[0145] ในบางรุรุ, rather than a probiotic itself, the nutritional composition(s) of the การเปิดเผยนี้ may ประกอบรวมด้วย a culture supernatant from a late-exponential growth phase of a probiotic batch-cultivation process (hereinafter referred to as the "culture supernatant"); in specific embodiments, the probiotic is LGG. Batch cultivation culture supernatant (which can also be referred to as "spent medium") may possesses protection against pathogen infection, ที่รวมถึง infection by C. sakazakii. Specifically the harvested culture supernatant may prevent the invasion of C. sakazakii to organs such as the brain and reduce mortality associated with C. sakazakii.
[0146] ในบางรุรุ, the nutritional composition ประกอบรวมด้วย a culture supernatant from a late-exponential growth phase of a probiotic batch-cultivation process, for use in the treatment or prevention of pathogen infection. In certain embodiments, the probiotic is LGG, and the
pathogen is C. sakazakii.
[0147] Without wishing to be bound by theory, it is believed that the activity of the culture
supernatant can be attributed to the mixture of components (ที่รวมถึง proteinaceous materials, and possibly ที่รวมถึง (exo)polysaccharide materials) as found released into the culture
medium at a late stage of the exponential (or "log") phase of batch cultivation of LGG. The
chemical composition of the culture supernatant is believed to be a mixture of a plurality of
amino acids, oligo- and polypeptides, and proteins, of various molecular weights. The culture supernatant may further ประกอบรวมด้วย polysaccharide structures and/or nucleotides. In some
embodiments the culture supernatant pertains to the entire, i.e. unfractionated culture
supernatant. Further, ในบางรุรุ the culture supernatant pertains to the entire, i.e. unfractionated culture supernatant.
[0148] The stages recognized in batch cultivation of bacteria are known to the skilled person.
These are the "lag," the "log" ("logarithmic" or "exponential"), the "stationary" and the "death" (or "logarithmic decline") phases. In all phases during which live bacteria are present, the bacteria metabolize nutrients from the media, and secrete (exert, release) materials into the culture
medium. The composition of the secreted material at a given point in time of the growth stages is not generally predictable.
[0149] ในบางรุรุ, a composition according to the การเปิดเผย and/or embodiments thereof is obtainable by a process ซึ่งประกอบรวมด้วย the steps of (a) subjecting a probiotic such as
LGG to cultivation in a suitable culture medium using a batch process; (b) harvesting the culture supernatant at a late exponential growth phase of the cultivation step, which phase is defined with reference to the second half of the time between the lag phase and the stationary phase of the batch-cultivation process; (c) optionally removing low molecular weight constituents from the supernatant so as to retain molecular weight constituents above 5 kiloดาลตัน (kDa) or even above 6kDa; (d) removing liquid contents from the culture supernatant so as to obtain the
composition.
[0150] In the การเปิดเผยนี้ and embodiments thereof, secreted materials are harvested from a late exponential phase. The late exponential phase occurs in time after the mid
exponential phase (which is halftime of the duration of the exponential phase, hence the
reference to the late exponential phase as being the second half of the time between the lag phase and the stationary phase). In particular, the term "late exponential phase" is used herein with reference to the latter quarter portion of the time between the lag phase and the stationary phase of the batch-cultivation process. ในบางรุรุ of the การเปิดเผยนี้,
harvesting of the culture supernatant is at a point in time of 75% to 85% of the duration of the exponential phase, and most preferably is at about 5/6 of the time elapsed in the exponential phase.
[0151] The term "cultivation" or "culturing" refers to the propagation of micro-organisms, in this case LGG, on or in a suitable medium. Such a culture medium can be of a variety of kinds, and is particularly a liquid broth, as customary in the art. A preferred broth, e.g., is MRS broth as generally used for the cultivation of lactobacilli. MRS broth generally ประกอบรวมด้วย polysorbate, acetate, magnesium and manganese, which are known to act as special growth factors for
lactobacilli, as well as a rich nutrient base. A typical composition ประกอบรวมด้วย (amounts in g/liter): peptone from casein 10.0; meat extract 8.0; yeast extract 4.0; D(+)-glucose 20.0; dipotassium hydrogen phosphate 2.0; Tween® 80 1.0; triammonium citrate 2.0; sodium acetate 5.0;
magnesium sulphate 0.2; manganese sulphate 0.04.
[0152] ในบางรุรุ, the culture supernatant of the การเปิดเผยนี้ may be
included in a nutritional composition that is an infant formula. The harvesting of secreted
bacterial products brings about a problem that the culture media cannot easily be deprived of undesired components. This specifically relates to nutritional products for relatively vulnerable subjects, such as infant formula or clinical nutrition. This problem is not incurred if specific
components from a culture supernatant are first isolated, purified, and then applied in a
nutritional product. However, it is desired to make use of a more complete cultural supernatant. This would serve to provide a composition better reflecting the natural action of the probiotic (i.e. LGG). One cannot, however, just use the culture supernatant itself as a basis for non-
viable probiotic materials to be specifically used in infant formula and the like.
[0153] ในบางรุรุ, the culture supernatan harvested from LGG cultivation does not contain components (as may present in the culture medium) that are not desired, or generally accepted, in nutritional compositions, such as an infant formula. With reference to polysorbate regularly present in MRS broth, media for the culturing of bacteria may include an emulsifying non-ionic surfactant, e.g. on the basis of polyethoxylated sorbitan and oleic acid (typically
available as Tween® polysorbates, such as Tween® 80). While these surfactants are
frequently found in food products, e.g. ice cream, and are generally recognized as safe, they are not in all jurisdictions considered desirable, or even acceptable for use in nutritional
products for relatively vulnerable subjects, such as infant formula or clinical nutrition.
[0154] The การเปิดเผยนี้ thus, ในบางรุรุ utilizes a culture media in which the aforementioned polysorbates can be avoided. To this end, a culture medium of the การเปิดเผย is devoid of polysorbates such as Tween 80. In a preferred embodiment of the การเปิดเผย
and/or embodiments thereof the culture medium may ประกอบรวมด้วย an oily ingredient selected from the group consisting of oleic acid, linseed oil, olive oil, rape seed oil, sunflower oil and mixtures thereof. It will be understood that the full benefit of the oily ingredient is attained if the presence of a polysorbate surfactant is essentially or entirely avoided.
[0155] The culture supernatant, ในบางรุรุ, may have a neutral pH, such as a pH of between pH 5 and pH 7, preferably pH 6.
[0156] In addition to the foregoing, it should be noted that the batch cultivation of lactobacilli, ที่รวมถึง LGG, is common general knowledge available to the person skilled in the art. These methods thus do not require further elucidation here. The culture supernatant of the การเปิดเผยนี้ can be harvested by any known technique for the separation of culture supernatant from a bacterial culture. Such techniques are well-known in the art and include, e.g.,
centrifugation, filtration, sedimentation, and the like.