Soy WHEY-based product enriched with vitamin b12 and method of producing the same

Abstract

The present disclosure relates to a soy whey-based product enriched with vitamin B12 and a method of improving vitamin B12 content in the soy whey-based product. The soy whey-based product comprises a fermented soy whey medium containing bifidobacteria and Propionibacterium sp., wherein the soy whey-based product is enriched with short-chain fatty acids and vitamin B12.

Description

SOY WHEY-BASED PRODUCT ENRICHED WITH VITAMIN B12 AND METHODOF PRODUCING THE SAMECROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority of Singapore application No. 10202303242X filed 16 November 2023, the contents of it being hereby incorporated by reference in its entirety for all purposes.TECHNICAL FIELD

[0002] The present disclosure generally relates to a soy whey-based product and a method of producing the same. In particular, the present disclosure relates to a soy whey-based product enriched with vitamin B12 and a method of producing the same.BACKGROUND

[0003] Plant-based diets, in various forms, have gained increasing popularity in the West. This trend has seen significant growth due to rising concerns about the consumption of animal products and their adverse effects on health and the environment. With a strong worldwide interest in plant-based foods, there has been an increase in vegans from 4 million in 2014 to nearly 20 million in 2017 in the United States of America alone. The adoption of a plant-based diet has been shown to have beneficial effects, including a lower incidence of colon cancer and type 2 diabetes mellitus. Yet, there are concerns about deficiencies in vitamin B12 and other minerals even when a diverse range of plant-based products is consumed. Plant foods do not naturally contain vitamin B 12. Vitamin B 12 is an essential micronutrient in the synthesis of DNA, with severe deficiencies resulting in megaloblastic anemia and dementia. To address the deficiencies, vegans and vegetarians often turn to vitamin B12 supplements or synthetically fortified foods. However, deficiencies may still exist if vegans and vegetarians do not take the supplements on a regular basis. Additionally, improper administration of vitamin B12supplements carries risk of an overdose or elevated B 12 levels, which may lead to side effects such as skin rash, fatigue, etc.

[0004] Hence, an alternative is to explore the creation of healthy plant-based beverages or food products with naturally produced vitamin B12. This could aid in increasing the vitamin B12 intake among vegans and vegetarians, and enriching plant-based meat products with vitamin B12 to meet nutritional requirements.

[0005] It is therefore desirable to provide a plant-based product enriched with vitamin B12 and a method of improving vitamin B12 content in the plant-based product which seek to address at least one of the problems described hereinabove, or at least to provide an alternative.SUMMARY

[0006] In one aspect, the present disclosure relates to a soy whey- based product comprising a fermented soy whey medium containing bifidobacteria and Propionihacterium sp., wherein the soy whey-based product is enriched with short-chain fatty acids and vitamin B12

[0007] In some embodiments, the soy whey-based product contains at least 4.94 pg / L to 9.91 pg / L of vitamin B12.

[0008] In another aspect, the present disclosure relates to a method of improving vitamin B 12 content in a soy whey-based product. The method comprises inoculating a soy whey medium with bifidobacteria and Propionihacterium sp. ; and fermenting the inoculated soy whey medium to obtain a soy whey-based product enriched with short-chain fatty acids and vitamin B 12.

[0009] In some embodiments, the step of inoculating further comprises co-culturing the bifidobacteria with the Propionihacterium sp.

[0010] In some embodiments, the step of inoculating comprises simultaneous inoculating the soy whey-based medium with the bifidobacteria and the Propionihacterium sp.

[0011] In some embodiments, the step of inoculating comprises inoculating the soy wheybased medium sequentially with the bifidobacteria and the Propionihacterium sp.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Various embodiments of the present disclosure are described hereinbelow in the detailed description with reference to the following drawings:FIG. 1A shows changes in B. lactis Bl-04 cell count during fermentation. (■•■): lactis Bl-04 fermented soy whey;); P. freudenreichii DSM 20271 fermented soy whey; ( “A-); B. lactis Bl-04 followed by P. freudenreichii DSM 20271 sequentially fermented soy whey; (“& ): P. freudenreichii DSM 20271 followed by B. lactis Bl-04 sequentially fermented soy whey; ( ): B. lactis Bl-04 and P. freudenreichii DSM 20271 simultaneously fermented soy whey. Error bars represent mean ± SD, and significant differences within days were determined by one way ANOVA within days, where *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001.FIG. 1B shows changes in P. freudenreichii DSM 20271 cell count during fermentation.B. lactis Bl-04 fermented soy whey; ("♦” ): P. freudenreichii DSM 20271 fermented soy whey; ( -Ar); B. lactis Bl-04 followed by P. freudenreichii DSM 20271 sequentially fermented soy whey; ): P. freudenreichii DSM 20271 followed by B. lactis Bl-04 sequentially fermented soy whey; ( ® ): B. lactis Bl-04 and P. freudenreichii DSM 20271 simultaneously fermented soy whey. Error bars represent mean ± SD, and significant differences within days were determined by one way ANOVA within days, where *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < Q.0001.FIG. 1C shows changes in pH in soy whey during fermentation. ("•"): B. lactis Bl-04 fermented soy whey; (■'♦" ); P. freudenreichii DSM 20271 fermented soy whey; ( ■A~): B. lactis Bl-04 followed by P. freudenreichii DSM 20271 sequentially fermented soy whey; ( ”§§“ ): P. freudenreichii DSM 20271 followed by B. lactis Bl-04 sequentially fermented soy whey; (~®“ ): B. lactis Bl-04 and P. freudenreichii DSM 20271 simultaneously fermented soy whey. Error bars represent mean ± SD, and significant differences within days were determined by one way ANOVA within days, where *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001.FIGs. 2A - 2C show changes in sugars in soy whey during fermentation. (FIG. 2A: fructose; FIG. 2B: glucose; FIG. 2C: sucrose). ("•■): B. lactis Bl-04 fermented soy whey; ("♦“ ): P. freudenreichii DSM 20271 fermented soy whey; ( 'A- ): B. lactis Bl-04 followed by P. freudenreichii DSM 20271 sequentially fermented soy whey; ( >8 ): P. freudenreichii DSM 20271 followed by B. lactis Bl-04 sequentially fermented soy whey; (*Ab ): B. lactis Bl-04 and P. freudenreichii DSM 20271 simultaneously fermented soy whey. Error bars represent mean ± SD, and significant differences within days were determined by one way ANOVA within days, where *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001.FIGs. 3A - 3C show changes in free organic acids in soy whey during fermentation. (FIG. 3A: Acetic acid; FIG. 3B: Lactic acid; FIG. 3C: Propionic acid). (”®~): B. lactis Bl-04 fermented soy whey;): P. freudenreichii DSM 20271 fermented soy whey; ( "Ar): B. lactis Bl-04 followed by P. freudenreichii DSM 20271 sequentially fermented soy whey; ("®* ): P. freudenreichii DSM 20271 followed by B. lactis Bl-04 sequentially fermented soy whey; (~^~ ): B. lactis Bl-04 and P. freudenreichii DSM 20271 simultaneously fermented soy whey. Error bars represent mean ± SD, and significant differences within days were determined by one way ANOVA within days, where *: P < 0.05, **: P < 0.01 , ***: P < 0.001, ****: P < 0.0001 .FIG. 4 shows heatmap of changes in amino acids in soy whey. Colour scale represents the intensities of substrates and metabolites, with red representing high concentrations, while blue represents low concentrations. UF: unfermented soy whey; B: B. lactis Bl-04 fermented soy whey; P: P. freudenreichii DSM 20271 fermented soy whey; BP: B. lactis Bl-04 followed by P. freudenreichii DSM 20271 sequentially fermented soy whey; PB: P. freudenreichii DSM 20271 followed by B. lactis Bl-04 sequentially fermented soy whey; BnP: B. lactis Bl-04 and P. freudenreichii DSM 20271 simultaneously fermented soy whey.FIG. 5 shows vitamin B12 changes in soy whey during fermentation. (“Bk): B. lactis Bl-04 fermented soy whey; ( ♦' ); p. freudenreichii DSM 20271 fermented soy whey; ( “Ar); B. lactis Bl-04 followed by P. freudenreichii DSM 20271 sequentially fermented soy whey; (“&): P. freudenreichii DSM 20271 followed by B. lactis Bl-04 sequentially fermented soy whey; ( "W“ ): B. lactis Bl-04 and P. freudenreichii DSM 20271 simultaneously fermented soy whey. Error bars represent mean ± SD, and significant differences within days were determined by one way ANOVA within days, where *: P < 0.05, **: P < 0.01, ***: P < 0.001, *”*: P < 0.0001. FIG. 6 shows principal component analysis biplot for non-volatile compounds of soy whey before and after fermentation. (□): Unfermented soy whey; ( 'W ): B. lactis Bl-04 fermented soy whey; (*♦"); P. freudenreichii DSM 20271 fermented soy whey; ("^”): B. lactis Bl-04 followed by P. freudenreichii DSM 20271 sequentially fermented soy whey;P. freudenreichii DSM 20271 followed by B. lactis Bl-04 sequentially fermented soy whey; ("&): B. lactis Bl- 04 and P. freudenreichii DSM 20271 simultaneously fermented soy whey.DESCRIPTION

[0013] The following description sets forth exemplary methods, parameters, and the like. The embodiments are described in sufficient detail to enable those skilled in the art to practise the invention. Other embodiments may be utilized, and structural and logical changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

[0014] Features that are described in the context of an embodiment may correspondingly be applicable to the same or similar features in the other embodiments. Features that are described in the context of an embodiment may correspondingly be applicable to the other embodiments, even if not explicitly described in these other embodiments. Furthermore, additions and / or combinations and / or alternatives as described for a feature in the context of an embodiment may correspondingly be applicable to the same or similar feature in the other embodiments.

[0015] In the context of various embodiments, the articles “a”, “an” and “the” as used regarding a feature or element include a reference to one or more of the features or elements.

[0016] In the context of various embodiments, the term “about” or “approximately” as applied to a numeric value encompasses the exact value and a reasonable variance, e.g. within 20% of the specified value.

[0017] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0018] By “comprising” it is meant including, but not limited to, whatever follows the word “comprising”. Thus, use of the term “comprising” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.

[0019] By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of’. Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0020] In one aspect, the present disclosure relates to a soy whey-based product comprising a fermented soy whey-based medium containing bifidobacteria and Propionibacterium sp., wherein the soy whey-based product is enriched with short-chain fatty acids and vitamin B12.

[0021] The term “soy whey-based medium” as used herein refers to the soy whey generated from the tofu and soy protein making process that contains necessary nutrients for supporting the growth of microorganism. The soy whey-based medium can be prepared in liquid, semisolid and solid form. In one exemplary embodiment, the soy whey-based medium is in liquid form.

[0022] In an exemplary embodiment, soy whey is the medium used for fermentation without the addition of reducing agents, such as cysteine, cysteine-HCl and ascorbic acid.

[0023] In some embodiments, the soy whey-based product contains at least 4.94 pg / L to 9.91 pg / L of vitamin B12. The concentration of the vitamin B12 will be dependent on the nutrients in the different sources of soy whey.

[0024] In some embodiments, the soy whey-based product contains bifidobacteria with a cell count ranges from 7 log CFU / mL to 8.2 log CFU / mL.

[0025] In some embodiments, the Propionibacterium sp. is selected from the species Propionibacterium freudenreichii. In further embodiments, the Propionibacterium sp. is a strain selected from Propionibacterium freudenreichii DSM 20271 (DSM 20271, also referred to as propionic acid bacteria “PAB”). The strain of Propionibacterium freudenreichii DSM 20271 is publicly available.

[0026] Tn some embodiments, the bifidobacteria is selected from the species Bifidobacterium animalis subsp. lactis. In further embodiments, the bifidobacterium is a strain selected from Bifidobacterium animalis subsp. lactis Bl-04 (Bl-04). The strain of Bifidobacterium is publicly available. Selected species can encompass other species including, but are not limited to, Bifidobacterium breve. Bifidobacterium longum, Bifidobacte riurn infanlis, and Bifidobacterium bifidum.

[0027] In some embodiments, the short-chain fatty acids are acetic acid and propionic acid.

[0028] In some embodiments, the soy whey-based product contains acetic acid of at least 2.9 g / L.

[0029] In some embodiments, the soy whey-based product contains propionic acid of at least 1.8 g / L.

[0030] In some embodiments, the concentration of short-chain fatty acids ranges from 1 g / L to 4.5 g / L, depending on the nutrients in the different sources of soy whey.

[0031] In another aspect, the present disclosure relates to a method of improving vitamin B 12 content in a soy whey-based product. The method comprises inoculating a soy whey medium with bifidobacteria and Propionibacterium sp.', and fermenting the inoculated soy whey medium to obtain a soy whey-based product enriched with short-chain fatty acids and vitamin B 12.

[0032] In some embodiments, the soy whey medium is subjected to pasteurization prior to inoculation with the bifidobacteria and the Propionibacterium sp. In some embodiments, the pasteurization process is carried out at a temperature ranging from 85 °C to 95 °C, from 89 °C to 92 °C or at about 90 °C for 5 to 60 minutes. In some embodiments, the pasteurization processis carried out at a higher temperature for a shorter duration. In some exemplary embodiments, the pasteurization process is carried out at temperature ranging from 120 °C to 150 °C or 138 °C to 150 °C, for several seconds or for 1 to 2 seconds.

[0033] In some embodiments, the step of inoculating the soy whey medium comprises coculturing the bifidobacteria with Propionibacterium sp.

[0034] Tn some embodiments, the step of inoculating the soy whey medium comprises simultaneous inoculating the soy whey medium with the bifidobacteria and the Propionibacterium sp. The bifidobacteria and the Propionibacterium sp. can be used in its provided or original form. In an exemplar}' embodiment, the bifidobacteria and the Propionibacterium sp. are sub-cultured in suitable nutrient broth, such as de Man, Rogosa and Sharpe broth supplemented with 0.05% L-cysteine HO (MRSC) and yeast extract lactate (YEL) broth, respectively, to achieve desired cell count as a starter culture to inoculate the soy whey medium.

[0035] In some embodiments, the step of inoculating the soy whey medium comprises inoculating the soy whey medium sequentially with the bifidobacteria and the Propionibacterium sp. In further embodiments, the step of inoculating the soy whey medium comprises inoculating the soy whey medium with the bifidobacteria to allow propagation for at least two days prior to inoculation with the Propionibacterium sp. In an alternate embodiment, the step of inoculating the soy whey medium further comprises inoculating the soy whey medium with the Propionibacterium sp. to allow propagation for at least six days prior to inoculation with the bifidobacteria.

[0036] In some embodiments, the soy whey-based product contains at least 4.94 pg / L to 9.91 pg / L of vitamin B12. This is resulting from the fermentation of the soy whey medium with the bifidobacteria and the Propionibacterium sp.

[0037] In various embodiments, the soy whey-based product contains bifidobacteria with a cell count ranges from 7 log CFU / mL to 8.2 log CFU / mL. Tn some embodiments, thebifidobacteria cell count reaches 7.8 log CFU / mL. In some embodiments, the bifidobacteria cell count reaches 8.2 log CFU / mL when the soy whey medium is inoculated with the bifidobacteria and the Propionibacterium sp. simultaneously.

[0038] In some embodiments, the method further comprises adjusting the pH of the soy whey medium 6.5 prior to inoculation. In some embodiments, the pH may be adjusted to within 5 to 8, 6 to 7.

[0039] In some embodiments, the step of fermenting the soy whey medium is conducted for up to 8 days. The duration of fermentation may be further adjusted for at least 3 to 5 days or at least 6 to 8 days.

[0040] In some embodiments, the step of fermenting the soy whey medium is conducted at 30 °C. In some embodiments, the fermentation can be carried out at a temperature ranging from 25 °C to 41 °C.

[0041] In some embodiments, the Propionibacterium sp. is selected from the species Propionibacterium freudenreichii. In further embodiments, the Propionibacterium sp. is a strain selected from Propionibacterium freudenreichii DSM 20271.

[0042] In some embodiments, the bifidobacteria is selected from the species Bifidobacterium animalis subsp. lactis. In further embodiments, the bifidobacterium is a strain selected from Bifidobacterium animalis subsp. lactis Bl-04 (Bl-04).

[0043] In some embodiments, the short-chain fatty acids are acetic acid and propionic acid.

[0044] In some embodiments, the soy whey-based product contains acetic acid of at least 2.9 g / L. In some embodiments, the soy whey-based product contains propionic acid of at least 1 .8 g / L.

[0045] In a further aspect, the present disclosure relates to a plant-based beverage comprising the soy whey-based product of the present disclosure. In yet another aspect, the present disclosure relates to a plant-based food product comprising the soy whey-based product of the present disclosure.

[0046] Advantageously, the method of the present disclosure can be applied to producing plant-based beverages enriched with vitamin B 12. It can also be applied to produce supplements or ingredients for use in producing plant -based meat products with an enhanced levels of vitamin B12. The inventors have surprisingly found that coculturing bifidobacteria and the Propionibacterium sp. improve vitamin B 12 contents in soy whey by fermentation. The inventors have also surprisingly found that certain bifidobacteria exhibit excellent growth when cocultured with Propionibacterium freudenreichii. The increase in vitamin B12 resulting from the fermentation of soy whey with bifidobacteria and the Propionibacterium sp. makes it possible for the development of beverages or functional ingredients to supplement dietary choices that may lack these nutrients. In an exemplary embodiment, a single serving of 375 mL of the plantbased beverage comprising the fermented soy whey-based product of the present disclosure is sufficient to provide the recommended daily intake of vitamin Bi 2. Furthermore, the fermented soy whey-based beverage contains bifidobacteria with cell count over 1,000,000 cells. The presence of both probiotics and postbiotics may help consumers to achieve various health benefits.

[0047] Furthermore, the fermentation process fully utilizes the soy whey generated from the tofu and soy protein making process. This helps to reduce waste from food manufacture.

[0048] To facilitate a better understanding of the present disclosure, the following examples of specific embodiments are given. In no way should the following examples be read to limit or define the entire scope of the disclosure. One skilled in the art will recognize that the examples set out below are not an exhaustive list of the embodiments of this disclosure.EXAMPLESExample 1

[0049] Bifidobacterial Cultivation and Enumeration

[0050] A freeze-dried culture of Bifidobacterium animalis subsp. laclis Bl-04 (Danisco,Copenhagen, Denmark) was reconstituted in sterilized peptone water (0.1% w / v peptone, OxoidLtd., Hampshire, UK), streaked on to de Man, Rogosa and Sharpe agar supplemented with 0.05% L-cysteine HC1 (MRSC) (Sigma Aldrich, St. Louis, MO, USA) and incubated anaerobically using anaerogens at 37 °C for 2 days to obtain pure colonies. Individual colonies were isolated and statically propagated in MRSC broth at 37 °C for 2 days before glycerol was added (final 15% v / v) and stored at -80 °C until use. Prior to inoculation into soy whey, frozen cultures were thawed and twice sub-cultured in MRSC broth at 37 °C for 24 h each. Cell pellets were then washed twice with a 0.85% w / v saline solution before being resuspended in soy whey and inoculated into soy whey.

[0051] Propionic Acid Bacterial Culture Preparation

[0052] Freeze-dried cultures of Propionibaclerium freudenreich.il (DSM 20271) was purchased from DSM (Heerlen, Netherlands). Freeze-dried DSM 20271 was reconstituted in yeast extract lactate (YEL) broth (1 g of tryptone (Oxoid), 1.67 g of 60 % sodium lactate syrup (Sigma Aldrich), 1 g of yeast extract (Oxoid), 0.25 g of KH2PO4 (Sigma Aldrich), and 0.0005 g of MgSO4 (Merck, Darmstadt, Germany) in 1 L of deionised water, adjusted to pH 7.0 with 2.5 M NaOH before being streaked on YEL agar (1.5% w / v agar) and incubated anaerobically for 6 days at 30 °C. Individual colonies were isolated and statically propagated in YEL broth for 4 days at 30 °C, then 15% glycerol (v / v) was added, and all pure cultures were stored at -80 °C until use. Prior to inoculation into soy whey, frozen cultures were thawed and twice sub-cultured in YEL broth at 30 °C for 3 days each. Cell pellets were then washed twice with 0.85% saline solution before resuspending in soy whey and inoculated into soy whey.

[0053] Preparation of Soy Whey Medium

[0054] Preparation of soy whey was done as described by Chua et al. (2018). Briefly, soybeans were soaked overnight in 1:6 (w / v) deionized water (beans: water). Soybeans were then strained and blended using a blender (Philips Blender Core Series 5000, Singapore) with deionized water in 1 :6 dry weight to volume ratio to obtain a slurry. Soymilk was filtered through a cheesecloth to separate the soy pulp. Then, 1 :2 (dry soybean weight: water volume) ofdeionized water was used to wash the soy pulp and the flow-through was pooled with the soymilk. The soymilk was then brought to the boil with continuous stirring and held for 5 min. The soymilk was cooled down to 87 °C before 2% (relative to soybean dry weight) gypsum (Home Brew' Ohio, Sandusky, United States) suspended in water in 10% ratio (w / v) was added to it. The soymilk and gypsum mixture was well stirred and left to curdle for 15 min before the coagulated tofu curd was pressed to release the soy w'hey. Soy whey w'as adjusted to 6.5 pH using 1 M potassium hydroxide and pasteurized at 105 °C for 30 min.

[0055] Lab Scale Fermentation

[0056] Five different static fermentation set-ups were prepared: a monoculture of B. lactis Bl-04 (B), a monoculture of P. freudenreichii DSM 20271 (P), a sequential culture of Bl-04 — ► DSM 20271 (BP), a sequential culture of DSM 20271 —>■ Bl-04 (PB), a simultaneous culture of DSM 20271 and Bl-04 (B&P). For the five fermentation set-ups, the inoculated soy whey w'as decanted in 40-mL aliquots into 50-mL centrifuge tubes. The tubes were kept at 30 °C for 8 days of fermentation. BP soy whey was first fermented with Bl-04 for two days, before inoculation with DSM 20271. PB was first fermented with DSM 20271 for 6 days, before inoculation with Bl-04. Whole culture sampling was done daily, cell counts, °Brix, and pH measurements w'ere done immediately, and samples were kept at - 20 °C until further analysis.

[0057] Sugar, Organic Acid and Isoflavone Analyses

[0058] Samples w'ere centrifuged at l l .OOOxg and filtered through a 0.22-pm syringe membrane prior to HPLC analysis. Sugars and organic acids were analysed using HPLC, coupled to refractive index detector (RTD) or a photo diode array (PDA) detector, respectively. The sugars analysed included sucrose, fructose, and glucose using a Zorbax carbohydrate column (150 x 4.6 mm, Agilent, Santa Clara, CA, USA). Organic acids were analysed w'ith a Supelco Gel C610H column (300 x 7.8 mm, Supelco, Bellefonte, PA, USA). HPLC coupled to PDA was used for isoflavone analysis, with a Zorbax Eclipse Plus C18 column (150 x 4.6 mm,Agilent). Analytes were quantified through external standard curves with R2> 0.99. Details ofHPLC parameters were based on Chua et al. (2018).

[0059] Amino Acid Analysis

[0060] A pre-set physiological chromatography program was used to analyse free amino acids on an ARACUS Amino Acid Analyzer (MembraPure, Berlin, Gennany) as per the manufacturer’s instructions. Unfermented and fermented samples were treated with 10% (v / v) sulfosalicylic acid for protein precipitation. Separation was done on a lithium cation exchange column with a post column derivatization with ninhydrin.

[0061] Vitamin B12 Analysis

[0062] Vitamin B12 analysis followed the methodology of Tindjau et al. (2023b). Briefly, 6 mL of a sample was transferred to a 15-mL centrifuge tube along with 0.1 mL of a 1 % KCN solution and 2.5 mL of a 0.4 M sodium acetate (pH 4) solution. The samples were then vortexed and autoclaved at 100 °C for 30 min before being cooled in an ice bath. Before extraction, samples were centrifuged at 1 l,000xg at 4 °C. Oasis HLB SPE columns (3-cc, 60-mg bed mass, Waters, MA, USA) were equilibrated with 2 mL of methanol, followed by 2 mL of distilled H2O. The sample was then loaded into the cartridge, followed by 2 mL of 5% (v / v) methanol wash and 1 mL of 90% methanol elution. Extracted samples were then filtered through a 0.22- pm syringe fdter and stored in a 2-tnL amber vial before analysis. The sample analysis was done using HPLC (Shimadzu, Kyoto, Japan) coupled with PDA. A Zorbax Eclipse Plus C18 column (150 x 4.6 mm; Agilent) was used for separation with gradient elution with a flow rate of 0.25 mL / min and column temperature at 40 °C with PDA set at 550 nm.

[0063] Statistical Analysis

[0064] Data were evaluated statistically using R-studio (R Foundation for Statistical Computing, Vienna, AT) for statistical significance (P < 0.05). One-way ANOVA (analysis of variance) was conducted with Tukey ’ s post hoc test to determine differences between treatments.Experimental data from two independent fermentations in duplicate (n = 4) were expressed as the mean ± standard deviation.Example 2

[0065] Changes in Bacterial Cell Counts

[0066] FIGs. 1A and IB show the changes in cell counts of bifidobacteria and PAB throughout soy whey fermentation. During the fermentation, PAB DSM 20271 grew well in soy whey, regardless of single or mixed cultures, as evident in the increases in cell counts. Interestingly, PAB in the B&P co-culture showed slightly slower growth from day 2-4 compared to PB and P, probably due to nutrient competition from bifidobacteria. By day 7, the PAB counts fell in the treatments of P, BP and B&P. The PAB cell counts in BP treatment did not decrease after day 7 possibly due to the 2 days’ delay in inoculation. Bifidobacterial strain Bl-04 in the monoculture did not grow and was no longer detectable after 6 days. In contrast, Bl-04 cell count stayed relatively constant in both sequential cultures (BP & PB). Although the viable counts of bifidobacterial Bl-04 were lower in the sequential culture treatments, Bl-04 maintained at 5.9 log CFU / mL throughout fermentation. In the simultaneous coculture, bifidobacterial Bl-04 cell counts grew beyond 7 log CFU / mL, peaking at 8.2 log CFU / mL at day 3 then dropping slightly to 7.8 log CFU / mL by day 8, from an initial cell count of 5.8 log CFU / mL. The growth of PAB in both mono- and cocultures is comparable to that in a previous study, whereby day 5, DSM 20271 reached the stationary phase with 10.73 ± 0.29 log CFU / mL in the present disclosure (Tindjau et al., 2023b). The slower growth of PAB from day 2 4 in the B&P co-culture was likely due to competition with Bl-04. Bifidobacteria exhibited clear and distinct growth patterns in different culture regimes with the monoculture having no growth and declining cell counts, the sequential cultures displaying a steady viable cell count, and the simultaneous culture revealing exponential growth. The lack of bifidobacterial growth in the monoculture could be par tially ascribed to the incubation temperature of 30 °C, instead of the optimum temperature of37 °C. Bifidobacteria were able to grow in soymilk at 30 °C, albeit slower compared to 37 °C.However, the minimal nutrients especially deficiency of bifidogenic factors in soy whey might be more likely responsible for the inability of bifidobacteria to grow in this medium. In the BP and PB sequential cultures, however, the presence of PAB prevented the dramatic decline of bifidobacteria in contrast to the bifidobacterial monoculture, indicating the beneficial effect of PAB on bifidobacterial survival. The significant improvement in bifidobacterial growth when grown simultaneously with PAB provided definitive evidence on the growth stimulation conferred by PAB on bifidobacteria.Example 3

[0067] Changes in pH, sugars, and organic acids

[0068] Changes in pH during fermentation are shown in FIG. 1C, while changes in sugars and organic acids are shown in FIGs. 2A-2C and 3A-3C, respectively. The bifidobacterial monoculture showed the least pH changes (pH 6.5 to pH 6) throughout the fermentation, followed by the PAB monoculture (pH 6.5 to pH 5), then sequential cultures (pH 6.5 to pH 5). The B&P co-culturc showed the sharpest decline in pH to pH 4.2. The insignificant pH decrease in the bifidobacterial monoculture was attributed to the lack of growth and early death of bifidobacteria. The pH reduction in both the DSM 20271 monoculture and sequential cultures correlated with the consumption of glucose and fructose as well as corresponding acid production. B&P coculturing resulted in significant sucrose reduction, suggesting elevated bifidobacterial activities through coculturing and the production of glycosidase, sucrase and / sucrose phosphorylase. The monoculture of PAB did not change the concentration of sucrose, as PAB do not have the ability to utilize sucrose. However, when PAB was gro 'n with Bl-04 simultaneously, sucrose significantly decreased from 2.14 ± 0.1 g / L to 0.78 ± 0.0 g / L. This was likely due to the saccharolytic activity of [3-fructofuranosidase (EC 3.2.1.26) in B. lactis. The decreases in pH resulted from the production of organic acids such as acetic acid, lactic acid, and propionic acid as a result of sugar consumption. Lactic acid showed a small but steady increase in the bifidobacterial monoculture (0.06 g / L), while it was not detected afterfermentation in other culture modes. Lactic acid was detected until day 2 in the BP sequential coculture, however, it was not detected 1 day after inoculation with PAB due to the conversion of lactic acid to propionic acid by PAB. Propionic acid was only detectable in samples inoculated with DSM 20271, with the B&P co-culture having significantly more propionic acid at 1.8 g / L compared to other culturing methods. PAB monoculture, BP and PB sequential cultures showed a similar trend in propionic acid accumulation with concentrations plateauing 3 days after PAB inoculation. Acetic acid showed a similar trend, and the B&P co-culture had the highest acetic acid level at 2.9 g / L.

[0069] Lactic acid and acetic acid are produced by bifidobacteria through the bifidus (fructose 6-phosphate) shunt. Bifidobacteria have an incomplete TCA cycle, with the inability to utilize citric acid and malic acid, and their concentrations did not change after fermentation. On the other hand, malic acid and citric acid were utilized in PAB monoculture and other culturing treatments. While some organic acids can be utilized in the TCA cycle by PAB, lactic acid is the preferred carbon source for PAB. Lactic acid formed from bifidobactcrial metabolism is utilized by PAB in the Wood-Werkman cycle to produce propionic acid. Overall, the B&P coculture had significantly more acids compared to the other 4 treatments. The improved growth of bifidobacterial via coculturing with PAB likely led to increased utilization of sugars and TCA cycle acids, resulting in higher acid production by the co-metabolism of PAB and bifidobacteria.Example 4

[0070] Changes in Free Amino Acids

[0071] The free amino acid contents in fermented and fermented soy whey are summarized in FIG. 4. The total free amino acids showed a significant decrease after fermentation in the PAB monoculture, BP and PB sequential cultures, and B&P co-culture. The bifidobacterial monoculture did not show changes in the total nor individual free amino acids, except for methionine. Methionine was suggested to be utilized in a reverse transsulfuration pathway to create cysteine and cystathionine by bifidobacteria. Cysteine and cystathionine did not changein the bifidobacterial monoculture, PAB monoculture, BP and PB sequential cultures. However, in the B&P co-culture, there was a significant decrease in cysteine, with an increase in cystathionine. The growth of bifidobacteria supports the hypothesis that cysteine, cystathionine and methionine could be linked through the transsulfuration pathway in bifidobacteria. Coculturing techniques have been employed to improve nutritional contents. However, coinoculation has not always been advantageous compared to monoculturing. Santos et al. (2014) reported a decrease in amino acids in co-cultured ferments, with an increase in monoculture. Our results show a dramatic decrease in the majority of essential amino acids (histidine, isoleucine, leucine, methionine and phenylalanine) in B&P co-culture. Valine interestingly increased in B&P co-culture, and lysine increased in all cultures that involved P. freudenreichii. These observations were previously reported, where B. longum BB536 fermentation of soy whey increased the valine content, and PAB fermentation of soy whey improved the lysine content (Tindjau et al., 2023a, 2023b). Amino acids such as glycine, and glutamic acid have been linked to vitamin B12 biosynthesis. Glycine is a precursor to 5 -aminolevulinic acid (ALA), a key compound in the heme biosynthetic pathway (Shemin pathway) formed through the condensation between glycine and succinyl -Co A. ALA can also be produced through the C5 pathway, where all glutamic acid carbon is incorporated in ALA. The utilization of these amino acids in P, BP, PB and B&P treatments could be attributed to vitamin B12 metabolism / biosynthesis by these bacterial cultures in soy whey.Example 5

[0072] Changes in isoflavonesTotal and individual isoflavone changes are presented in Table 1. All five culturing treatments reduced the total isoflavone glycosides, where the bifidobacterial monoculture led to 50% reduction, PAB monoculture, BP and PB sequential cultures and B&P co-culture had at least 90% reduction. Isoflavone aglycones showed no significant changes compared to the unfermented soy whey (10.80 ± 0.78 mg / L) in the bifidobacterial monoculture and B&P co-culture, while inthe PAB monoculture, BP and PB sequential cultures, aglycones significantly increased to 24.43 ± 2.09 mg / L, 18.80 ± 2.63 mg / L, and 19.28 ± 0.80 mg / L, respectively. The reduction in isoflavone glycosides and an increase in isoflavone aglycones suggest the conversion of glycosides to aglycones catalysed by 0-glycosidase, cleaving the glycosidic bond (Chua et al., 2018). One unanticipated result was the insignificant change in isoflavone aglycones in the B&P co-fermented soy whey after fermentation. A possible explanation is the catabolism of isoflavones to bacterial equol. Mustafa et al. (2020) demonstrated that B. longum BB536 and B. breve ATCC 15700 produced equol within 12 h of soymilk fermentation, and by 24 h, isoflavone aglycone levels started decreasing. The co-culture of PAB DSM 20271 with bifidobacterial Bl- 04 might have led Bl-04 to convert isoflavone aglycones, particularly daidzein to equol.Table 1Isoflavone and vitamin B12 contents in soy whey before and after fermentation with single or mixed cultures of B. lactis Bl-04 and P. freudenreichii DSM 20271.a-e Statistical analysis using ANOVA (n = 4) at 95% confidence interval with Tukey’s post hoc test.a(B): B. lactis Bl-04 fermented soy whey; (P): P. freudenreichii DSM 20271 fermented soy whey; (BP): B. lactis Bl-04 followed by P. freudenreichii DSM 20271 sequentially fermented soy whey; (PB): P. freudenreichii DSM 20271 followed by B. lactis Bl-04 sequentially fermented soy whey; (B&P): B. lactis Bl-04 and P. freudenreichii DSM 20271 simultaneously fermented soy whey.Example 6

[0073] Production of vitamin B12

[0074] FIG. 5 shows the vitamin B12 content in fermented soy whey during co-culturing or monoculturing of PAB DSM 20271 and bifidobacterial Bl-04. Bifidobacteria fermented soy whey did not produce vitamin B 12 throughout the fermentation, and this could be a result of the lack of growth. Vitamin B12 was at detectable levels 2 days after inoculation with DSM 20271. PAB accumulated vitamin B12 of up to 6 pg / L after 6 days, and by day 8 there was not significantly more vitamin B12 production. The BP sequential fermentation produced a comparable amount of vitamin B 12 to that in the PAB monoculture by the end of fermentation. Interestingly, the PB sequential fermentation increased B12 content from day 6 to day 8. Furthermore, the B&P simultaneous fermentation generated 30% more vitamin B 12 compared to all other treatments. Co-culturing lactic acid bacteria (LAB) and PAB has been previously researched. Hugenschmidt et al. (2011) and Xie et al. (2019) showed that co-culturing did not improve the vitamin B12 content compared to single PAB cultures. PAB production of vitamin B12 has been shown to be improved through the addition of cobalt, riboflavin, nicotinamide and 5,6-dimethylbenzimidazole (DMBI) (Chamlagain et al., 2016). Cobalt is the important mineral in the center of B12. Riboflavin has been postulated to be converted to DMBI, a lower ligand in B12 and utilized in the production of vitamin B12. While bifidobacteria can also produce B12, its production may be limited by the necessity of an anaerobic environment and the addition ofcysteine (Tindjau et al., 2023c). In a closed system, PAB may create an anaerobic environment through CO2 production, but it is more likely that bifidobacteria produce the precursors to Bn, leading to an increase in Bn content. The similar contents of Bn in single PAB and sequential cultures suggest that sequential culturing did not provide an advantage to vitamin B production.

[0075] Chamlagain et al. (2016, 2018) demonstrated a strain-dependent effect of riboflavin and nicotinamide supplementation (27 mM) in a cheese whey-based medium that led to an increase in vitamin Bn production by PAB. B. lactis was reported to be incapable of producing riboflavin, but it can produce nicotinamide (Deguchi et al., 1985). This prompted us to hypothesize the possibility of nicotinamide production by Bl-04, thereby stimulating riboflavin production by PAB (Piwowarek et al., 2018), hence, indirectly facilitating vitamin Bn production by PAB. However, the level of nicotinamide production by Bl-04 will need to be assessed to prove this hypothesis. A more likely explanation could be the better growth of Bl- 04 in simultaneous culture that led to Bl-04 production of vitamin B - Piwowarek et al. (2022) also evaluated the utilization of apple pomace and potato wastewater for DSM 20271 and achieved vitamin B values ranging from 90 to 290 pg / 100 g of cell biomass. The presence of B vitamins and trace elements, especially riboflavin, nicotinamide, and cobalt likely increased Bn productivity by DSM 20271 in apple pomace and potato wastewater. Therefore, soy whey should be examined for key micronutrients in vitamin Bn production. Improvements in Bn productivity by PAB could be promoted by incorporating food side-streams that are reservoirs of riboflavin, nicotinamide, and cobalt.

[0076] Example 7

[0077] Principal component analysis

[0078] A principal component analysis (PCA) was carried out using total sugars, lactic acid, acetic acid, propionic acid, total amino acids, total isoflavone aglycones, total isoflavone glycosides, and vitamin Bn- The PCA biplot is presented in FIG. 6 where PCI accounted for65.21 %, and PC2 for 21 .83%. There was clear separation between samples, particularly betweenthe B&P co-culture and the other samples. The unfermented sample and bifidobacterial monoculture did not show distinct separation, while the PAB monoculture, PB and BP sequential cultures clustered together. The separation between the B&P co-culture and the other samples was driven by the levels of acetic acid, propionic acid, and vitamin B12. For the bifidobacterial monoculture and unfermented cluster, it was driven by total isoflavone glucosides, lactic acid, and free amino acids. However, the separation of the PAB monoculture, BP and PB sequential cultures from other samples was led by total isoflavone aglycones.

[0079] Conclusions

[0080] The Examples and results demonstrated the viability of utilizing B. lactis Bl-04 and P. freudenreichii DSM 20271 by co-culturing to improve growth of Bl-04, and production of vitamin B12 and short-chain fatty acids in soy whey. In monocultures, Bl-04 was unable to grow nor survive at above 4 log CFU / mL within 4 days. In co-culture with PAB, bifidobacteria exhibited adequate growth at over 7 log CFU / mL. Co-culturing also improves the vitamin B12 content, at over 8 pg / L. Short-chain fatty acids such as acetic acid and propionic acid were greatly improved in cocultured soy whey, compared to mono- and sequential cultures, suggesting a mutualistic relationship. Co-culturing could improve not only the probiotic viability, but also the metabolite content in mix-cultured soy whey.

[0081] Although embodiments of the invention have been shown and described, the invention is not limited to the described embodiments. Instead, it would be appreciated by those skilled in the art that various modifications and variations can be made to the embodiments of the invention without departing from the scope of the invention, the scoop of which is set forth in the following claims.

Claims

CLAIMS1. A soy whey-based product comprising: a fermented soy whey medium containing bifidobacteria and Propionibacterium sp., wherein the soy whey-based product is enriched with short-chain fatty acids and vitamin Bn2. The soy whey -based product of claim 1, wherein the soy whey-based product contains at least 4.94 pg / L to 9.91 pg / L of vitamin B12.

3. The soy whey-based product of claim 1 , wherein the bifidobacteria has a cell count ranges from 7 log CFU / mL to 8.2 log CFU / mL.

4. The soy whey-based product of claim 1, wherein the Propionibacterium sp is Propionibacterium freudenreichii.

5. The soy whey-based product of claims 1 or 4, wherein the Propionibacterium sp is Propionibacterium freudenreichii DSM 20271.

6. The soy whey-based product of claim 1, wherein the bifidobacteria is Bifidobacterium animalis subsp. lactis.

7. The soy whey-based product of claim 1 or 6, wherein the bifidobacteria is Bifidobacterium animalis subsp. lactis Bl-04.

8. The soy whey-based product of claim 1, wherein the short-chain fatty acids are acetic acid and propionic acid.

9. The soy whey-based product of claim 8, wherein the soy whey-based product contains acetic acid of at least 2.9 g / L.

10. The soy whey-based product of claim 8, wherein the soy whey-based product contains propionic acid of at least 1.8 g / L.

11. A method of improving vitamin B12 content in a soy whey-based product comprising: inoculating a soy whey medium with bifidobacteria and Propionibacterium sp. and fermenting the inoculated soy whey medium to obtain a soy whey-based product enriched with short-chain fatty acids and vitamin B 12.

12. The method of claim 1 1 , wherein the soy whey-based product contains at least 4.94 pg / L to 9.91 pg / L of vitamin B12.

13. The method of claim 11 or 12, wherein the soy whey-based product contains bifidobacteria with a cell count ranges from 7 log CFU / mL to 8.2 log CFU / mL.

14. The method of claim 11, wherein the step of inoculating further comprises co-culturing the bifidobacteria with Propionibacterium sp.

15. The method of claim 11, wherein the step of inoculating comprises simultaneous inoculating the soy whey medium with the bifidobacteria and the Propionibacterium sp.

16. The method of claim 11, wherein the step of inoculating comprises inoculating the soy whey medium sequentially with the bifidobacteria and the Propionibacterium sp.

17. The method of claim 11, wherein the step of inoculating comprises inoculating the soy whey medium with the bifidobacteria to allow propagation for at least two days prior to inoculation with the Propionibacterium sp.

18. The method of claim 11, wherein the step of inoculating comprises inoculating the soy whey medium with the Propionibacterium sp. to allow propagation for at least six days prior to inoculation with the bifidobacteria.

19. The method of any one of claims 11 to 18, wherein the bifidobacteria is Bifidobacterium animalis subsp. lactis.

20. The method of claim 19, wherein the bifidobacteria is Bifidobacterium animalis subsp. lactis Bl-04.

21. The method of any one of claims 11 to 18, wherein the Propionibacterium sp is Propionibacterium freudenreichii.

22. The method of claim 21 , wherein the Propionibacterium sp is Propionibacterium freudenreichii DSM 20271.

23. The method of claim 11, wherein the step of fermenting the soy whey medium is conducted for up to 8 days.

24. The method of claim 11, wherein the step of fermenting the soy whey medium is conducted at 30 °C.

25. The method of claim 11, further comprising adjusted the pH of the soy whey medium to 6.5 prior to inoculation.

26. A plant-based beverage comprising the soy whey-based product of any one of claims 1 to 10.

27. A plant-based food product comprising the soy whey-based product of any one of claims 1 to 10.

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