Manuel Krewinkel

Biodiversity of refrigerated raw milk microbiota and their enzymatic spoilage potential.

The refrigerated storage of raw milk selects for psychrotolerant microorganisms, many of which produce peptidases and lipases. Some of these enzymes are heat resistant and are not sufficiently inactivated by pasteurisation or even ultra-high temperature (UHT) treatment. In the current study, 20 different raw cows milk samples from single farms and dairy bulk tanks were analysed close to delivery to the dairies or close to processing in the dairy for their cultivable microbiota as well as the lipolytic and proteolytic potential of the isolated microorganisms. Altogether, 2906 isolates have been identified and assigned to 169 species and 61 genera. Pseudomonas, Lactococcus and Acinetobacter were the most abundant genera making up 62% of all isolates, whereas 46 genera had an abundance of <1% and represent only 6.6%. Of all isolates, 18% belong to hitherto unknown species, indicating that a large fraction of the milk microbiota is still unexplored. The potential of the isolates to produce lipases or peptidases followed in many cases a genus or group specific pattern. All isolates identified as members of the genus Pseudomonas exhibited mainly lipolytic and proteolytic activity or solely proteolytic activity. On the other hand, nearly all isolates of the genus Acinetobacter were lipolytic but not proteolytic. Only 37% of all tested lactic acid bacteria (LAB) showed enzymatic activity at 6 °C and the type of activity was proteolytic in 97% of these cases.

Enzymatic production of lactulose and epilactose in milk

The enzymatic production of lactulose was described recently through conversion of lactose by a thermophilic cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE). In the current study, we examined the application of CsCE for lactulose and epilactose production in milk (1.5% fat). The bioconversions were carried out in stirred reaction vessels at 2 different temperatures (50 and 8°C) at a scale of 25 mL volume. At 50°C, 2 highly different CsCE amounts were investigated for the time course of formation of lactulose and epilactose. The conversion of milk lactose (initial lactose content of 48.5 ± 2.1 g/L) resulted in a final yield of 57.7% (28.0 g/L) lactulose and 15.5% (7.49 g/L) epilactose in the case of the approximately 9.5-fold higher CsCE amount (39.5 µkat epilactose, 50°C) after 24 h. Another enzymatic lactose conversion was carried out at low 8°C, an industrially relevant temperature for milk processing. Although the CsCE originated from a thermophilic microorganism, it was still applicable at 8°C. This enzymatic lactose conversion resulted in 56.7% (27.5 g/L) lactulose and 13.6% (6.57 g/L) epilactose from initial milk lactose after 72 h. The time courses of lactose conversion by CsCE suggested that first epilactose formed and afterward lactulose via epilactose. To the best of our knowledge, this is the first time that an enzyme has produced lactulose directly in milk in situ at industrially relevant temperatures.

Novel cellobiose 2-epimerases for the production of epilactose from milk ultrafiltrate containing lactose

A selected number of enzymes have recently been assigned to the emerging class of cellobiose 2-epimerases (CE). All CE convert lactose to the rare sugar epilactose, which is regarded as a new prebiotic. Within this study, the gene products of 2 potential CE genes originating from the mesophilic bacteria Cellulosilyticum lentocellum and Dysgonomonas gadei were recombinantly produced in Escherichia coli and purified by chromatography. The enzymes have been identified as novel CE by sequence analysis and biochemical characterizations. The biochemical characterizations included the determination of the molecular weight, the substrate spectrum, and the kinetic parameters, as well as the pH and temperature profiles in buffer and food matrices. Both identified CE epimerize cellobiose and lactose into the C2 epimerization products glucosylmannose and epilactose, respectively. The epimerization activity for lactose was maximal at pH 8.0 or 7.5 and 40°C in defined buffer systems for the CE from C. lentocellum and the CE from D. gadei, respectively. In addition, biotransformations of the foodstuff milk ultrafiltrate containing lactose were demonstrated. The CE from D. gadei was produced in a stirred-tank reactor (12 L) and purified using an automatic system. Enzyme production and purification in this scale indicates that a future upscaling of CE production is possible. The bioconversions of lactose in milk ultrafiltrate were carried out either in a batch process or in a continuously operated enzyme membrane reactor (EMR) process. Both processes ran at an industrially relevant low temperature of 8°C to reduce undesirable microbial growth. The enzyme was reasonably active at the low process temperature because the CE originated from a mesophilic organism. An epilactose yield of 29.9% was achieved in the batch process within 28 h of operation time. In the continuous EMR process, the epilactose yield in the product stream was lower, at 18.5%. However, the enzyme productivity was approximately 6 times higher because the continuous epilactose formation was carried out for about 6 d without further addition of biocatalyst. Within this time, 24g of epilactose in 2.8 L of permeate were produced. The batch and the EMR process showed that the milk ultrafiltrate, which is a sidestream of the milk protein production, might be upgraded to a dairy product of higher value by the enzymatic in situ production of epilactose.

Pseudomonas helleri sp. nov. and Pseudomonas weihenstephanensis sp. nov., isolated from raw cow's milk.

Analysis of the microbiota of raw cows milk and semi-finished milk products yielded seven isolates assigned to the genus Pseudomonas that formed two individual groups in a phylogenetic analysis based on partial rpoD and 16S rRNA gene sequences. The two groups could be differentiated from each other and also from their closest relatives as well as from the type species Pseudomonas aeruginosa by phenotypic and chemotaxonomic characterization and average nucleotide identity (ANIb) values calculated from draft genome assemblies. ANIb values within the groups were higher than 97.3 %, whereas similarity values to the closest relatives were 85 % or less. The major cellular lipids of strains WS4917T and WS4993T were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol; the major quinone was Q-9 in both strains, with small amounts of Q-8 in strain WS4917T. The DNA G+C contents of strains WS4917T and WS4993T were 58.08 and 57.30 mol%, respectively. Based on these data, strains WS4917T, WS4995 ( = DSM 29141 = LMG 28434), WS4999, WS5001 and WS5002 should be considered as representatives of a novel species of the genus Pseudomonas, for which the name Pseudomonas helleri sp. nov. is proposed. The type strain of Pseudomonas helleri is strain WS4917T ( = DSM 29165T = LMG 28433T). Strains WS4993T and WS4994 ( = DSM 29140 = LMG 28438) should be recognized as representing a second novel species of the genus Pseudomonas, for which the name Pseudomonas weihenstephanensis sp. nov. is proposed. The type strain of Pseudomonas weihenstephanensis is strain WS4993T ( = DSM 29166T = LMG 28437T).

Epilactose production by 2 cellobiose 2-epimerases in natural milk

It was reported recently that cellobiose 2-epimerases (CE) from various aerobic microorganisms convert lactose to epilactose in defined buffer systems. In this study, we showed that CE from 2 mesophilic microorganisms, Flavobacterium johnsoniae and Pedobacter heparinus, were capable of converting lactose to prebiotic epilactose not only in buffer but also in a complex milk system. First, the 2 enzymes were separately cloned, recombinantly expressed in Escherichia coli, and purified by column chromatography. The production of F. johnsoniae CE was carried out in a stirred-tank reactor, indicating that future upscaling is possible. The bioconversions of milk lactose were carried out at an industrially relevant low temperature of 8°C to avoid undesired microbial contaminations or chemical side reactions. Both enzymes were reasonably active at this low temperature, because of their origin from mesophilic organisms. The enzymes showed different operational stabilities over a 24-h time-course. A conversion yield of about 30 to 33% epilactose was achieved with both enzymes. No side products were detected other than epilactose. Therefore, CE may introduce an added value for particular dairy products by in situ production of the prebiotic sugar epilactose.

Pseudomonas lactis sp. nov. and Pseudomonas paralactis sp. nov., isolated from bovine raw milk

Five strains, designated WS 4672T, WS 4998, WS 4992T, WS 4997 and WS 5000, isolated from bovine raw milk formed two individual groups in a phylogenetic analysis. The most similar species on the basis of 16S rRNA gene sequences were Pseudomonas azotoformans IAM 1603T, Pseudomonas gessardii CIP 105469T and Pseudomonas libanensis CIP 105460T showing 99.7-99.6 % similarity. Using rpoD gene sequences Pseudomonas veronii LMG 17761T (93.3 %) was most closely related to strain WS 4672T and Pseudomonas libanensis CIP 105460T to strain WS 4992T (93.3 %). The five strains could be differentiated from their closest relatives and from each other by phenotypic and chemotaxonomic characterization and ANIb values calculated from draft genome assemblies. ANIb values of strains WS 4992T and WS4671T to the closest relatives are lower than 90 %. The major cellular polar lipids of both strains are phosphatidylethanolamine, phosphatidylglycerol, a phospholipid and diphosphatidylglycerol, and their major quinone is Q-9. The DNA G+C content of strains WS 4992T and WS 4672T were 60.0  and 59.7  mol%, respectively. Based on these genotypic and phenotypic traits two novel species of the genus Pseudomonas are proposed: Pseudomonas lactis sp. nov. [with type strain WS 4992T (=DSM 29167T=LMG 28435T) and the additional strains WS 4997 and WS 5000], and Pseudomonasparalactis sp. nov. [with type strain WS 4672T (=DSM 29164T=LMG 28439T) and additional strain WS 4998].