 75SBS. All samples, except 75SBS, showed highest increase in soluble protein content at 48 h and this was highest in 100S (49%) followed by 90SBS (15%), while increases in 100SBS, 90S, and 75S were about 12%. Significant (P < 0.05) increases in total amino acids throughout fermentation were attributed to cysteine in 100S and 90S; and methionine in 100S and 90SBS. A 3.2% increase in sum of total amino acids was observed in 75SBS at 72 h, while decreases up to 7.4% in 100SBS at 48 and 72 h, 6.8% in 100S at 48 h and 4.7% in 75S at 72 h were observed. Increases in free amino acids throughout fermentation were observed in glutamate (NFP and 75SBS), GABA and alanine (LFP). Lactic acid was 2.5- to 3.5-fold higher in LFP than in NFP, and other organic acids detected were acetate and succinate. Maltose levels were the highest among the reducing sugars and were two to four times higher in LFP than in NFP at the beginning of the fermentation, but at 72 h, only fructose levels were significantly (P < 0.05) higher in LFP than in NFP. Enzyme activities were higher in LFP at 0 h, but at 72 h, the enzyme activities were higher in NFP. Both fermentation processes improved nutritional quality through increased protein and amino acid solubility and degradation of phytic acid (85% in NFP and 49% in LFP by 72 h).

Using NIR Spectroscopy for the Prediction of Free Amino Acids during Cheese Ripening

Introduction T his paper is a summary of the paper by Skeie et al. in which the correlation between near infrared (NIR) spectra of grated cheese and the development of selected free amino acids during ripening of rindless Norvegia and Präst cheese was evaluated. Traditionally, cheese ripening has been studied by regularly sampling cheeses at a cheese store and then performing sensory and/or chemical analysis on these samples. However, by this method, the cheeses will be punctured, followed by a modifi cation of the micro climate which again infl uences further ripening, so there is a need for non-destructive prediction methods. Near infrared spectroscopy has previously shown good prediction abilities with regard to fat, moisture and protein in natural and processed cheese, correlating well with chemical measurements. However, quantitative measurements of these components are not good indicators of how cheese ripening will proceed since they are more or less constant throughout the ripening process. On the other hand, changes in peptides and free amino acids provide very good indices of the ripening process. The types and amounts of free amino acids in cheese will infl uence fl avour and describe how far the ripening has proceeded; the composition of free amino acids will vary according to cheese variety and age. The objective of this work was to evaluate the ability of NIR spectra to predict the development of selected free amino acids during ripening of rindless Norvegia and Präst cheese. Rindless Norvegia is a Norwegian Dutch-type cheese produced in rindless blocks, while Präst is a Swedish open texture semi-hard circular cheese with rind. The earliest Norvegia is often sold after 45 days of ripening, while the earliest Präst is sold after 90 days of ripening; both are considered well-ripened after 270 days. The cheeses used for this experiment were part of an extensive study on similarities between cheeses from various dairy plants in Scandinavia producing the same cheese varieties. Cheeses were sampled monthly during part of 1999 from three Norwegian dairies producing Norvegia and three Swedish dairies producing Präst. The cheeses were analysed after 30, 90, 180 and 270 days of ripening.

Draft Genome Sequence of Enterococcus hirae Strain INF E1 Isolated from Cultured Milk.

ABSTRACT Here, we present the draft genome of Enterococcus hirae INF E1, found as a contaminant in cultured milk and studied for its ability to metabolize milk fat globule membrane glycoconjugates.