Milk booster product comprising concentrated blends of breast milk from different donors

Abstract

The present invention relates to a human breast milk booster product, comprising combined breast milk from 2 or more individual providers and around 200 000 or more microbiome sequences present in the product (ASVs), in particular around 200 000 or more sequences from viable microbiota. Also provided is a food product comprising the human breast milk booster, use of the human breast milk booster as a food supplement, and the human breast milk booster for use in the treatment of under nourishment, malnourishment or malnutrition. Further provided is a method for producing a human breast milk booster product, comprising combining breast milk obtained from 2 or more individual providers, and concentrating the combined breast milk, thereby producing a concentrated human breast milk booster product comprising around 200 000 or more microbiome sequences present in the product (ASV), in particular around 200 000 or more sequences from viable microbiota.

Description

[0001] MILK BOOSTER PRODUCT

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a human breast milk booster product, comprising combined breast milk from 2 or more individual providers and around 200 000 or more microbiome sequences present in the product (ASVs), in particular around 200 000 or more sequences from viable microbiota. Also provided is a food product comprising the human breast milk booster, use of the human breast milk booster as a food supplement, and the human breast milk booster for use in the treatment of under nourishment, malnourishment or malnutrition. Further provided is a method for producing a human breast milk booster product, comprising combining breast milk obtained from 2 or more individual providers, and concentrating the combined breast milk, thereby producing a concentrated human breast milk booster product comprising around 200 000 or more microbiome sequences present in the product (ASV), in particular around 200 000 or more sequences from viable microbiota.

[0004] BACKGROUND OF THE INVENTION

[0005] Breastfeeding, the feeding of infants with human breast milk, is one of the most effective ways to ensure child health and survival, according to the World Health Organisation. Breast milk contains all the energy and nutrients that the infant needs for the first months of life, and it continues to provide up to half or more of a child’s nutritional needs during the second half of the first year, and up to one third during the second year of life. Breastfed children perform better on intelligence tests, are less likely to be overweight or obese and less prone to diabetes later in life. Women who breastfeed also have a reduced risk of breast and ovarian cancers (WHO). Despite these numerous benefits, fewer than half of all infants under 6 months of age are exclusively breastfed. Reasons for this low trend are numerous, but include social norms and pressures to use marketed milk formula alternatives, as well as the inability of some mothers to easily produce breast milk and feed their child.

[0006] Human breast milk is known to contain antibodies and a bacterial “microbiome” which can be passed from the mother to the child. In particular, the microbiome of human breast milk has been shown to pass to and influence the microbiomes of children fed with breast milk compared to formula milk (Banic et al. (2022) Int. J. Mol. Sci., 23(22): 14382, doi: htps: / / doi.org / 10.3390 / ijms232214382; and Wang et al. (2023) Foods, 12(9): 1740, doi: htps: / / doi.org / 10.3390 / foods12091740). Furthermore, a diverse breast milk microbiome which positively affects the infant microbiome has been demonstrated to be beneficial to both short term and long term child health (Notarbartolo et al. (2022) Pediatr. Gastroenterol. Hepatol. Nutr, 25(3):194-210, doi: https: / / doi.Org / 10.5223 / pghn.2022.25.3.194), consistent with knowledge of the gut microbiome in adults and its known relationship with multiple diseases and general health throughout the body.

[0007] There is therefore a need to provide a human breast milk booster and / or supplement product which can provide, or even improve on, the benefits of breast milk. Such a product may then be used to ‘boost’ human breast milk or supplement a food product, such as formula milk.

[0008] SUMMARY OF THE INVENTION

[0009] According to a first aspect of the invention, there is provided a human breast milk booster product, comprising combined breast milk obtained from 2 or more individual providers and around 200 000 or more microbiome sequences present in the product, as determined using an amplicon sequence variant (ASV), exact sequence variant (ESV), zero-radius OTU (ZOTU), sub-OTU (sOTU), haplotype or oligotype method, and wherein the human breast milk booster product is concentrated relative to the breast milk obtained from the individual providers.

[0010] In particular embodiments, the 200 000 or more microbiome sequences are sequences from viable microbiota. In some embodiments, the human breast milk booster comprises around 225 000 or more microbiome sequences, in particular around 225 000 or more sequences from viable microbiota. In further embodiments, the human breast milk booster comprises around 250 000 or more microbiome sequences, in particular around 250 000 or more sequences from viable microbiota.

[0011] The human breast milk booster product may be concentrated by removing water from the combined and / or obtained breast milk, such as by centrifugation, freezing and / or lyophilisation. In certain embodiments, the human breast milk booster product is lyophilised.

[0012] In a further aspect of the invention, there is provided a food product comprising the human breast milk booster product described herein. In another aspect, there is provided a use of the human breast milk booster product described herein as a food supplement. The food product or food to be supplemented may be formula milk or human breast milk.

[0013] In a yet further aspect, there is provided the human breast milk booster product described herein, for use in the treatment of under nourishment, malnourishment or malnutrition.

[0014] In a still further aspect of the invention, there is provided a method for producing a human breast milk booster product, the method comprising the steps of: (i) combining breast milk obtained from 2 or more individual providers; and

[0015] (ii) concentrating the combined breast milk, thereby producing a concentrated human breast milk booster product comprising around 200 000 or more microbiome sequences present in the product, as determined using an amplicon sequence variant (ASV), exact sequence variant (ESV), zero-radius OTU (ZOTII), sub-OTU (sOTU), haplotype or oligotype method.

[0016] In particular embodiments, the 200 000 or more microbiome sequences are sequences from viable microbiota. In some embodiments, the produced concentrated human breast milk booster product comprises around 225 000 or more microbiome sequences, in particular around 225 000 or more sequences from viable microbiota. In further embodiments, the produced concentrated human breast milk booster product comprises around 250000 or more microbiome sequences, in particular around 250 000 or more sequences from viable microbiota.

[0017] In yet further embodiments, combining step (i) may be performed after concentrating step (ii) or after an optional lyophilising step (iii).

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Microbiome diversity is increased in the human breast milk booster product (the Vyarna Bio Information Booster; VBIB). Average amplicon sequence variant (ASV) microbiome diversity as measured in ‘fresh’ (i.e. extracted, not lyophilised or combined) human breast milk, lyophilised (not combined) or the concentrated and combined human breast milk booster product described herein (VBIB).

[0020] Figure 2: The human breast milk booster product comprises large microbiome diversity. A) Barplots of microbiome composition at the species level of the gram-positive (P) and gram-negative (N) bacteria in each indicated ‘fresh’ (F), lyophilised (L) or combined / mixed (M) sample. Each coloured section of each bar represents a distinct sequence identified.

[0021] Figure 3: The microbiome is diverse between individual providers. Beta diversity plot of microbiome diversity differences between samples, represented as principle coordinate analysis (PCoA) of gram-positive (P) and gram-negative (N) bacterial microbiome diversity between ‘fresh’ (F) samples as in Figure 2.

[0022] DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention is based on the development by the inventors of a human breast milk booster product, methods for producing and uses of the same, wherein the breast milk booster product (the terms “human breast milk booster product”, “human breast milk booster”, “breast milk booster product”, “breast milk booster”, “booster product” and “booster” may all be used interchangeably herein to refer to the breast milk booster product described) is concentrated and comprises a greater viable microbiome diversity relative to breast milk obtained from a single individual provider (i.e. relative to ‘natural’ breast milk), by virtue of being produced by combining breast milk from 2 or more individual providers. The human breast milk booster product is called the Vyarna Bio Information Booster (VBIB).

[0024] Thus, according to a first aspect of the invention, there is provided a human breast milk booster product, comprising combined breast milk obtained from 2 or more individual providers and around 200 000 or more microbiome sequences present in the product, as determined using an amplicon sequence variant (ASV), exact sequence variant (ESV), zero-radius OTU (ZOTII), sub-OTU (sOTU), haplotype or oligotype method, and wherein the human breast milk booster product is concentrated relative to the breast milk obtained from the individual providers.

[0025] The term “microbiota / microbiome” refers to a population of microorganisms that exists within a niche in the human body, with a mutualistic relationship with the host and includes bacteria, viruses, fungi, parasites and archaea. Herein, the term “microbiome” and associated diversity values refers to bacterial content of the breast milk and produced booster product, since such diversity may be readily measured according to the methods described herein. As will be readily appreciated, references herein to the microbiome and microbiota may refer in particular to viable microbiota, such that the sequence diversity described herein is from viable microbiota / the diversity is of viable microbiome sequences. When considering the human microbiome, the gut is often focussed on and the role of a healthy and diverse, mostly non- pathogenic intestinal microbiome in human health is well known. For example, the relationship between the microbiome and host is associated with immunity, and dysbiosis has been linked to various human diseases, such as anxiety, depression, hypertension, cardiovascular diseases, obesity, diabetes, inflammatory bowel disease and cancer (reviewed in Afzaal et al.

[0026] (2022) Front. Microbiol., 13:999001 , doi: htps: / / doi.org / 10.3389 / fmicb.2022.9990Q1 ; Hou et al. (2022) Sig. Transduc. Target Then, 7(1):135, doi: htps: / / doi.org / 10.1038 / s41392-Q22-

[0027] 00974-4; and Fan & Pedersen (2021) Nat. Rev. Microbiol., 19(1):55-71 , doi: Thus, changes in gut microbiota lead not only to issues of the gut and intestines, but also other organs and tissues (reviewed in Ahlawat & Sharma (2021) Lett. Appl. Microbiol., 72(6):636-668, doi:

[0028] Diet can greatly influence the diversity of the gut microbiome (Simoes, Maganinho & Sousa (2022) Eur. J. Nutr, 61 (3): 1187-1198, doi: https: / / doi.org / 10.1007 / s00394-021-02755-1). In particular, the microbiome of human breast milk has been shown to pass to and influence the microbiomes of children fed with breast milk compared to formula milk, and a diverse breast milk microbiome which positively affects the infant microbiome has been demonstrated to be beneficial to both short term and long term child health (Notarbartolo et al. (2022); Banic et al. (2022); and Wang et al. (2023)).

[0029] The presence of microbiota in human breast milk is known and the composition of the microbiome in terms of the bacterial species and genera has been well studied to date, including the effects of diet and socioeconomic factors on the breast milk microbiome (see the review article: Fernandez et al. (2020) Front. Cell. Infect. Microbiol., 10, doi: as well as Londono-Sierra et al. (2023)

[0030] Microorganisms, 11(7):1812, doi:

[0031] However, as demonstrated previously and herein (see Figure 1 herein) human breast milk from individuals may contain up to 190 000, and more specifically from around 120 000 to around 185 000 sequences from viable microbiota (as determined using an amplicon sequence variant (ASV) method). By contrast, the present invention provides a breast milk booster product with significantly greater diversity of viable microbiome, specifically of around 200 000 or more, around 225 000 or more, or around 250 000 or more sequences (as determined by ASV). In particular, demonstrated herein is a breast milk booster product comprising around 198000 microbiota sequences (as determined by ASV, i.e. around 198 000 sequences from viable microbiota). Such increased diversity is maintained after the booster product has been concentrated by removing water, in particular by lyophilisation, while in ‘fresh’ breast milk from individual providers diversity is decreased after lyophilisation.

[0032] Increased diversity in the herein provided breast milk booster product and production methods result from the combining of breast milk from 2 or more individual providers. Thus, in certain embodiments the 200 000 or more microbiota sequences comprised in the breast milk booster product described herein is due to the combination of breast milk from multiple providers, in particular 2 or more individual providers (e.g. 3 or more). The diversity may thus be a sum of the diversity of each individual provider. For example, the total number of sequences from viable microbiota may be the sum of individual 1’s viable microbiome sequences plus individual 2’s viable microbiome sequences. Further individual providers’ viable microbiome sequences may simply be added to this total. Alternatively, the total number of sequences from viable microbiota may be a function of the sum of the diversity of each individual provider, such as to allow for any diversity loss that may occur during processing of the milk to produce the booster product. The term “providers” herein refers to the individual human subjects (i.e. women) who are lactating and donate / provide breast milk, such that the breast milk is obtained from said providers. Such milk obtained from an individual may be referred to as ‘fresh’ prior to any combining and / or concentrating as described herein. The breast milk may be obtained from an individual using any suitable method, such as by hand expression or using a breast pump. In a specific example, obtaining the breast milk from an individual provider may be as follows:

[0033] 1) select one breast for extraction;

[0034] 2) clean the nipple and areola;

[0035] 3) extract breast milk using a sterile breast pump; and

[0036] 4) place the extracted breast milk on ice immediately after extraction.

[0037] A suitable volume to be extracted / expressed / obtained from the provider may be between 100ml and 200ml (approx. 3.4-6.8 fluid ounces), such as around 150ml (approx. 5 fluid ounces).

[0038] In a certain embodiment, breast milk obtained from 2 or more individual providers is combined. Thus, according to this embodiment the breast milk booster product comprises breast milk combined from 2 or more individual providers. In a further particular embodiment, breast milk obtained from 3 or more individual providers, such as from 3 individual providers, is combined. Thus, according to this particular embodiment the breast milk booster product comprises breast milk combined from 3 or more individual providers, such as from 3 individual providers.

[0039] The terms “combining”, “combined” and the like herein refer to the “mixing” of breast milk from individual providers. Such combining may be performed prior to or after concentrating as described herein. In embodiments wherein concentrating is performed by lyophilisation, the concentrated / lyophilised breast milk from individual providers may be combined after said lyophilising. Thus, in some embodiments combining is performed prior to concentrating and / or lyophilisation. According to these embodiments, it will be appreciated that combining of the liquid and unconcentrated ‘fresh’ breast milk is performed. In other embodiments, combining is performed after concentrating. According to these embodiments, combining is of the liquid concentrated breast milk to produce the human breast milk booster product described herein. In yet further embodiments, combining is performed after lyophilising. According to these embodiments, combining is of the dry lyophilised breast milk to produce the booster product.

[0040] As mentioned hereinbefore and will be readily appreciated by the skilled person, the microbiome of breast milk varies between individuals from different geographical locations (Pace et al. (2021) Microorganisms, 9(6):1153, doi: https: / / doi.org / 10.3390 / microorganisms9061153). This is the same as the well known finding that the gut microbiome can vary across geographical locations. Thus, in order to further increase the diversity of the obtained breast milk and breast milk booster product, each of the individual providers may be selected from different geographical locations. Different locations may be distant, e.g. different countries or continents, or may be relatively geographically proximal (e.g. within the same country). Such geographically proximal locations may vary in other ways than by location.

[0041] As described hereinbefore, the microbiome in human breast milk is transferred to the child during feeding. However, as well as the bacterial microbiome, viruses and other pathogens that the mother / woman may be infected with can also transfer. Thus, it is preferable that the individual providers described herein are negative for any viral infection. Additionally or alternatively, the individual providers may be negative for any bacterial infection. As such, in further embodiments the individual providers may be screened for diseases and / or disorders, in particular viral infections. Examples of viral infections include, but are not limited to HIV or CMV, which is particularly harmful in very young or premature babies / neonates. Thus, in a yet further embodiment the breast milk booster product is substantially free of viruses. “Substantially free” and “negative” refer to a level at which no or minimal detectable virus or viral infection is present in the booster product, breast milk obtained from the individual providers or other sample obtained from the individual providers for screening. Additionally or alternatively, the individual providers may be negative for a bacterial infection, in particular a harmful bacterial infection. As will be readily appreciated, determining ‘harmful’ bacteria may be challenging and thus the booster product may not be substantially free of harmful or disease-causing bacteria. However, in circumstances where such harmful and / or diseasecausing bacterial are identifiable, they should not be present in the booster product, the breast milk obtained from the individual providers or any other sample obtained from the individual providers for screening.

[0042] Viable Microbiome Diversity

[0043] As described herein, the human breast milk booster product comprises around 200 000 or more sequences from viable microbiota. In further embodiments, the booster product comprises around 225 000 or more microbiome sequences, in particular around 225 000 or more sequences from viable microbiota. In particular embodiments, the booster product comprises around 250 000 or more microbiome sequences, in particular around 250 000 sequences from viable microbiota. As demonstrated herein, the booster product and the methods of producing the same according to the present invention comprise such high viable microbiota diversity, which is greater than that found in ‘fresh’ or uncombined human breast milk.

[0044] Various methods for identifying and determining sequences from the microbiome, in particular from viable microbiota, will be recognised by the skilled person. However, by way of nonlimiting example sequencing may particularly be used, wherein the bacterial 16S ribosome RNA (rRNA) can be sequenced to identify bacteria of different species or genera, or which are of the same species but comprise distinct 16S rRNA sequences that may lead to functional and / or characteristic differences. The 16S rRNA sequence is particularly suited to the detection of bacteria from different species / genera and the reconstruction of phylogenies as it has been shown to have a slow rate of evolution (Woese & Fox (1977) PNAS, 74(11):5088- 5090, doi: ). A sequence may suitably be determined as an amplicon sequence variant (ASV; also known as exact sequence variant (ESV), zeroradius OTU (ZOTII), sub-OTU (sOTU), haplotype or oligotype) of the sequenced 16S rRNA. The analysis of sequencing data by an ASV method comprises initially determining which exact sequences were read and how many times. This information is then combined with an error model for the sequencing run, determining the probability that any given read at any given frequency is not due to sequencing error, effectively generating a p-value for each exact sequence and the likelihood that the sequence was generated due to sequencing error. Sequences are then filtered using a threshold confidence value, with those sequences falling below the threshold confidence value due to e.g. the low number of occurrences for that sequence, excluded. As ASVs are exact unique sequences, with no clustering based on similarity (e.g. as done with operating taxonomic units (OTUs)), they can distinguish single nucleotide differences between sequences and can be readily compared across experiments and studies, with no dependence on the same reference database being used or reanalysis to re-cluster. Thus ASVs are particularly preferred for determining the number of microbiota sequences according to the invention because they allow high resolution to identify single nucleotide differences in sequences from different bacteria, such as potentially different bacterial cells, different sub-species, different species or different genera. Thus, in particular embodiments the number of sequences, in particular the number of sequences from viable microbiota, is determined using an amplicon sequence variant (ASV), exact sequence variant (ESV), zero-radius OTU (ZOTU), sub-OTU (sOTU), haplotype or oligotype method. Alternatively but less preferred herein, another method for the number of sequences, such as from viable microbiota, is using an operational taxonomic unit (OTU). An OTU method uses clustering to combine sequences with high levels of similarity (e.g. 97% or greater), with clustering being performed de novo or based on a reference database (including ‘openreference clustering’ using a combination of reference-based and de novo clustering, and ‘closed-reference clustering’ where only the reference database is used). However, as is understood by the skilled person, ASV methods provide higher resolution, more accurate and more diverse sequences, giving the best representation of diversity.

[0045] Concentration

[0046] The breast milk booster product described herein is concentrated relative to the breast milk obtained from the individual providers (i.e. relative to the ‘fresh’ breast milk as referred to herein). Concentration involves the removing of water from the obtained or combined breast milk, such that the resulting concentrated breast milk or concentrated breast milk booster product (i.e. wherein concentration has been performed before combining or after combining, respectively) has less water and thus less volume than the starting material (i.e. the ‘fresh’ breast milk). As will be readily appreciated, in order to provide the herein described viable microbiome diversity, methods of concentration must be selected which are suitable for maintaining a viable microbiome in the breast milk and booster product. For example, heating to evaporate water from the breast milk will not be suitable as the microbiome will not survive. Thus, in certain embodiments the breast milk obtained from the individual providers or booster product is not heat treated or treated in any related method comprising heat, such as pasteurisation. In particular, the breast milk obtained from the individual providers or booster product is not pasteurised. In further embodiments, any concentration method which maintains the viable microbiome and its diversity may be used to concentrate the breast milk obtained from the individual providers or booster product. In non-limiting examples, suitable concentration methods include centrifugation, freezing, skimming and lyophilisation. Thus, in some embodiments the breast milk obtained from the individual providers or booster product is concentrated by removing water from the combined and / or obtained breast milk, such as by centrifugation, freezing, skimming and / or lyophilisation. Centrifugation concentrates the breast milk / booster product by separating the solid and more dense liquid components from the water contained in the breast milk. Following centrifugation, water may be removed from the ‘top’, leaving behind the concentrated more dense components, such as fats etc. Freezing concentrates by turning the water component of breast milk / the booster product from liquid to solid, allowing it to be removed from the remaining components / the other components may be removed from the solid, frozen water. The process of skimming concentrates by removing the water from the cream of the breast milk / booster product following separation, such as by allowing the milk / booster to sit and settle. Lyophilisation (also known as “freeze drying” and “cryodesiccation”) dehydrates at a low temperature by removing water / ice by sublimation. Lyophilisation is known for preserving the product and its structure and properties. Herein, lyophilisation maintains the nourishing properties and viable microbiota of the breast milk / booster product as described herein while removing water and thus concentrating. Methods of lyophilisation are well known in the art. In some embodiments, concentration may comprise any two or more suitable methods, such as two or more of centrifugation, freezing and / or lyophilisation. In a particular embodiment, the breast milk obtained from the individual providers or booster product is concentrated by lyophilisation. Depending on the chosen method of concentration, it will be appreciated that the amount of water removed may vary. For example, when lyophilisation is used to concentrate the breast milk / booster product, substantially all of the water will be removed. Substantially all of the water may also be removed when concentrating the breast milk / booster product by centrifugation, freezing and / or skimming. Thus, in one embodiment concentrating removes substantially all of the water from the obtained breast milk or booster product. In further embodiments, concentrating removes 90% or more of the water from the obtained breast milk or booster product, such as 95% or more or 99% or more. In alternative embodiments, concentrating removes 50% or more, 60% or more, 70% or more or 80% or more of the water from the obtained breast milk or booster.

[0047] Production Methods

[0048] According to a further aspect of the present invention, there is provided a method for producing a human breast milk booster product, the method comprising the steps of:

[0049] (i) combining breast milk obtained from 2 or more individual providers; and

[0050] (ii) concentrating the combined breast milk, thereby producing a concentrated human breast milk booster product comprising around 200 000 or more microbiome sequences present in the product, as determined using an amplicon sequence variant (ASV), exact sequence variant (ESV), zero-radius OTU (ZOTII), sub-OTU (sOTU), haplotype or oligotype method.

[0051] According to this aspect and as described hereinbefore, the sequence diversity may be of viable microbiota such that the produced human breast milk booster product comprises around 200 000 or more sequences from viable microbiota. In further embodiments, the produced booster product comprises around 225 000 or more microbiome sequences, in particular around 225 000 or more sequences from viable microbiota. In particular embodiments, the produced booster product comprises around 250 000 or more microbiome sequences, in particular around 250 000 or more sequences from viable microbiota.

[0052] Combining according to the method of producing the booster product may be performed at any stage of the method described herein. For example, combining may be performed prior to concentrating (e.g. by lyophilisation) or after concentration of the breast milk. Thus, in one embodiment combining step (i) is performed prior to concentrating step (iii). In another embodiment, combining step (i) is performed after concentrating step (ii). Concentrating may be by any method described hereinbefore. In particular, concentrating is by a method which maintains the viability of the microbiome whilst removing water from the breast milk or combined breast milk. Thus, in one embodiment concentrating in the described method for producing a breast milk booster comprises removing water from the breast milk. As described hereinbefore, such concentration is performed without heat treatment or any related method comprising heat, such as pasteurisation. Thus, in embodiments the method for producing a human breast milk booster product described herein does not comprise any heat treatment and / or pasteurisation step(s). In further embodiments, any concentration method which maintains the viable microbiome and its diversity may be used. In a yet further embodiment, concentrating is by centrifugation. In another embodiment, concentration is by freezing. In a yet other embodiment, concentration is by lyophilisation. In further embodiments, concentration may comprise any two or more suitable methods, such as two or more of centrifugation, freezing and / or lyophilisation. In further embodiments wherein the method further comprises step (iii) of lyophilising the booster product, combining step (i) may be performed after lyophilising step (iii).

[0053] Thus, in one embodiment, the method for producing a human breast milk booster product comprises the steps of:

[0054] (i) combining breast milk obtained from 2 or more individual providers; then

[0055] (ii) concentrating the combined breast milk, thereby producing a human breast milk booster product as described herein.

[0056] In another embodiment, the method for producing a human breast milk booster product comprises the steps of:

[0057] (i) concentrating breast milk obtained from 2 or more individual providers; then

[0058] (ii) combining the concentrated breast milk, thereby producing a human breast milk booster product as described herein.

[0059] As described hereinbefore, the breast milk booster product may be lyophilised. Thus, in some embodiments, the method for producing a human breast milk booster product further comprises step (iii) of lyophilising the human breast milk booster product. Such lyophilisation may be performed after / as well as concentrating or may be used as the concentration method as described hereinbefore. Thus, in a further embodiment concentrating step (ii) comprises lyophilisation. Lyophilisation may be performed prior to or after combining and / or concentrating. In a further embodiment, the method for producing a human breast milk booster product comprises the steps of:

[0060] (i) combining breast milk obtained from 2 or more individual providers;

[0061] (ii) concentrating the combined breast milk; and

[0062] (iii) lyophilising the concentrated and combined breast milk, thereby producing a lyophilised human breast milk booster product as described herein.

[0063] In a yet further embodiment, the method for producing a human breast milk booster product comprises the steps of:

[0064] (i) concentrating breast milk obtained from 2 or more individual providers;

[0065] (ii) combining the combined breast milk; and

[0066] (iii) lyophilising the concentrated and combined breast milk, thereby producing a lyophilised human breast milk booster product as described herein.

[0067] In a still further embodiment, the method for producing a human breast milk booster product comprises the steps of:

[0068] (i) combining breast milk obtained from 2 or more individual providers; and

[0069] (ii) concentrating the combined breast milk by lyophilisation, thereby producing a lyophilised human breast milk booster product as described herein.

[0070] Thus, in one embodiment the method for producing a human breast milk booster product comprises the steps of:

[0071] (i) combining breast milk obtained from 2 or more individual providers; and

[0072] (ii) lyophilising the combined breast milk.

[0073] In a further embodiment, the method for producing a human breast milk booster product comprises the steps of:

[0074] (i) concentrating breast milk obtained from 2 or more individual providers by lyophilisation; and

[0075] (ii) combining the lyophilised breast milk, thereby producing a lyophilised human breast milk booster product as described herein.

[0076] Thus, in one embodiment the method for producing a human breast milk booster product comprises the steps of:

[0077] (i) lyophilising breast milk obtained from 2 or more individual providers; and

[0078] (ii) combining the lyophilised breast milk. Food Products, Supplements and Uses

[0079] Human breast milk is a nourishment for developing infants postpartum. Therefore, it will be appreciated that the breast milk booster product described herein may be used as a food product / in the manufacture of a food product or as a food supplement. Such food product or supplemented food will thus comprise greater viable microbiome diversity than ‘fresh’ breast milk obtained from any one individual provider, as described hereinbefore. Without being bound by theory, it is expected that a food or supplemented food herein will provide improved outcomes for health by providing such increased microbiome and resulting in an increased diversity of microbiome for the infant receiving the food product / supplemented food, since diet can greatly influence the diversity of the gut microbiome (Simoes, Maganinho & Sousa (2022)) and a diverse breast milk microbiome which positively affects the infant microbiome has been demonstrated to be beneficial to both short term and long term child health (Notarbartolo et al. (2022); Banic et al. (2022); and Wang et al. (2023)).

[0080] Thus, in a further aspect of the present invention there is provided a food product comprising the human breast milk booster described herein. In a related aspect, there is provided a method of producing a food product comprising the breast milk booster product described herein, comprising adding the breast milk booster product to a food product or providing the booster product for addition to a food product. The food product may be milk, in particular milk for infants, children and babies such as formula milk, cow’s milk, human breast milk or cow’s milk alternatives (e.g. soy and nut milks etc.). Thus, in one embodiment the food product is formula milk. In another embodiment, the food product is human breast milk.

[0081] In another aspect, there is provided the use of the human breast milk booster product described herein as a food supplement. In a related aspect, there is provided a method of supplementing food, comprising adding the breast milk booster product described herein to a food or providing the booster product for addition to a food. In embodiments, the food to be supplemented is milk, in particular milk for infants, children and babies such as formula milk, cow’s milk (often referred to simply as “milk”), human breast milk or cow’s milk alternatives (e.g. soy and nut milks etc.). Thus, in one embodiment the food to be supplemented is formula milk. In another embodiment, the food to be supplemented is human breast milk.

[0082] Since the breast milk booster described herein is hypothesised to increase microbiome diversity and have a positive impact on infant health, it may also provide positive effects in situations of under nourishment, malnourishment and / or malnutrition. Such conditions are problematic in developing infants, and it is imperative that children / babies in their early years of life are provided with sufficient nutrition. Thus, in a yet further aspect there is provided the human breast milk booster product described herein for use in the treatment, amelioration or prevention of under nourishment, malnourishment or malnutrition. In another aspect, there is provided a method of treating, ameliorating or preventing under nourishment, malnourishment or malnutrition, comprising administering the breast milk booster product described herein to a subject in need thereof, wherein said administration is by mouth or orally. According to these aspects, administration of the breast milk booster for the treatment, amelioration or prevention is by mouth / orally and in particular as part of or supplemented to a food product as described hereinbefore. In a certain embodiment, treatment, amelioration or prevention comprises feeding the subject with a food product comprising the breast milk booster product, in particular milk, formula milk or human breast milk supplemented with the booster product. In further embodiments, the subject is a child, baby or infant who is under nourished, malnourished or suffering from malnutrition. In other embodiments, the subject is a child, baby or infant who is at risk of suffering from or developing under nourishment, malnourishment or malnutrition.

[0083] It will be appreciated that references herein to “treatment” and “amelioration” include such terms as “prevention”, “reversal” and “suppression”. Such references include provision of the breast milk booster product or food product comprising / supplemented with the booster as described herein prior to the onset of any symptoms or signs of under nourishment, malnourishment or malnutrition, e.g. wherein the subject is at risk of under nourishment, malnourishment or malnutrition. Administration of the breast milk booster product or food product comprising / supplemented with the booster as described herein may also be anticipated after the onset of under nourishment, malnourishment or malnutrition, either before clinical presentation or after symptoms / signs manifest.

[0084] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As may be used herein, the term “about” includes up to and including 10% greater and up to and including 10% lower than the value specified, suitably up to and including 5% greater and up to and including 5% lower than the value specified, especially the value specified. The term “between” as may be used herein includes the values of the specified boundaries.

[0085] Throughout the specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations thereof such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps. In addition, as used herein and the appended claims, the singular forms “a”, “an” and “the” include plural referents, and vice versa, unless the content clearly dictates otherwise. Thus, for example references to “a food product” may include two or more such food products.

[0086] It will be understood that all embodiments described herein may be applied to all aspects of the invention and vice versa, and such combinations would be readily apparent from the description provided herein and to those skilled in the art.

[0087] Other features and advantages of the present invention will be apparent from the description provided herein. It should be understood, however, that the description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications will become apparent to those skilled in the art. The invention will now be described using the following, non-limiting examples:

[0088] EXAMPLES

[0089] Example 1: Collection of Human Breast Milk for Analysis of Microbiome Diversity & Production of the Vyarna Bio Information Booster (VBIB)

[0090] Consent of Provider

[0091] Brief the procedure for milk extraction and the purpose of the procedure to the provider in English and the native language of the provider.

[0092] Take consent and instruct the provider to fill out the information and consent form.

[0093] Guide the provider to the extraction room and make her feel comfortable.

[0094] Collection of Milk Sample

[0095] Select one breast for the extraction.

[0096] Clean the nipple and areola with baby wipes.

[0097] Use a sterile breast pump for human breast milk (HBM) extraction.

[0098] Extract approx. 150ml (approx. 5 ounces) of HBM and immediately place it on ice.

[0099] Distribution of HBM into Groups

[0100] Distribute the extracted HBM into different groups by making small aliquots.

[0101] Take seven 15ml falcon tubes and label them as follows:

[0102] Falcon tube 1 : Fresh (001)

[0103] Falcon tube 2: Lyophilised (001)

[0104] Note: Add these labels on the falcon tube cap and also on the white area at the side of the falcon tube. Make sure to write group type, provider code (001), and date of HBM extraction. Pour 10ml HBM into each falcon tube 1 (‘Fresh’) Pour 15ml into falcon tube 2 (‘Lyophilised’).

[0105] Fresh HBM

[0106] Label two 1.5ml microcentrifuge tubes. One tube as “+” and the other as with sample ID (e.g. 001+ or 001-)

[0107] Take extracted milk and directly pour 1 ml into each tube (make sure not to touch the neck of the bottle with the falcon tube’s mouth).

[0108] Centrifuge the tubes at 5000 g for 10 minutes prior to experiment initiation.

[0109] Remove the supernatant by quick inverting and gentle tapping Make sure the pellet is not dislodged.

[0110] Store in freezer.

[0111] Move tube 2 into lyophiliser, still frozen. Set for 48 hours.

[0112] Combining

[0113] Remove tubes from lyophiliser.

[0114] Combine equal amounts of lyophilised HBM from each of the providers to produce the Vyarna Bio Information Booster (VBIB).

[0115] Example 2: Analysis of Microbiome Diversity in Fresh, Lyophilised & Lyophilised Combined (VBIB) Breast Milk

[0116] Take 2 falcon tubes and add 1 gram of lyophilised HBM with 9ml purified water in each tube to reconstitute.

[0117] Centrifuging at 4000 g for 15 minutes at 4°C.

[0118] Remove the upper and middle layers. Retain the sediments in the bottom.

[0119] - Add 600pl PBS and centrifuge for 10 minutes at 10 000 g at 4°C.

[0120] Discard the supernatant and keep the pellet.

[0121] Take two 1.5ml microcentrifuge tubes and label one tube as “+” and the other as with sample ID (e.g. 001+ or 001-).

[0122] Transfer the pellet into the microcentrifuge tubes and proceed with the experiment.

[0123] Combined

[0124] Make combinations of lyophilised milk as follows:

[0125] Sample 1+2+3+4+5 Take 2 falcon tubes and add 1 gram of combined lyophilised HBM with 9ml purified water in each tube to reconstitute.

[0126] Centrifuging at 4000 g for 15 minutes at 4°C.

[0127] Remove the upper and middle layers. Retain the sediments in the bottom.

[0128] - Add 600pl PBS and centrifuge for 10 minutes at 10 000 g at 4°C.

[0129] Discard the supernatant and keep the pellet.

[0130] Take two 1.5ml microcentrifuge tubes and label one tube as “+” and the other as with sample ID (e.g. 001+ or 001-).

[0131] Transfer the pellet into microcentrifuge tubes and proceed with the experiment.

[0132] Take Resuspension solution A and add it to a lysozyme-containing tube.

[0133] Make small fractions of 500pL and store the unused portions at -20°C until needed.

[0134] - Add 100pL of Resuspension Solution A (with lysozyme) into the “001 +” labelled tube. Incubate at 37°C for 45 minutes.

[0135] Take out the sample every 15 minutes and vortex for 30 sec and put it back in the incubator till the incubation time is completed.

[0136] - After incubation, add 300pL of Buffer SK and 10pL of reconstituted Proteinase K to the digestion mixture.

[0137] Mix well by vortexing (for 1 min).

[0138] - Again incubate the lysate at 55°C for 45 minutes.

[0139] Take out the sample every 15 minutes and vortex for 30 sec and put it back in the incubator till the incubation time is completed. (Gram-Negative)

[0140] - Add 400pL of Buffer SK and mix well by vortexing for 1 minute.

[0141] Binding to Column

[0142] - Add 200pL of absolute ethanol to the lysis mixture, and mix by vortexing for 30 seconds.

[0143] - Assemble a column with one of the provided collection tubes (ZymoBIOMICS DNA miniprep / microprep kit). Place the collection tube on a column.

[0144] Using a micropipette, carefully transfer the lysate mixed with ethanol to the spin column.

[0145] Centrifuge the column assembly for 2 minutes at 14 000 RPM (~20 000 g) to bind the bacterial DNA. If all the liquid does not pass through the column, spin for an additional 2 minutes at 14 000 RPM (-20 000 g).

[0146] Column Wash

[0147] - Add 500pL of Buffer SK to the column and centrifuge for 2 minutes at 14 000 RPM (-20 000 g).

[0148] Discard the flowthrough and reassemble the column and the collection tube.

[0149] - Add 500pL of Wash Solution A to the column and centrifuge for 1 minute at 14 000 RPM (-20 000 g).

[0150] Spin for an additional minute.

[0151] Discard the flowthrough and reassemble the spin column with its collection tube.

[0152] - Apply 500pL of Wash Solution A to the column and centrifuge for 1 minute at 14 000 RPM (-20 000 g).

[0153] Discard the flowthrough and reassemble the spin column with its collection tube.

[0154] Centrifuge for 2 minutes at 14 000 RPM (~20 000 g) to ensure the column is completely dry.

[0155] Discard the collection tube.

[0156] DNA Elution

[0157] T ransfer the spin column to a provided 1 ,7mL Elution tube.

[0158] - Apply 50pL of Elution Buffer B to the column and centrifuge at 2 000 RPM (-425 g) for 2 minutes.

[0159] Spin for an additional 1 minute at 14 000 RPM (-20 000 g) to complete the DNA elution.

[0160] The purified DNA sample may be stored at 4°C for a few days or it is recommended that samples be placed at -20°C for long-term storage.

[0161] DNA Quantification

[0162] Quantification of extracted DNA was preformed on a NanoDrop as per standard protocols. In particular, the A280 / A260 ratio for detection is:

[0163] - RNA: 1.8-2.2

[0164] - DNA: 1.8-2.0

[0165] Protein: 0.6-1

[0166] Record the concentration and purity values, and clean the pedestal with a small amount of ethanol before measuring the next sample. Targeted

[0167] The DNA samples were prepared for targeted sequencing with the Qt / / c -16S™NGS Library Prep Kit (Zymo Research, Irvine, CA). These primers were custom designed by Zymo Research to provide the best coverage of the 16S gene while maintaining high sensitivity. The primer sets used in this project are Quick-16S™Primer Set V1-V3 (Zymo Research Europe, Freiburg, Germany).

[0168] The sequencing library was prepared using an innovative library preparation process in which PCR reactions were performed in real-time PCR machines to control cycles and therefore limit PCR chimera formation. The final PCR products were quantified with qPCR fluorescence readings and pooled together based on equal molarity. The final pooled library was cleaned up with the Select-a-Size DNA Clean & Concentrator™ (Zymo Research, Irvine, CA), then quantified with TapeStation® (Agilent Technologies, Santa Clara, CA) and Qubit® (Thermo Fisher Scientific, Waltham, WA).

[0169] Control

[0170] The ZymoBIOMICS® Microbial Community Standard (Zymo Research, Irvine, CA) was used as a positive control for each DNA extraction, if performed. The ZymoBIOMICS® Microbial Community DNA Standard (Zymo Research, Irvine, CA) was used as a positive control for each targeted library preparation. Negative controls (i.e. blank extraction control, blank library preparation control) were included to assess the level of bioburden carried by the wet-lab process.

[0171] The final library was sequenced on Illumina® NextSeq™1000 with a P1 reagent kit (600 cycles). The sequencing was performed with 40% PhiX spike-in.

[0172] Bioinformatics

[0173] Unique amplicon sequences (AVSs) were inferred from raw reads using the Dada2 pipeline (Callahan et al. (2016) Nat. Methods., 13:581-583, doi: Chimeric sequences were also removed with the Dada2 pipeline. Taxonomy assignment was performed using Uclust from Qiime v.1.9.1. Taxonomy was assigned with the Zymo Research Database, a 16S database that is internally designed and curated, as reference.

[0174] Composition visualization, alpha-diversity, and beta-diversity analyses were performed with Qiime v.1.9.1 (Caporaso etal. (2010) Nat. Methods, 7(5):335-336, doi: https: / / doi.org. >8 /

[0175] . If applicable, taxonomy that have significant abundance among different groups were identified by LEfSe (Segata et al. (2011) Genome Biol., 12(6):R60, doi: using default settings. Other analyses such as heatmaps, Taxa2SV_deomposer, and PCoA plots were performed with internal scripts.

[0176] Absolute Abundance Quantification

[0177] A quantitative real-time PCR was set up with a standard curve. The standard curve was made with plasmid DNA containing one copy of the 16S gene and one copy of the fungal ITS2 region prepared in 10-fold serial dilutions. The primers used were the same as those used in Targeted Library Preparation. The equation generated by the plasmid DNA standard curve was used to calculate the number of gene copies in the reaction for each sample. The PCR input volume (4pl) was used to calculate the number of gene copies per microliter in each DNA sample.

[0178] The number of genome copies per microliter DNA sample was calculated by dividing the gene copy number by an assumed number of gene copies per genome. The value used for 16S copies per genome is 4. The value used for ITS copies per genome is 200. The amount of DNA per microliter DNA sample was calculated using an assumed genome size of 4.64 x 106 bp, the genome size of Escherichia coli, for 16S samples, or an assumed genome size of 1.20 x 107 bp, the genome size of Saccharomyces cerevisiae, for ITS samples. This calculation is shown below:

[0179] Calculated Total DNA = Calculated Total Genome Copies x Assumed Genome Size (4.64 x 106 bp) x Average Molecular Weight of a DNA bp (660 g / mole / bp) Avogadro’s Number (6.022 x 1023 / mole) x DNA Dilution Factor

[0180] Results

[0181] Results of the microbiome diversity as measured by ASV from the sequencing data (Dada2) and assignment of taxonomy (llclust from Qiime v.1.9.1 and the Zymo Research 16S database) is shown Figures 1-3.

[0182] Figure 1 is a bargraph of the average ASV microbiome diversity as measured in ‘fresh’ (i.e. extracted, not lyophilised or combined) human breast milk, lyophilised (not combined) or the concentrated and combined human breast milk booster product described herein (the Vyarna Bio Information Booster; VBIB). This summary data clearly shows the increased viable microbiome diversity in the booster product (VBIB) compared to ‘fresh’, uncombined human breast milk from any individual provider and the same following lyophilisation. Importantly, while lyophilisation appears to reduce viable microbiome diversity compared to fresh human breast milk, this is increased in the combined VBIB product and such increase is to a significantly higher ASV than in the breast milk from any individual provider. Therefore, the human breast milk booster product described herein (VBIB) comprises significantly greater viable microbiome diversity than either fresh or lyophilised human breast milk obtained for any individual provider.

[0183] Figure 2 shows a representation of the gram-positive (P) and gram-negative (N) bacterial microbiome species composition of each indicated ‘fresh’ (F), lyophilised (L) or combined / mixed (M) samples tested.

[0184] Figure 3 shows a beta diversity plot of principle coordinate analysis (PCoA) of grampositive (P) and gram-negative (N) bacterial microbiome diversity between ‘fresh’ (F) samples as in Figure 2.

Claims

CLAIMS1. A human breast milk booster product, comprising combined breast milk obtained from 2 or more individual providers and around 200 000 or more microbiome sequences present in the product, as determined using an amplicon sequence variant (ASV), exact sequence variant (ESV), zero-radius OTU (ZOTU), sub-OTU (sOTU), haplotype or oligotype method, and wherein the human breast milk booster product is concentrated relative to the breast milk obtained from the individual providers.

2. The human breast milk booster product of claim 1, wherein the 200 000 or more microbiome sequences are sequences from viable microbiota.

3. The human breast milk booster product of claim 1 or claim 2, comprising around 225 000 or more microbiome sequences, in particular around 225 000 or more sequences from viable microbiota.

4. The human breast milk booster product of any one of claims 1 to 3, comprising around 250 000 or more microbiome sequences, in particular around 250 000 or more sequences from viable microbiota.

5. The human breast milk booster product of any one of claims 1 to 4, wherein the human breast milk booster product is concentrated by removing water from the combined and / or obtained breast milk, such as by centrifugation, freezing, skimming and / or lyophilisation.

6. The human breast milk booster product any one of claims 1 to 5, wherein the human breast milk booster product is lyophilised.

7. The human breast milk booster product of any one of claims 1 to 6, wherein the breast milk obtained from the individual providers or human breast milk booster product is not heat treated or pasteurised.

8. The human breast milk booster product of any one of claims 1 to 7, comprising breast milk obtained 3 or more individual providers.

9. The human breast milk booster product of any one of claims 1 to 8, wherein each of the individual providers are from different geographical locations.2210. The human breast milk booster product of any one of claims 1 to 9, wherein the human breast milk booster product is substantially free of viruses.

11. A food product comprising the human breast milk booster product of any one of claims 1 to 10.

12. The food product of claim 11 , wherein the food product is formula milk or human breast milk.

13. Use of the human breast milk booster product of any one of claims 1 to 10 as a food supplement.

14. The use of claim 13, wherein the food to be supplemented is formula milk or human breast milk.

15. The human breast milk booster product of any one of claims 1 to 10, for use in the treatment, amelioration or prevention of under nourishment, malnourishment or malnutrition.

16. A method for producing a human breast milk booster product, the method comprising the steps of:(i) combining breast milk obtained from 2 or more individual providers; and(ii) concentrating the combined breast milk, thereby producing a concentrated human breast milk booster product comprising around 200 000 or more microbiome sequences present in the product, as determined using an amplicon sequence variant (ASV), exact sequence variant (ESV), zero-radius OTU (ZOTU), sub-OTU (sOTU), haplotype or oligotype method, optionally wherein the 200 000 or more microbiome sequences are sequences from viable microbiota.

17. The method of claim 16, wherein the produced concentrated human breast milk booster product comprises around 225 000 or more microbiome sequences, in particular around 225 000 or more sequences from viable microbiota.

18. The method of claim 16 or claim 17, wherein the produced concentrated human breast milk booster product comprises around 250 000 or more microbiome sequences, in particular around 250 000 or more sequences from viable microbiota.

19. The method of any one of claims 16 to 18, wherein the breast milk or human breast milk booster product is not heat treated or pasteurised.

20. The method of any one of claims 16 to 19, wherein concentrating is by removing water from the breast milk, such as by centrifugation, freezing skimming and / or lyophilisation.

21. The method of any one of claims 16 to 20, wherein the method further comprises step (iii) of lyophilising the human breast milk booster product.

22. The method of any one of claims 16 to 21, wherein combining step (i) is performed after concentrating step (ii) or after lyophilising step (iii).

23. The method of any one of claims 16 to 22, wherein breast milk obtained from 3 or more individual providers is combined.

24. The method of any one of claims 16 to 23, wherein each of the individual providers are from different geographical locations.

25. The method of any one of claims 16 to 24, wherein the individual providers are negative for a bacterial and / or viral infection.

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