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FEMS Microbiology Letters logoLink to FEMS Microbiology Letters
. 2025 Aug 29;372:fnaf091. doi: 10.1093/femsle/fnaf091

Golden oats - Natural fortification of oat milk with riboflavin through fermentation

Emmelie Joe Freudenberg Rasmussen 1, Jesper Holck 2, Peter Ruhdal Jensen 3, Christian Solem 4,✉
PMCID: PMC12416276  PMID: 40880078

Abstract

Plant-based beverages are often fortified with different vitamins, especially B-vitamins, as the raw materials used for their production have a low content of these. Recently, we reported a simple and natural approach for obtaining vitamin B2 (riboflavin) secreting derivatives of the lactic acid bacterium (LAB) Lactococcus lactis, based on the observation that riboflavin can alleviate heat-induced oxidative stress. Here, we explore the potential of these strains for enriching plant-based beverages based on soy and oats, with riboflavin. Three riboflavin producing L. lactis strains were selected for the study: ER10, ALE13, and LDH13, where the latter is a lactate dehydrogenase-deficient derivative of ALE13. We found that ER10 produced more than 50 % more riboflavin in soy milk than ALE13 under static conditions (i.e. with no active aeration). Aerated culturing, in general, increased riboflavin production, especially for LDH13. The protein in oat milk is mostly insoluble and thus unavailable for the L. lactis strains used. To address this, oat milk was treated with food grade proteases, Alcalase® and Flavourzyme®, generating soluble peptides. When LDH13 was grown in the enzymatically treated oat milk with aeration, this resulted in a 600 % increase in riboflavin content (∼6 mg/L), demonstrating that the bioavailability of amino acids limits riboflavin production in oat milk. Here, we found that arginine played a special role in riboflavin production. By supplementing enzymatically treated oat milk with arginine, the riboflavin content could be further increased to 8 mg/L.

Keywords: biofortification, riboflavin, lactic acid bacteria, nutrition, amino acid profiling, oligosaccharide profiling

Highest reported riboflavin titer for oat-based beverages after biofortification with Lactococcus lactis, enabling clean labeling and potentially masking off-flavors.

Introduction

Plant-based milk alternatives (PBMA) are becoming increasingly popular for various reasons. They are often claimed to be more sustainable (Khanpit et al. 2024), compatible with a vegetarian or vegan lifestyle and do not contain lactose, which some tolerate poorly (Walther et al. 2022). However, the nutritional content of PBMA is a health concern as they are often low in bioavailable protein, minerals, and vitamins. In general, the vitamin content in food can be a challenge, since humans lack the ability to synthesize most of these. The main source of vitamins is the food we eat and to some extent the microorganisms in the gut (Hossain et al. 2022). Mammalian milk, on the other hand, is designed by nature to be a superior source of nutrition able to sustain life of mammals during their first critical period, and is rich in nutrients, including vitamins (O’Neil et al. 2018, Olanbiwoninu et al. 2023). To compensate for the low vitamin content, PBMA are often supplemented to ensure they meet nutritional requirements (Turck et al. 2017) and consumer demands (Walther et al. 2022).

One group of vitamins, often present in low amounts in PBMA and plant foods in general, are the B-vitamins (Hunt 1975, Titcomb and Tanumihardjo 2019). Of these, riboflavin, or vitamin B2, is particularly important as its derivatives flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are needed for the function of important metabolic enzymes (Leblanc et al. 2011), including the ones involved in the biosynthesis of other B-vitamins such as niacin (B3), pyridoxine (B6), folate (B9) (Saedisomeolia and Ashoori 2018), and cobalamin (B12) (Powers 2003). Riboflavin is also a strong antioxidant (Ashoori and Saedisomeolia 2014), which in its reduced form can help eliminate hydroperoxides (Saedisomeolia and Ashoori 2018).

Lactic acid bacteria (LAB) are known for their important role in food fermentation, where they contribute to organoleptic properties, like taste and texture, and help prolong the shelf life of the fermented product. PBMA fermented with LAB has also reached the market in the form of plant-based yoghurt analogues, where LAB can help improve the flavor. One example is butter aroma formed by certain LAB, which can help mask undesirable flavors (Gu et al. 2022, Yu et al. 2023), and make the product resemble yoghurt (Collier et al. 2023).

Vitamin biofortification using LAB is a promising approach, as it can help compensate for vitamin deficiencies and enable manufacturers to achieve clean label status, while adding to taste and texture (Leblanc et al. 2011). Despite the great potential of biofortification, which has been demonstrated clearly in various fermentation processes (Juarez del Valle et al. 2014, Chen et al. 2017, Spacova et al. 2022), no commercially available products intentionally fortified with riboflavin can be found on the market.

As an example, naturally occurring riboflavin-producing LAB found in pickled vegetables, have been estimated to produce between 0.096 and 0.7 mg/L of riboflavin, where Lactobacillus plantarum was found to be among the most efficient producers (Ge et al. 2020). Furthermore, Lactobacillus acidophilus has been seen to naturally produce riboflavin, if certain B-vitamins are already present, reaching an increase from 1 mg/L to 6.57 mg/L in soy milk (Ewe et al. 2010). Roseoflavin is a toxic riboflavin analogue that can be used to select for riboflavin overproduction (Leblanc et al. 2011, Averianova et al. 2020). A roseoflavin-resistant L. plantarum strain enabled biofortification of soy milk with as much as 1.55 mg/L (from 0.309 to 1.86 mg/L) (Juarez del Valle et al. 2014, Levit et al. 2021).

In the current study, we investigate whether riboflavin producing Lactococcus lactis strains, isolated after exposure to heat-induced oxidative stress, can be used to fortify plant-based milks based on soy and oat with riboflavin. Soy milk was selected as a reference PBMA due to its high availability of soluble proteins, which closely resemble the protein profile of cow’s milk. Since oat milk contains mostly insoluble protein, which is unavailable to L. lactis, we attempt to increase the bioavailability by treating the oat milk with commercial proteases. We report a dramatic effect hereof on riboflavin production. Finally, we determine the overall amino acid content in enzymatically treated oat milk prior to and after fermentation and discuss why certain amino acids are preferentially metabolized and promote riboflavin production.

Materials and methods

Bacterial strains

Lactococcus lactis ALE13, was isolated after adaptive laboratory evolution (ALE), under gradually increasing high temperatures (Rasmussen et al. 2024). LDH13 is a derivative of ALE13, that is deficient in lactate dehydrogenase (LDH) activity (Rasmussen et al. 2024). ER10 is a riboflavin secreting derivative of L. lactis MG1363, obtained after overnight growth with aeration and at high temperature (37°C) in defined SAL medium lacking riboflavin (Rasmussen et al. 2024).

Cultivation of strains

The wild-type L. lactis MG1363, and the derived strains ALE13, LDH13, and ER10 were grown on solid M17 medium (2 % agar) supplemented with 1 % glucose, before transferring to liquid SAL medium containing 0.5 % glucose or 0.5 % maltose. The culture was then transferred to oat milk (Oatly, Malmø, Sweden) or soy milk (Naturli, Vejen, Denmark) with 1 % maltose. For liquid cultivation, either static conditions (i.e. no active aeration) or aerated conditions (200 RPM shaking) were used for 24 h, with an incubation temperature of 30°C and start OD600 nm of 0.01.

Quantification of riboflavin by fluorescence

Riboflavin was quantified based on its fluorescent properties. For this purpose, a Tecan Infinite M200 Pro microplate reader was used in conjunction with black-walled white bottom 96-well plates equipped with 200 µL wells. Excitation wavelength was set at 485 nm, and the emission wavelength was set at 528 nm (Spacova et al. 2022), with a gain optimized to 60 for optimal quantification and sensitivity. Samples analysed for riboflavin content were withdrawn at early stationary phase, adjusted to pH 4.3 ± 0.2 with lactic acid, centrifuged at 7000 × g and filtered (0.2 µm pore size) prior to fluorescence measurements. A standard curve was prepared using pure riboflavin (Sigma-Aldrich, 83–88-5).

Enzymatic treatment

The proteins in oat milk were hydrolyzed using the following procedure. The procedure was chosen based on a study that in-depth analysed the effective hydrolysis of oat protein using food grade Alcalase® (Fuentes et al. 2021) and Flavourzyme® (Esfandi et al. 2019, Fuentes et al. 2021). The pH of the oat milk was first adjusted to 10. For each liter of oat milk, 800 µL of Alcalase® a protease from Bacillus licheniformis (Sigma-Aldrich, 9014–01-1, ≥ 2.4 U/g) was added (Fuentes et al. 2021), and incubated at 37°C for 4 h with 100 RPM shaking. This was followed by a heat inactivation step (85°C for 30 min), and pH adjustment to 7. Subsequently 500 µL/L of Flavourzyme® a protease from Aspergillus oryzae was added (Sigma-Aldrich, 232–752-2, ≥ 500 U/g) (Fuentes et al. 2021), and the oat milk was further hydrolyzed for 2 h at 50°C with 100 RPM shaking. Finally, to inactivate the protease, the oat milk was heated to 85°C for 30 min.

Amino acid analysis

The amino acid content of acid-hydrolyzed oat milk samples (regular oat milk, enzymatically treated and fermented) was quantified using Liquid Chromatography-Mass Spectrometry (LC–MS). For each sample, 500 µL was hydrolyzed using 500 µL of 12 M HCl at 110°C for 18 h. The headspace was filled with nitrogen. After hydrolysis, the samples were filtered through a 0.22 μm CA syringe filter. The filtrate (100 μL) was mixed with 1500 μL 0.2 M KOH to neutralize the acid and 1600 μL of buffer (100 mM ammonium formate) was added to normalize the pH with the mobile phase. The LC machine was a 1260 Infinity II (Agilent technologies, USA) containing a bioZen 2.6 μm Glycan column (Phenomenex, USA) connected to a Quadrupole 6120 MS (Agilent technologies) with an ESI ion source. The following settings were used; a flow rate of 0.5 mL/min, a column temperature of 40°C, 1 μL injection volume, and 16 min run time. A gradient mix of two mobile phases, A (10 mM ammonium formate in acetonitrile) and B (10 mM ammonium formate in water) was used as follows: 0–2 min 0 % phase B, 2–7 min 5 % phase B, 7–12 min 20 % phase B, and 12.1–16 min 0 % phase B. A mix of amino acid standards containing 17 amino acids (excluding glutamine, tryptophan, and asparagine) was run to make the standard curves. Samples were analysed and amino acids quantified using MassHunter Quantitative analysis version 7.0 software. Due to the harsh hydrolysis conditions used certain amino acids are degraded (glutamine, asparagine, and tryptophan) and thus cannot be quantified.

Oligosaccharide profiling

Identification and quantitation of oligosaccharides was performed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD; Dionex™ ICS6000 system, Dionex Corp., Sunnyvale, CA, USA). Samples of 10 µL were injected on a CarboPac PA200 column (3 mm × 250 mm + 3 mm × 50 mm guard, Thermo Scientific, Waltham, MA, USA). The chromatography was performed at 0.5 mL/min at 30°C on a three-eluent system with eluent A (milliQ water), eluent B (sodium hydroxide), and C (sodium acetate). The elution profile was as follows: 0–5 min, isocratic 10 mM C; 5–20 min, linear gradient to 200 mM C; 20–20.1 min, isocratic 500 mM C; 20.1–25 min, exponential gradient (Chromeleon type 1) to 10 mM C; and 25–30 min, isocratic 10 mM C. Eluent B was kept at 80 mM throughout the profile. External standards of glucose, malto-oligos DP 2–7, and cello-oligos DP 2-5 were used for identification and quantitation. Beta-glucan oligos in samples were quantified as cello-oligo DP 5 equivalents.

Results and discussion

Enhancing riboflavin levels in PBMA

To enhance the riboflavin content, we fermented soy and oat milk under static conditions (no active aeration) with the riboflavin producing L. lactis strains, ALE13, ER10 and the lactate dehydrogenase-deficient derivative of ALE13 (LDH13). These are natural derivatives of L. lactis MG1363 obtained after exposure to heat-induced oxidative stress (Rasmussen et al. 2024). It was possible to increase the riboflavin content in both soy and oat milk, with the more nutrient-rich soy milk supporting the highest titer for both strains, 0.58 mg/L, 0.90 mg/L, and 0.71 mg/L for ALE13, ER10, and LDH13, respectively (Fig. 1). Since soy milk is low in fermentable carbohydrates, we supplemented it with 1 % maltose (29 mM), a slowly fermented carbohydrate, which we have shown to be beneficial for riboflavin production (Rasmussen et al. 2024).

Figure 1.

Figure 1.

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Riboflavin content in statically fermented soy and oat milk. Soy and oat milk were fermented with three riboflavin producing L. lactis strains: ALE13, ER10, and a lactate dehydrogenase-deficient derivative of ALE13 (LDH13). Static cultivation conditions were used (i.e. no active aeration) at 30°C. Riboflavin content is shown in mg/L. Samples were analysed after 24 h of fermentation. Non-fermented oat and soy milk is shown as dark gray solid bars. ALE13 titer is represented by dark gray solid bars with diagonal lines, while soy and oat fermented with ER10 are shown as light gray solid bars, and lastly the LDH13 titer is visualized as light gray with cross-hatched lines. Experiments were carried out in triplicate, and the averages ± SD are indicated.

For these fermentations we chose soy and oat milk products without added riboflavin, which contained 0.035 mg/L and 0.020 mg/L riboflavin, respectively (Fig. 1). The population reference intake (PRI) of riboflavin is 1.3 mg/day and 1.1 mg/day for men and women over 19, respectively, although some population groups have higher requirements, e.g. pregnant and lactating women (Turck et al. 2017). Thus, the riboflavin content in PBMA is quite low, especially when compared to cow’s milk, which on average has a content of 2 mg/L (ranging from 0.78 to 4.58 mg/L) (Laverroux et al. 2020).

While sucrose is the major carbohydrate found in soy milk (Gu et al. 2022, Walther et al. 2022), oat milk, depending on how extensively the starch has been hydrolyzed, contains glucose, maltose, and higher oligomers of glucose (Gu et al. 2022). Table 1 shows that the oat milk used in this study has maltose as the main carbohydrate, which is favorable for riboflavin overproduction. From previous work we know that excessive acidification due to lactic acid production decreases riboflavin production, but that aerated culturing conditions can reduce lactic acid production to some extent (Duwat et al. 2001, Rasmussen et al. 2024). Thus, we decided to investigate whether aerated culturing of ALE13 and ER10 in soy and oat milk could boost riboflavin production. A small improvement was observed for soy milk for ALE13, however for oat milk the riboflavin titer increased more than 70 % by aerated culturing for both ALE13 and ER10 as seen in Fig. 2. This prompted us to test the performance of the lactate dehydrogenase deficient derivative of ALE13 (LDH13), which yield higher biomass under aerobic conditions, due to NAD+ regeneration through the NADH oxidase and by the production of diacetyl and acetoin that can help mask undesirable flavors (Gu et al. 2022, Yu et al. 2023). The LDH13 boosted the riboflavin titer to 0.85 mg/L and 1.42 mg/L in oat milk and soy milk, respectively (Fig. 2.).

Table 1.

Oligosaccharide profiling of regular and protease-treated oat milk (ProOat) before and after fermentation with L . lactis LDH13, aerated culturing. The carbohydrate content is shown in µM for regular oat milk (oat), regular oat milk fermented with LDH13 (oat fermented), enzymatically treated oat milk (ProOat), and lastly enzymatically treated oat milk fermented with LDH13 (ProOat fermented). Glu1 = Glucose, Glu2 = Maltose, Glu3 = Maltotriose, Glu4 = Maltotetraose, Glu5 = Maltopentaose, Glu6 = Maltohexose, and Glu7 = Maltoheptaose.

Glu1 Glu2 Glu3 Glu4 Glu5 Glu6 Glu7
Oat 2861 98 324 21 817 476 32 47 88
Oat fermented 200 95 041 20 559 414 18 79 94
ProOat 11 789 107 151 30 832 3105 1174 577 119
ProOat fermented 3337 66 853 18 684 891 587 519 63
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Figure 2.

Figure 2.

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Riboflavin content in soy and oat milk fermented with aeration. Soy and oat milk were fermented with three riboflavin producing L. lactis strains: ALE13 and ER10, and a lactate dehydrogenase-deficient derivative of ALE13 (LDH13). The fermentations were carried out in conical shake flasks at 30°C with 200 RPM shaking. Riboflavin content is shown in mg/L. Samples were analysed after 24 h of fermentation. Non-fermented oat and soy milk is shown as dark gray solid bars. ALE13 titer is indicated by the dark gray solid bars with diagonal lines, whereas those corresponding to ER10 titer is shown as light gray solid bars, and lastly the LDH13 titer is visualized as light gray with cross-hatched lines. The experiment was carried out in triplicate, and the averages ± SD are indicated.

Amino acid availability in oat milk restricts riboflavin production

Even though aeration increases the content of riboflavin in oat milk significantly, the mostly insoluble protein in oat milk is expected to be a major bottleneck for growth and riboflavin production by L. lactis. Soy milk has a high soluble protein content, similar to that of cow’s milk, whereas the protein content of oat is only 10 % of that of cow’s milk (Walther et al. 2022) and most of these proteins are not soluble, and thus unavailable to L. lactis. Therefore, we decided to use commercial food-grade proteases to generate peptides and free amino acids that can be assimilated readily by L. lactis. For this study, we selected the two best performing strains, ER10 and LDH13, based on their highest riboflavin titer under their respective optimal condition, aerated culturing. When the enzymatically treated oat milk (ProOat) was fermented with the LDH13 strain with aeration, we observed a massive 600 % increase in riboflavin production (Fig. 3), clearly demonstrating that the amino acid availability is a major bottleneck for riboflavin production in oat milk.

Figure 3.

Figure 3.

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Riboflavin content in fermented protease-treated oat milk (ProOat), with aeration. Oat milk, and oat milk treated with Flavourzyme® and Alcalase® (ProOat), was fermented with ER10 and the lactate dehydrogenase-deficient L. lactis strain LDH13. The fermentations were carried out in conical shake flasks at 30°C with 200 RPM shaking. Riboflavin content is shown in mg/L. Samples were analysed after 24 h of fermentation. Non-fermented oat milk and ProOat are shown as dark gray solid bars. ER10 titer is indicated by the light gray solid bars, and lastly the LDH13 titer is visualized as light gray solid bars with cross-hatched lines. The experiment was carried out in triplicate, and the averages ± SD are indicated.

To investigate amino acid consumption, we determined the total amino acid content of enzymatically treated oat milk (ProOat) and oat milk before and after fermentation. We noticed that 90 % of the arginine and a significant part of the serine had been consumed by L. lactis in the enzymatically treated oat milk (Fig. 4.). These two amino acids play a unique role for L. lactis as they can be metabolized by L. lactis, especially under energy starved conditions, and their catabolism is linked to adenosine triphosphate (ATP) production (Crow and Thomas 1982). In the oat milk used, maltose is the main fermentable carbohydrate available, and since maltose is fermented slowly by L. lactis, the cells indeed experience energy starvation when grown on this carbohydrate (Sjoberg and Hahn-Hagerdal 1989). This has previously been shown to benefit riboflavin production, which makes the enzymatically treated oat milk a great substrate for riboflavin biofortification (Rasmussen et al. 2024).

Protease treatment facilitates carbohydrate utilization

We also studied the carbohydrate utilization in oat milk and enzymatically treated oat milk (ProOat), since carbohydrate utilization is directly linked to riboflavin production. Prior to protease treatment, the oat milk mostly contained maltose (Glu2), smaller amounts of maltotriose (Glu3) and a low amount of glucose (Glu1) (Table 1). Treatment with commercial proteases appeared to have released additional sugars, especially glucose. The carbohydrate analysis furthermore revealed that sugar utilization was greatly enhanced for the protease-treated oat milk (ProOat). LDH13 utilized almost 40 % of the available maltose in enzymatically treated oat milk (ProOat), whereas in oat milk, it consumed mainly glucose (93 %) and only 3 % of the maltose. The LDH13 strain consumed ~70 % of the available glucose in ProOat.

Arginine catabolism, which converts arginine via citrulline into ornithine, concurrently producing ATP, is expressed to a higher level when L. lactis is grown on slowly fermentable sugars, such as maltose (Crow and Thomas 1982, Douwenga et al. 2023). It is also known that L. lactis can switch easily from low-quality sugars, such as maltose to high-quality sugar like glucose without much delay (Douwenga et al. 2023). When the preculture is grown solely on maltose, this may explain why not all maltose and arginine are consumed, but a large portion of the now available glucose is. However, it appears that the presence of arginine may preferentially enhance the utilization of maltose, which in turn favors riboflavin production. Glucose is a readily metabolized carbohydrate for L. lactis, which supports faster growth than the slowly metabolized maltose. On glucose, fast growth results in enhanced expression of nucleotide metabolism, i.e. purine and pyrimidine biosynthesis, which has a negative effect on riboflavin production since purines repress riboflavin biosynthesis (Chen et al. 2017, Rasmussen et al. 2024). Maltose, on the other hand, which is slowly metabolized, does not have this stimulatory effect on nucleotide metabolism. In conjunction with the mixed-acid fermentation pattern observed on maltose, which yields more ATP (Sjöberg et al. 1995), oat milk makes a great choice for producing riboflavin using L. lactis.

Adding extra arginine can boost riboflavin production

The fact that arginine was almost depleted during growth on enzymatically treated oat milk (ProOat) (Fig. 4), indicated that additional ATP generated through arginine catabolism is beneficial for riboflavin production. To test this hypothesis, we supplemented the protease-treated oat milk (ProOat) with additional arginine, which clearly benefited riboflavin production, as seen in Fig. 5(A). In the protease-treated oat milk (ProOat), by adding 20 mM arginine, the riboflavin titer could be increased by almost 2.5 mg/L, corresponding to more than a 40 % increase. In a previous study it was found that 20 mM arginine supported the highest biomass yield for L. lactis grown on the slowly fermented sugar galactose (Crow and Thomas 1982). Since the effect of added arginine was moderate, and supplementing arginine adds cost to a final product, we did not explore the effect of arginine further. We did however notice that arginine was fully consumed in the arginine supplemented ProOat milk (Fig 5B), and it is likely that additional arginine could increase the riboflavin titer further.

Figure 4.

Figure 4.

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Total amino acid profile of oat milk and protease-treated oat milk (ProOat) before and after fermentation with L. lactis LDH13, with aeration. The amino acid concentration is shown in mg/g of sample. Amino acids in non-fermented oat milk are represented by gray bars, fermented oat milk by light gray bars, enzymatically treated oat milk (ProOat) by dark gray bars, and fermented enzymatically treated oat milk (ProOat) by black bars.

Figure 5.

Figure 5.

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Riboflavin content in fermented protease-treated oat milk with 20 mM arginine and corresponding amino acid consumption profile. A) Oat milk, oat milk treated with Flavourzyme® and Alcalase® (ProOat), and ProOat with 20 mM arginine were fermented with the lactate dehydrogenase-deficient L. lactis strain LDH13. The fermentations were carried out in conical shake flasks at 30°C with 200 RPM shaking. Riboflavin content is shown in mg/L. Samples were analysed after 12 h of fermentation, expressed as solid dark gray bars with diagonal lines, and 24 h of fermentation, expressed as light gray bars. B) The amino acid concentrations of serine (SER) and arginine (ARG) are shown in mg/g of sample after 24 h of fermentation. The concentration in oat milk is represented by gray bars, enzymatically treated oat milk (ProOat) by light gray bars, and ProOat with 20 mM arginine by dark gray bars. Their corresponding fermented sample with LDH13 is represented by filled black dots.

While yogurt and other fermented milk products remain popular, there is a growing consumer demand for non-dairy products to accommodate the increasing prevalence of lactose intolerance and vegetarianism. As a result, the current emphasis is on producing non-dairy beverages that incorporate new ingredients, offering both high functionality and consumer acceptability. Therefore, designing naturally biofortified PBMA’s with riboflavin will help accommodate nutritional requirements and clean labeling. Soy-based products have previously been biofortified using riboflavin producing Lactobacillus strains (Levit et al. 2021), to a level similar to what we obtained for soy milk using L. lactis. However, to the best of our knowledge, this is the first report demonstrating biofortification of oat milk with riboflavin, and we reached the highest titer ever reported for a fermented plant-based beverage.

Summing up, by treating oat milk with commercial proteases it is possible to increase the availability of amino acids, especially arginine, and stimulate growth and production of riboflavin by L. lactis. We observed that serine was consumed as well. Serine is involved in nucleotide metabolism, where it provides one-carbon units for purine base biosynthesis (Kilstrup et al. 2005). Serine is also a precursor for other amino acids such as glycine helping maintain the cellular amino acid pool (Kilstrup et al. 2005) and can be directly converted into pyruvate (Fernández and Zúñiga 2006). This preliminary data suggests the need for more investigation of serine’s role in metabolism and specifically riboflavin production in PBMA to provide more comprehensive insights.

We have shown that it is possible to use enzymatically treated oat milk as a superior growth substrate for producing riboflavin, due to its high maltose and arginine content, where the latter supports generation of additional ATP for growth and riboflavin biosynthesis. By using an LDH-deficient strain for producing riboflavin, we could minimize lactic acid production and thereby promote riboflavin production and mask off-flavors (Gu et al. 2022, Yu et al. 2023, Rasmussen et al. 2024).

Contributor Information

Emmelie Joe Freudenberg Rasmussen, National Food Institute, Technical University of Denmark, DK-2800 Kgs., Lyngby, Denmark.

Jesper Holck, DTU Bioengineering, Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kgs., Lyngby, Denmark.

Peter Ruhdal Jensen, National Food Institute, Technical University of Denmark, DK-2800 Kgs., Lyngby, Denmark.

Christian Solem, National Food Institute, Technical University of Denmark, DK-2800 Kgs., Lyngby, Denmark.

Conflict of interest

All authors declare no conflict of interest.

Funding

This work was supported by Independent Research Fund Denmark (DFF), Technology and Production Sciences (grant number 2035-00059B).

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