Enhancing the Sweetness of Yoghurt through Metabolic Remodeling of Carbohydrate Metabolism in Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus

Abstract

Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus are used in the fermentation of milk to produce yoghurt. These species normally metabolize only the glucose moiety of lactose, secreting galactose and producing lactic acid as the main metabolic end product. We used multiple serial selection steps to isolate spontaneous mutants of industrial strains of S. thermophilus and L. delbrueckii subsp. bulgaricus that secreted glucose rather than galactose when utilizing lactose as a carbon source. Sequencing revealed that the S. thermophilus strains had mutations in the galKTEM promoter, the glucokinase gene, and genes encoding elements of the glucose/mannose phosphotransferase system (PTS). These strains metabolize galactose but are unable to phosphorylate glucose internally or via the PTS. The L. delbrueckii subsp. bulgaricus mutants had mutations in genes of the glucose/mannose PTS and in the pyruvate kinase gene. These strains cannot grow on exogenous glucose but are proficient at metabolizing internal glucose released from lactose by ␤-galactosidase. The resulting strains can be combined to ferment milk, producing yoghurt with no detectable lactose, moderate levels of galactose, and high levels of glucose. Since glucose tastes considerably sweeter than either lactose or galactose, the sweetness of the yoghurt is perceptibly enhanced. These strains were produced without the use of recombinant DNA technology and can be used for the industrial production of yoghurt with enhanced intrinsic sweetness and low residual levels of lactose. IMPORTANCEBased on a good understanding of the physiology of the lactic acid bacteria Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, we were able, by selecting spontaneously occurring mutants, to change dramatically the metabolic products secreted into the growth medium. These mutants consume substantially more of the lactose, metabolize some of the galactose, and secrete the remaining galactose and most of the glucose back into the milk. This allows production of yoghurt with very low lactose levels and enhanced natural sweetness, because humans perceive glucose as sweeter than either lactose or galactose. Modern yoghurt production begins with pasteurization of milk, followed by inoculation and fermentation by commercially prepared starter cultures containing strains of the lactic acid bacteria Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (1). In order to satisfy consumer demands regarding appearance, sweetness, texture, and flavor, a number of additional ingredients may be added, including natural or artificial colors, sucrose or artificial sweeteners, texturizing agents, and fruit preparations (2). Today, yoghurt may contain 5 to 12% (wt/vol) added sucrose in addition to the natural sugars (lactose and galactose) already present, resulting in sugar contents that rival those of soft drinks and tarnishing the image of yoghurt as a healthy food. In addition, the potential for gastrointestinal discom...

New Insights into Various Production Characteristics of Streptococcus thermophilus Strains

“…The gal promoter plays an important role in the galactose phenotype of S. thermophilus [42,50,51,52,53,54]. It has been found that the point mutations in the promoter region of gal operon lead to low expression level of the Leloir pathway enzymes for galactose utilization [42,50].…”

“…Recently, it was found that spontaneous galactose-fermenting mutant St1-Ga + -1 has the same mutation in the −10 region of galK , i.e., TACAAT, which is closer to the consensus sequence TATAAT. Strain St1-Ga + -1 can ferment galactose, while its mother strain St1-WT is unable to utilize galactose, although it has intact galKTEM genes [54]. At the same time, Sørensen et al found that another spontaneous galactose-fermenting mutant St2-Ga + -1 had a C-to-A mutation between the −10 region and the galK transcription initiation site [54].…”

“…Strain St1-Ga + -1 can ferment galactose, while its mother strain St1-WT is unable to utilize galactose, although it has intact galKTEM genes [54]. At the same time, Sørensen et al found that another spontaneous galactose-fermenting mutant St2-Ga + -1 had a C-to-A mutation between the −10 region and the galK transcription initiation site [54]. It was speculated that this mutation might increase the “opening potential” for the −9 to +3 region, and then lead to creating a stronger promoter [54].…”

Effect of dandelion extract, sucrose and starter culture on the viscosity, water-holding capacity and pH of plain yogurt

“…It is considered by nutritionists to have a high nutritional value and positive bioactive effects. To satisfy consumer demands, a number of additional ingredients may be added to yogurt, including natural or artificial colors, sucrose or artificial sweeteners, texturizing agents (Sørensen et al, 2016), prebiotic ingredients and probiotic bacteria (Oliveira et al, 2015). In recent years, some vegetable (Kiros et al, 2016) and fruit preparations (Oliveira et al, 2015) along with plants extracts (Balthazar et Taraxacum officinale, known as the dandelion, is a member of the Asteraceae/Compositae family.…”

Symposium review: Genomic investigations of flavor formation by dairy microbiota

“…In addition to using WGS as a means to uncovering new pathways for flavor generation, the technology can also be used as a screening tool to monitor changes in known pathways in the generation of spontaneous mutants with enhanced flavor capabilities. This approach was taken by Sørensen et al (2016) in an elegant study where WGS analysis of spontaneous mutants derived from strains of S. thermophilus and Lb. delbrueckii ssp.…”