Lactic acid bacteria are used extensively in the food and feed industry in the manufacturing of fermented products including most dairy products such as cheese, yoghurt and butter; meat products; bakery products; wine or vegetable products. When used for such purposes, cultures of lactic acid bacteria are generally referred to as starter cultures and they impart specific features to various fermented products by performing a number of metabolic and other functions herein.
In the present context, the expression “lactic acid bacteria” designates a group of Gram positive, catalase negative, non-motile, microaerophilic or anaerobic bacteria which ferment sugar with the production of acids including lactic acid as the predominantly produced acid, acetic acid, formic acid and propionic acid. The industrially most useful lactic acid bacteria are found among Lactococcus species, Lactobacillus species, Streptococcus species, Enterococcus species, Leuconostoc species, Oenococcus species and Pediococcus species.
When lactic acid bacteria are cultivated in a medium like milk or any other starting material in the manufacturing of food and feed products, the medium becomes acidified as a natural consequence of the growth and metabolic activity of the lactic acid bacterial starter cultures. In addition to the production of lactic acid/lactate from citrate, lactose or other sugars several other metabolites such as e.g. acetaldehyde, α-acetolactate, acetoin, acetate, ethanol, carbon dioxide, diacetyl and 2,3-butylene glycol (butanediol) are produced during the growth of the lactic acid bacteria.
Generally, the growth rate and the metabolic activity of lactic acid bacterial starter cultures can be controlled by selecting appropriate growth conditions for the strains of the specific starter culture used such as appropriate growth temperature, oxygen tension and content of nutrients.
Although milk is generally an ideal medium for the growth of lactic acid bacteria, a high content of oxygen in the milk affects the growth of the bacteria adversely and it is known in the dairy industry that a reduction of the oxygen content of the milk raw material may result in a more rapid growth of the added bacteria which in turn results in a more rapid acidification of the inoculated milk. Currently, such a reduction of the oxygen content is carried out by heating the milk in open systems, by deaerating the milk in vacuum or by a sparging treatments. Alternative means of reducing the oxygen content include the addition of oxygen scavenging compounds or the use of mixed cultures comprising two or more lactic acid bacterial species, at least one of which is less sensitive to oxygen.
WO 98/54337 discloses a method of enhancing the growth rate of lactic acid bacteria by cultivating the lactic acid bacteria in association with a metabolically engineered lactic acid bacterial helper organism which has a defect in its pyruvate metabolism, resulting in an increased oxygen consumption by the helper organism. However, this method of reducing the oxygen content in a medium is limited to the use of specific modified lactic acid bacterial strains and thus, there is a need to find a biological method of oxygen reduction in a food or feed starting material which does not involve the use of specifically mutated or metabolically modified lactic acid bacteria.
As mentioned above, when grown anaerobically lactic acid bacterial cells ferment sugars principally to lactic acid/lactate via pyruvate. NADH produced in the cells during this catabolism is reoxidised via lactate dehydrogenase. Under aerobic conditions, however, NADH can partly become reoxidised by NADH peroxidase and oxidase. Thus, under aerobic conditions pyruvate can be converted into other end products than those produced under anaerobic conditions which in turn results in an increase in biomass yield. Most lactic acid bacteria are catalase negative when grown in a haeme or haematin free medium. NADH peroxidase may therefore act to remove H2O2 produced by aerobic cultures of species unable to form a pseudo-catalase.
It is known that some lactic acid bacteria can form catalase and cytochromes when the aerobic growth medium is supplemented with haematin, blood or a haemoprotein. Sijpesteijn (1970) showed that the fermentation of strains of Lactococcus lactis and Leuconostoc mesenteroides in the presence of 10 ppm of haemin induced profound changes in the aerobic breakdown of glucose by resting cells of both organisms. This was observed when cells were cultivated under aerobic condition and, in the presence of haemin and glucose, were transferred into a resting cell medium, i.e. a medium wherein the cells are not capable of growth. Furthermore, an increased O2 uptake was observed and less lactic acid and more acetic acid and acetoin was produced. It was shown that cytochromes were formed in these organisms when cultured under the above culture conditions and that the respiration became more sensitive to KCN. It was suggested by this author that, after growth in the presence of haemin, a cytochrome-mediated respiration regulated by haemin was mainly responsible for the oxidation of NADH and that NADH oxidase only played a minor role under these conditions.
In a study on a cytochrome-like system in lactic acid bacteria, Ritchey & Seeley (1976) reported similar results. When grown on a haematin-containing medium with glucose some strains such as strains of Streptococcus faecalis or L. lactis, e.g. L. lactis subsp. lactis biovar. diacetylactis produced cytochromes whereas S. faecium did not. It was stated by these authors that strains like S. faecalis or L. lactis are blocked in the steps of haeme synthesis but possess the genetic determinants to establish a membrane-bound cytochrome electron transport chain under appropriate condition.
However, Kaneko et al. (1990) could not observe cytochromes when culturing a Citr+ (i.e. citrate metabolising) strain of L. lactis under aerobic conditions and in the presence of haemin and/or Cu+. Furthermore, they did not observe any difference in NADH oxidase, diacetyl reductase or lactate dehydrogenase activity when culturing that strain under these culture conditions. However, they observed an increase in the production of diacetyl and acetoin due to an activation of diacetyl synthase by haemin and/or Cu+. Japanese Patent Application JP 04-36180 and EP 430 406<both filed in the name of Meiji Milk Production Co. Ltd., proposed using these culture conditions to improve the production of diacetyl and acetoin by using the Citr+ strain of L. lactis. 
The above studies all show that the addition of haemin or haematin to the fermentation medium under aerobic conditions results in changes in the aerobic breakdown of glucose and that a possible cytochrome dependent aerobic respiration is induced in strains cultured under the above conditions. However, the above documents do not address the problem of reducing the oxygen content of the milk raw material or any other starting material in order to increase the growth of the added bacteria of a starter culture. Although an increased O2 uptake in resting cell systems under aerobic conditions in the presence of haemin and glucose has been reported, the ability of such cultured strains to be used in a starter culture for the manufacturing of a food or feed product has not been suggested.
The present invention is based on the discovery that lactic acid bacterial strains, when cultured or fermented under aerobic conditions in the presence of haemin and other porphyrin compounds, are capable of maintaining their increased oxygen reducing activity/capability when inoculated into milk or any other media under appropriate conditions and without addition of a porphyrin compound. It is thus possible to provide a generally applicable biological method for reducing the oxygen content in milk or any other food or feed starting material, whereby the growth and metabolic activity of lactic acid bacterial starter cultures herein can be substantially enhanced. It is therefore a primary objective of the present invention to provide such a method and culturally modified cells, which are useful in such a method.
Accordingly, these findings have opened up for a novel approach for providing useful culturally modified lactic acid bacterial starter cultures, which approach is based on relatively simple classical fermentation methods and which does not involve genetic engineering or classical mutagenesis. From a practical technological point of view this is advantageous, since the use of genetic engineering or classical mutagenesis in the construction of new strains is very labour intensive and costly and the use of genetically modified organisms in food production may give rise to consumer concerns.
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