A study of potential energy sources used by cheese ripening bacteria

Non-starter lactic acid bacteria (NSLAB) continue to grow in cheese during ripening even though lactose and its monosaccharide moieties (glucose and galactose (Gal)) are depleted. This indicates that the NSLAB are able to utilize other energy sources present in the cheese during ripening. Components from the starter bacteria, amino acids (AA), citrate and carbohydrates from the milk fat globule membrane have all been suggested as potential sources of energy for NSLAB, however, the substrates used during cheese ripening are currently not clearly defined. The objective of this work was to study potential energy sources used by cheese ripening bacteria during cheese ripening, with the hypothesis that carbohydrates from the glycoconjugates found in the milk fat globule membrane (MFGM) are essential for the development of bacteria during cheese ripening. The growth and metabolism of selected Lactobacillus (Lb.) paracasei, Lb. plantarum INF15D, Lactococcus lactis subsp. lactis ML8 and Enteroccus (Ec.) hirae INFE1 when grown in a model system added MFGM or its monosaccharide components as the only potential carbon sources was studied. Analysis of catabolic- as well as anabolic products have been conducted during growth of the bacteria, using high performance liquid chromatography for organic acids, carbohydrates and AA. Gas chromatography was used to monitor volatile compounds. Two dimensional gel electrophoresis was used to chart the protein expressions of Lb. plantarum INF15D when grown on the single monosaccharides found in the MFGM. Matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was ultimately used to identify proteins of interest from the two dimensional gels. The effect of the difference in MFGM monosaccharide utilization between NSLAB was explored in a cheese making trial using MFGM rich buttermilk powder as an addition to the cheese milk. The lactic acid bacteria (LAB) studied, all relevant to cheese production and ripening, were able to grow and survive for an extended period of time with a bovine MFGM isolate as the only potential carbohydrate source. All isolates were able to effectively utilize most of the monosaccharides found in MFGM glycoconjugates. Growth of Lb. plantarum INF15D on the acylated aminosugar N-acetylglucosamine (GlcNAc) led to induction of enzymes involved in the degradation of this sugar through the GlcNAc pathway, and this yielded large amounts of acetate as a degradation product. Presumably, the large amounts of acetate released from the degradation of N-acetylgalactosamine (GalNAc) and GlcNAc induced a decrease of cultivable number of bacteria at an earlier stage than when incubated on any of the other monosaccharides. The potential cheese contaminant Ec. hirae INFE1 showed superior growth and survival abilities compared with the other LAB when grown on the MFGM media, and on several of the MFGM monosaccharides. Enzymes involved in two different pathways yielding energy from serine (Ser) were found in Lb. plantarum INF15D, whereas none of the studied Lb. paracasei strains decreased the levels of Ser when grown on any of the monosaccharides. Growth of Lb. plantarum INF15D on Gal resulted in a metabolic shift expressed as different fates of the produced pyruvate compared to growth on the other monosaccharides, as well as initiation of degradation of some AA known to potentially take part in energy production, specifically arginine and tyrosine. Enzymes involved in ribose metabolism as well as phosphoketolase pathway enzymes were upregulated, leading to the production of ethanol and acetate. These findings indicate that Lb. plantarum INF15D are able to utilize several different substrates for energy at the same time to secure growth and survival during sub-optimal conditions. The cell shape determining protein MreB, that leads to elongation of the cells was also found to be upregulated during growth of Lb. plantarum INF15D on Gal. Cheeses were made with the addition of butter milk, and low initial numbers (log 2 cfu/mL cheese milk) of two different Lb. paracasei adjunct cultures (INF448 and INF456) known to be different in their utilization of the MFGM monosaccharide GalNAc. A low initial number was used to mimic the content of NSLAB normally found in cheese milk. Even if the Lb. paracasei adjuncts have the ability to utilize the MFGM components, they did not dominate the NSLAB microflora in the cheese in the later stages of ripening. The starter bacteria were located within the protein networks in clusters which were distributed homogeneously throughout the cheese matrix regardless of additive, while lactobacilli were rare, and when found, they were in huge clusters of clearly elongated cells