Nisin Production Research Articles

AcrR1, a novel TetR/AcrR family repressor, mediates acid and antibiotic resistance and nisin biosynthesis in Lactococcus lactis F44

Lactococcus lactis, widely used in the manufacture of dairy products, encounters various environmental stresses both in natural habitats and during industrial processes. It has evolved intricate machinery of stress sensing and defense to survive harsh stress conditions. Here, we identified a novel TetR/AcrR family transcription regulator, designated AcrR1, to be a repressor for acid and antibiotic tolerance that was derepressed in the presence of vancomycin or under acid stress. The survival rates of acrR1 deletion strain ΔAcrR1 under acid and vancomycin stresses were about 28.7-fold (pH 3.0, HCl), 8.57-fold (pH 4.0, lactic acid) and 2.73-fold (300 ng/mL vancomycin) greater than that of original strain F44. We also demonstrated that ΔAcrR1 was better able to maintain intracellular pH homeostasis and had a lower affinity to vancomycin. No evident effects of AcrR1 deletion on the growth and morphology of strain F44 were observed. Subsequently, we characterized that the transcription level of genes associated with amino acids biosynthesis, carbohydrate transport and metabolism, multidrug resistance, and DNA repair proteins significantly upregulated in ΔAcrR1 using transcriptome analysis and quantitative reverse transcription-PCR assays. Additionally, AcrR1 could repress the transcription of the nisin post-translational modification gene, nisC, leading to a 16.3% increase in nisin yield after AcrR1 deletion. Our results not only refined the knowledge of the regulatory mechanism of TetR/AcrR family regulator in L. lactis, but presented a potential strategy to enhance industrial production of nisin.

Nisin-based therapy: a realistic and eco-friendly biocontrol strategy to contrast Xylella fastidiosa subsp. pauca infections in planta.

The lack of sustainable strategies for combating Xylella fastidiosa (Xf) highlights the pressing need for novel practical antibacterial tools. In this study, Lactococcus lactis subsp. lactis strain ATCC 11454 (L. lactis), known for its production of nisin A, was in vitro tested against Xf subsp. pauca. Preliminary investigations showed that nisin A was involved in a strong antagonistic activity exhibited by L. lactis against Xf. Thus, the efficacy of nisin A was comprehensively assessed through a combination of in vitro and in planta experiments. In vitro investigations employing viable-quantitative PCR, spot assay, turbidity reduction assay, fluorescence microscopy, and transmission electron microscopy demonstrated nisin's robust bactericidal effect on Xf at a minimal lethal concentration of 0.6 mg/mL. Moreover, results from fluorescence and transmission electron microscopies indicated that nisin directly and rapidly interacts with the membranes of Xf cells, leading to the destruction of bacterial cells in few minutes. In in planta tests, nisin also demonstrated the ability to tackle Xf infections within Nicotiana benthamiana plants that remained asymptomatic 74 days post inoculation. Furthermore, RPLC-ESI-MS/MS analyses showed that nisin translocated to all parts of the plants and remains intact for up to 9 days. For the first time, this study underscores the nisin-based strategy as a realistic and eco-friendly approach to be further investigated against Xf infections in the field.

Rerouting the Metabolic Pathway in Lactococcus lactis ATCC 11454 to Improve Nisin Productivity by Disrupting Lactate Dehydrogenase (ldh)

ABSTRACT Nisin is an effective biopreservative, however its wide application in food is restricted due to its low productivity. Therefore, the present work aimed to increase its yield by disrupting lactate dehydrogenase (ldh) and re-directing the carbon flow towards nisin production. The ldh-deficient Lactococcus lactis ATCC 11454 (ldhΔ) strain was created by inserting ampicillin (amp) within the ldh to disrupt its function. Under controlled fermentation conditions (30°C, pH 6, 1.0% glucose), the ldhΔ strain exhibited negligible decrease in pH (2.5%) over 48 h, in contrast to the wild-type (WT) strain, which experienced a 23.6% decrease in pH (pH 4.2). Overall, the ldhΔ strain showed a significant (p < 0.05) increase in growth and nisin activity by 24.9% and 62% compared to the WT strain, respectively. This strategy could serve as a practical approach to further enhance the production of bacteriocin or other compounds by modulating their metabolic pathways.

Evaluation of Safety and Probiotic Traits from a Comprehensive Genome-Based In Silico Analysis of Ligilactobacillus salivarius P1CEA3, Isolated from Pigs and Producer of Nisin S.

Ligilactobacillus salivarius is an important member of the porcine gastrointestinal tract (GIT). Some L. salivarius strains are considered to have a beneficial effect on the host by exerting different probiotic properties, including the production of antimicrobial peptides which help maintain a healthy gut microbiota. L. salivarius P1CEA3, a porcine isolated strain, was first selected and identified by its antimicrobial activity against a broad range of pathogenic bacteria due to the production of the novel bacteriocin nisin S. The assembled L. salivarius P1CEA3 genome includes a circular chromosome, a megaplasmid (pMP1CEA3) encoding the nisin S gene cluster, and two small plasmids. A comprehensive genome-based in silico analysis of the L. salivarius P1CEA3 genome reveals the presence of genes related to probiotic features such as bacteriocin synthesis, regulation and production, adhesion and aggregation, the production of lactic acid, amino acids metabolism, vitamin biosynthesis, and tolerance to temperature, acid, bile salts and osmotic and oxidative stress. Furthermore, the strain is absent of risk-related genes for acquired antibiotic resistance traits, virulence factors, toxic metabolites and detrimental metabolic or enzymatic activities. Resistance to common antibiotics and gelatinase and hemolytic activities have been discarded by in vitro experiments. This study identifies several probiotic and safety traits of L. salivarius P1CEA3 and suggests its potential as a promising probiotic in swine production.

Production of bacterial cellulose (BC)/nisin composite with enhanced antibacterial and mechanical properties through co-cultivation of Komagataeibacter xylinum and Lactococcus lactis subsp. lactis

By employing co-cultivation technique on Komagataeibacter xylinum and Lactococcus lactis subsp. lactis, bacterial cellulose (BC)/nisin films with improved antibacterial activity and mechanical properties were successfully produced. The findings demonstrated that increased nisin production is associated with an upregulation of gene expression. Furthermore, results from Scanning electronic microscopy (SEM), Fourier transform infrared (FTIR), X-ray diffraction (XRD), and Thermogravimetric analysis (TG) confirmed the integration of nisin within BC. While being biocompatible with human cells, the BC/nisin composites exhibited antimicrobial activity. Moreover, mechanical property analyses showed a noticeable improvement in Young's modulus, tensile strength, and elongation at break by 161, 271, and 195 %, respectively. Additionally, the nisin content in fermentation broth was improved by 170 % after co-culture, accompanied by an 8 % increase in pH as well as 10 % decrease in lactate concentration. Real-time reverse transcription PCR analysis revealed an upregulation of 11 nisin-related genes after co-cultivation, with the highest increase in nisA (5.76-fold). To our knowledge, this is the first study which demonstrates that an increase in secondary metabolites after co-culturing is modulated by gene expression. This research offers a cost-effective approach for BC composite production and presents a technique to enhance metabolite concentration through the regulation of relevant genes.

Nisin Purification from a Cell-Free Supernatant by Electrodialysis in a Circular Economy Framework.

Nisin, an antimicrobial peptide produced by Lactococcus lactis strains, is a promising natural preservative for the food industry and an alternative to antibiotics for the pharmaceutical industry against Gram-positive bacteria. Nisin purification is commonly performed using salting out and chromatographic techniques, which are characterized by their low yields, the use of solvents and the production of large volumes of effluents. In the present work, the purification of nisin from a cell-free supernatant (CFS), after the production of nisin by fermentation on a whey permeate medium, was studied using ammonium sulfate precipitation and electrodialysis (ED) as a promising eco-friendly process for nisin purification. Results showed an increase in nisin precipitation using a 40% ammonium sulfate saturation (ASS) level with a purification fold of 73.8 compared with 34.5 and no purification fold for a 60% and 20% ASS level, respectively. The results regarding nisin purification using ED showed an increase in nisin purification and concentration fold, respectively, of 21.8 and 156 when comparing the final product to the initial CFS. Nisin-specific activity increased from 75.9 ± 4.4 to 1652.7 ± 236.8 AU/mg of protein. These results demonstrated the effectiveness of ED coupled with salting out for nisin purification compared with common techniques. Furthermore, the process was noteworthy for its relevance in a circular economy scheme, as it does not require any solvents and avoids generating polluting effluents. It can be employed for the purification of nisin and the recovery of salts from salting out, facilitating their reuse in a circular economy.

Extraction and Purification Nisin from Lactobacillus lactis and Determine the Optimal Conditions for its Production and Evaluation its Effectiveness in Food Preservation

The aim of the study was to purify and extract nisim from Lactobacillus lactis that diagnosed in the Laboratory of Microbiology in the Department of pathological analysis/College of Applied Sciences-Samara University. The optimal conditions for the production of nisin were determined, the optimal medium was De Man Rogosa Sharpe (MRS). As for the PH, the results showed that the best pH for the production of nisin was at pH 5, the optimum temperatures was 30° C. As for the incubation period and circumference, the highest productivity of the nisin was 18 hours. The nisin was purified using ammonium sulfate to precipitate nisin, which is one of the initial steps to purify proteins, and then the deposited protein was passed after its dilution and concentrated in the Ion Exchange column. The ability of the extracted and purified nisin in the biopreservation of meat was determined when it was added at different concentrations (25%, 50%, 100%). The concentration of 100% was the most appropriate concentration in biopreservation based on the absence of contamination in the meat during the first three days and the low numbers of bacteria from the fourth day to the seventh day, there were significant differences between the numbers of bacteria at the level of probability (P < 0.05) for samples of meat stored in refrigeration.

Combination approach of paired starter culture and lactic acid on inhibiting autochthonous lactic acid bacteria for extending kimchi shelf life

Excessive kimchi fermentation by lactic acid bacteria (LAB) during distribution and storage may negatively influence the organoleptic properties of kimchi. Thus, this study aimed to extend kimchi shelf life by inhibiting the growth of autochthonous lactic acid bacteria by applying paired starter culture, consisting of nisin-producing Lactococcus lactis WiKim0098 and nisin-resistant Leuconostoc citreum WiKim0096, and lactic acid. To maximize nisin production in the L. lactis WiKim0098 culture medium, the food grade medium composition and culture conditions were optimized using response surface methodology. Leu. citreum WiKim0096 exhibited resistance to nisin owing to high positive surface charge and was successfully used as a paired starter. In the kimchi application experiments, paired starter culture addition delayed the over-acidification (more than 1% as acidity) of kimchi for approximately 5–6 days, and when combined with lactic acid, delayed the ripening for more than 8 days compared to control kimchi fermented at 10 °C. The results of bacterial community and metabolite analysis via principal component analysis showed that paired starter culture and lactic acid inhibited the LAB growth at the early stage of kimchi fermentation. Our results suggest that combining paired starter culture with lactic acid can effectively extend the kimchi shelf life. Specifically, it is expected to be effectively applicable to kimchi products intended for export, which demand extended shelf life.

Production of nisin from Lactococcus lactis in acid-whey with nutrient supplementation

The production of Nisin, an FDA-approved food preservative, was attempted by Lactococcus lactis subsp. lactis ATCC® 11454 using the underutilized milk industry effluent, acid-whey, as a substrate. Nisin production was further improved by studying the effect of supplementation of nutrients and non-nutritional parameters. The addition of yeast extract (6% w/v) as nitrogen source and sucrose (4% w/v) as carbon source were found to be suitable nutrients for the maximum nisin production. The changes in the medium pH due to lactic acid accumulation during batch fermentation and its influence on the production of nisin were analyzed in the optimized whey medium (OWM). The production characteristics in OWM were further compared with the nisin production in MRS media. The influence of nisin as an inducer for its own production was also studied and found that the addition of nisin at 0.22 mg/ml promote the nisin production. The analysis of consumption of various metal ions present in the OWM during the nisin production was also analyzed, and found that the copper ions are the most consumed ion. The highest nisin yield of 2.6 × 105 AU/mL was obtained with OWM.

Nisin Production from Waste: A Review

Nisin is a bacteriocin produced by microbial fermentation of the well-known starter culture for dairy products, Lactococcus lactis. A major challenge in food safety and security is the prevention of the emergence of unwanted microbes. Artificial preservatives added to prolong the shelf life of foods may have negative effects on consumer health. An alternative approach is to use bio-preservatives. However, the cost of production of nisin is relatively higher than other chemical-based alternatives, because of which it has low marketability. Traditional production techniques are expensive and harmful to the environment. This review investigates sustainable alternatives employing waste products as fermentation substrates, including whey, corn stover, cane molasses, soya-bean cake, bean pulp and mussel processing waste. These eco-friendly strategies minimize negative environmental effects while providing resource and cost-efficient solutions. This review demonstrates the possibility for India to become self-sufficient in nisin production and reduce reliance on imports by emphasizing waste-to-value techniques. Keywords: Nisin, Bio-preservatives, Waste-to-value, Sustainable, Eco-friendly

Extending shelf life of fresh noodles with nisin producer Lactococcus lactis subsp. lactis fermented sourdough and characteristic changes during storage

In this study, the effect of sourdough fermented with nisin producer Lactococcus lactis subsp. lactis CGMCC1.2030 on the shelf life and quality characteristics of fresh noodles fortified with it was investigated. The shelf life of fresh noodles with sourdough increased from 12 days to 22 days compare to the blank control during storage at 4 °C. Slight antimould activity was observed in sourdough fortified fresh noodles, and the brightened color of these noodles improved their appearance, which related to a decrease in the b* value of the noodle sheet from 12.35 to 8.47 over the storage period. Furthermore, the hardness and chewiness of sourdough-fortified fresh noodles decreased by 25.96% and 18.57%, respectively, which was lower than the 35.59% and 36.50% reduction observed in the blank control after the whole storage period. Confocal laser scanning microscopy (CLSM) analysis revealed less aggregated and more continuous gluten networks, which in combination with mitigated water migration might result in the delayed deterioration of texture quality of sourdough-fortified fresh noodles.

Synthesis, high yield strategy and application of nisin: A review

SummaryNisin is a peptide with antibacterial activity against a variety of pathogens. It was originally obtained from a Lactococcus lactis. It is classified as a class I bacteriocin, as it is synthesised by ribosomes and post‐translationally modified. It has been studied extensively and has a wide range of applications in the food industry, biomedicine, veterinary, and research fields. It is approved as a food preservative by the Food and Agriculture Organization (FAO). What's more, nisin, the only antimicrobial peptide that has generally considered as safe (GRAS) status from both World Health Organization (WHO) and Food and Drug Administration (FDA), has the potential to become an important natural antibiotic as bacteria become more resistant to traditional antibiotics. However, nisin purified from biological sources usually results in low yields and high production costs, limiting industrial‐scale manufacturing and application in other fields. Hence, more methods need to be used to optimise the production of nisin. Therefore, this paper aimed to understand the structure and biosynthesis mechanism of nisin and to summarise the innovative methods for increasing the production of nisin using biosynthesis regulation.

Bioengineering of a Lactococcus lactis subsp. lactis strain enhances nisin production and bioactivity.

Lactococcus lactis subsp. lactis is a food bacterium that has been utilized for decades in food fermentation and the development of high-value industrial goods. Among these, nisin, which is produced by several strains of L. lactis subsp. lactis, plays a crucial role as a food bio-preservative. The gene expression for nisin synthesis was evaluated using qPCR analysis. Additionally, a series of re-transformations of the strain introducing multiple copies of the nisA and nisRK genes related to nisin production were developed. The simultaneous expression of nisA and nisZ genes was used to potentiate the effective inhibition of foodborne pathogens. Furthermore, qPCR analysis indicated that the nisA and nisRK genes were expressed at low levels in wild-type L. lactis subsp. lactis. After several re-transformations of the strain with the nisA and nisRK genes, a high expression of these genes was obtained, contributing to improved nisin production. Also, co-expression of the nisA and nisZ genes resulted in extremely effective antibacterial action. Hence, this study would provide an approach to enhancing nisin production during industrial processes and antimicrobial activity.

Nisin E Is a Novel Nisin Variant Produced by Multiple Streptococcus equinus Strains

Nisin A, the prototypical lantibiotic, is an antimicrobial peptide currently utilised as a food preservative, with potential for therapeutic applications. Here, we describe nisin E, a novel nisin variant produced by two Streptococcus equinus strains, APC4007 and APC4008, isolated from sheep milk. Shotgun whole genome sequencing and analysis revealed biosynthetic gene clusters similar to nisin U, with a unique rearrangement of the core peptide encoding gene within the cluster. The 3100.8 Da peptide by MALDI-TOF mass spectrometry, is 75% identical to nisin A, with 10 differences, including 2 deletions: Ser29 and Ile30, and 8 substitutions: Ile4Lys, Gly18Thr, Asn20Pro, Met21Ile, His27Gly, Val32Phe, Ser33Gly, and Lys34Asn. Nisin E producing strains inhibited species of Lactobacillus, Bacillus, and Clostridiodes and were immune to nisin U. Sequence alignment identified putative promoter sequences across the nisin producer genera, allowing for the prediction of genes in Streptococcus to be potentially regulated by nisin. S. equinus pangenome BLAST analyses detected 6 nisin E operons across 44 publicly available genomes. An additional 20 genomes contained a subset of nisin E transport/immunity and regulatory genes (nseFEGRK), without adjacent peptide production genes. These genes suggest that nisin E response mechanisms, distinct from the canonical nisin immunity and resistance operons, are widespread across the S. equinus species. The discovery of this new nisin variant and its immunity determinants in S. equinus suggests a central role for nisin in the competitive nature of the species.

Process modelling and environmental assessment on the valorization of lignocellulosic waste to antimicrobials

The Sustainable Development Goals defined by the United Nations established a roadmap on the way to a sustainable society based on a circular economy in which the residual fractions of process streams emerge as raw materials for the provision of material goods and services. In this context, the approach of biorefineries as the technological scheme for processing integrally renewable resources stands out. Among the different target products, "bioactive" compounds have aroused great interest given their high added value. This work evaluates the environmental profile associated with the valorization of residual lignocellulosic waste streams: olive pruning and eucalyptus residues for the biotechnological production of nisin, an antimicrobial compound produced by Lactoccocus lactis with application in the food preservation and biomedical sectors. Process modelling in combination with Life Cycle Analysis allowed addressing the conceptual design and the environmental assessment of a full-scale production process. As a main conclusion, the technological feasibility of the valorization of waste fractions as sources of fermentable sugars in the biotechnological process has been demonstrated. Moreover, the integral use of the raw materials can allow not only the production of nisin as an economic driver, but also of other biotechnological products such as lactic acid and energy for the self-supply of the process. The main novelty of this manuscript is based on rethinking the valorization of lignocellulosic waste beyond energy recovery to obtain bioproducts with high added value in the market with applicability in the food sector. Although energy recovery is the most widely implemented end-of-life option, the use of lignocellulosic waste as fermentable sugars has been shown to improve sustainability and circularity outcomes.

Effects of Individual and Combined Fermentation Factors on Antimicrobial Activity of Nisin by Lactococcus lactis ATCC 11454.

There is an increasing demand for natural food preservatives due to consumers' concern on the negative effects of chemical preservatives in food products. Nisin (bacteriocin) is an effective food biopreservative that has been approved globally. However, its low yield proves to be a limiting factor and must be addressed to meet the increasingly high demand from the food industry. The present work thus investigated the effects of individual and combined fermentation factors on Lactococcus lactis ATCC 11454 growth and nisin activity using the one-factor-at-a-time (OFAT) method. The level of each factor that gave the highest nisin production was then selected and combined to further improve its activity. The best combined conditions for highest cell growth and nisin activity were 30°C, pH 6.0, and mild agitation with the addition of 1.0% w/v glucose, 1.0% w/v skim milk, and 0.5% v/v Tween 20. This increased nisin production by 22.7% as compared to control (basic condition). The present work provided critical information on the relationship between fermentation conditions, growth, and nisin activity of L. lactis ATCC 11454 that could be explored to understand the potential and limitation of the strain. This fermentation strategy can also serve as a benchmark to further enhance the production of bacteriocin or other biopreservative compounds.

Recovery of nisin from culture supernatants of Lactococcus lactis by ultrafiltration: Flux properties and separation efficiency

Nisin is a class I bacteriocin, which is produced by lactic acid bacteria (LAB) belonging to Lactococcus and Streptococcus genera. Nisin is approved by the FDA (Food and Drug Administration) as a natural biopreservative agent under reference E234. Besides food application, nisin has also a potential for therapeutic application to treat a range of infectious diseases. Nisin, which is commercially available, is mainly produced by liquid fermentation. The production of commercial high-quality nisin is hampered by the high cost of downstream processing, which often involves subsequent steps of salt precipitation, centrifugation, and chromatography. Membrane processes offer a set of advantages. Indeed, these processes are simple and effective technologies for protein concentration and separation; One of the biggest limitations is the decrease in permeate flux. In this study, the performance of three UF membranes (10, 50, and 100 kDa) for the separation and concentration of nisin obtained from Lactococcus lactis (L. lactis) culture supernatants was evaluated. Permeate flux decline, fouling resistances, fouling index, nisin recovery in the permeate and retentate streams were evaluated. The 10 kDa membrane showed the best nisin recovery properties with the highest recovery yield and purification factor, achieving 100% and 4 in the retentate stream, respectively.

Breeding of a High-Nisin-Yielding Bacterial Strain and Multiomics Analysis

Nisin is a green, safe and natural food preservative. With the expansion of nisin application, the demand for nisin has gradually increased, which equates to increased requirements for nisin production. In this study, Lactococcus lactis subsp. lactis lxl was used as the original strain, and the compound mutation method was applied to induce mutations. A high-yielding and genetically stable strain (Lactobacillus lactis A32) was identified, with the nisin titre raised by 332.2% up to 5089.29 IU/mL. Genome and transcriptome sequencing was used to analyse A32 and compare it with the original lxl strain. The comparative genomics results show that 107 genes in the A32 genome had mutations and most base mutations were not located in the four well-researched nisin-related operons, nisABTCIPRK, nisI, nisRK and nisFEG: 39 single-nucleotide polymorphisms (SNPs), 34 insertion mutations and 34 deletion mutations. The transcription results show that the expression of 92 genes changed significantly, with 27 of these differentially expressed genes upregulated, while 65 were downregulated. Our findings suggest that the output of nisin increased in L. lactis strain A32, which was accompanied by changes in the DNA replication-related gene dnaG, the ABC-ATPase transport-related genes patM and tcyC, the cysteine thiometabolism-related gene cysS, and the purine metabolism-related gene purL. Our study provides new insights into the traditional genetic mechanisms involved nisin production in L. lactis, which could provide clues for a more efficient metabolic engineering process.

Evaluation and Mathematical Analysis of a Four-Dimensional Lotka–Volterra-like Equation Designed to Describe the Batch Nisin Production System

Nisin, an antibacterial compound produced by Lactococcus lactis strains, has been approved by the US Food and Drug Administration to be used as a safe food additive to control the growth of undesirable pathogenic bacteria. Nisin is commonly described as a pH-dependent primary metabolite since its production depends on growth and culture pH evolution. However, the relationships between bacteriocin synthesis (BT), biomass production (X), culture pH, and the consumption of the limiting nutrient (total nitrogen: TN) have not been described until now. Therefore, this study aims to develop a competitive four-dimensional Lotka–Volterra-like Equation (predator-prey system) to describe these complex relationships in three series of batch fermentations with L. lactis CECT 539 in diluted whey (DW)-based media. The developed four-dimensional predator-prey system accurately described each individual culture, providing a good description of the relationships between pH, TN, X, and BT, higher values for R2 and F-ratios, lower values (&lt;10%) for the mean relative percentage deviation modulus, with bias and accuracy factor values approximately equal to one. The mathematical analysis of the developed equation showed the existence of one asymptotically stable equilibrium point, and the phase’s diagram obtained did not show the closed elliptic trajectories observed in biological predator-prey systems.

Harnessing cross-resistance – Sustainable nisin production from low-value food side streams using a Lactococcus lactis mutant with higher nisin-resistance obtained after prolonged chlorhexidine exposure

Nisin has a tendency to associate with the cell wall of the producing strain, which inhibits growth and lowers the ceiling for nisin production. With the premise that resistance to the cationic chlorhexidine could reduce nisin binding, variants with higher tolerance to this compound were isolated. One of the resistant isolates, AT0606, had doubled its resistance to nisin, and produced three times more free nisin, when cultured in shake flasks. Characterization revealed that AT0606 had an overall less negatively charged and thicker cell wall, and these changes appeared to be linked to a defect high-affinity phosphate uptake system, and a mutation inactivating the oleate hydratase. Subsequently, the potential of using AT0606 for cost efficient production of nisin was explored, and it was possible to attain a high titer of 13181 IU/mL using a fermentation substrate based on molasses and a by-product from whey protein hydrolysate production.