Variation of Amino acid composition in dried bovine dairy powders from a range of product streams

Variation of Amino acid composition in dried bovine dairy powders from a range of product streams | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Variation of Amino acid composition in dried bovine dairy powders from a range of product streams Simon Robert Gilmour, Stephen E. Holroyd, Maher D. Fuad, Dave Elgar, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4356289/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Multiple samples of a range of dairy powders were analysed for their amino acid (AA) content, allowing an in-depth analysis of the differences between their AA profiles and how various manufacturing processes give rise to the differences between product types. The products analysed were whole milk powder (WMP), skim milk powder (SMP), cheese whey protein concentrate (WPC-C), lactic acid whey protein concentrate (WPC-L), high fat whey protein concentrate (WPC-HF), hydrolysed whey protein concentrate (WPH) and demineralised whey protein (D90). Analysis demonstrated that WMP and SMP share broadly similar AA profiles with minor differences that were most probably due to the small levels of protein in milk fat, which is close to absent in SMP. When comparing WPC-C and WPC-L, there were higher levels of threonine, serine, glutamic acid, and proline in the former, but lower levels of tyrosine, phenylalanine and tryptophan. This is due to these products being separated from casein via different methods. WPI and WPC-HF show differences in the levels of every AA with the exception of histidine; they originate from similar sweet whey streams, but then processing diverges, resulting in the AA variation. D90 was consistently lower in every AA when compared with WPC-C; while both originate from sweet whey streams, D90 has a nanofiltration step in its manufacture that increases its non-protein nitrogen content, impacting its AA levels. Dairy Protein Amino Acid Whey Protein Concentrate Milk Powder Variability Protein Fractionation Nutritional Composition Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Dairy powders are a nutrient dense source of high-quality nutrition that are used as ingredients in a wide range of foods consumed by a variety of people across all age ranges. As public awareness of the importance of good nutrition in maintaining health and wellness continues to grow globally (Teodoro 2023 ), so too does demand for dairy worldwide, and this growing demand is expected to continue over the next decade (OECD/FAO 2022 ). Dairy proteins are amongst the highest quality available in the food supply due to a combination of their high digestibility and essential amino acid content (Mathai et al. 2017 ). Moreover, there is a wide variety of dairy powders that consist of different protein fractions, produced by a variety of processes, resulting in different amino acid (AA) profiles across these products. Dairy proteins are often categorised into two main types based solely on relative solubility, i.e., casein (contained in colloidal micelles) that makes up approximately 80% of the total protein, and whey that makes up the remaining 20%. However, there is more diversity in dairy proteins than suggested by this high-level categorisation. There are four principal caseins, α S1 -, α S2 -, β- and κ-casein, and four principal whey proteins, α-lactalbumin (α-Lac), β-lactoglobulin (BLG), bovine serum albumin (BSA) and immunoglobulins. In addition to these main proteins there are numerous minor proteins, including those associated with the milk fat globular membrane (MFGM), a phospholipid tri-layer that encapsulates milk fat (Fong and Norris 2009 ). Every one of these proteins has a unique AA profile that contributes to the overall AA profile of total milk protein; when these proteins are separated by the various processes utilised in the manufacture of dairy products the resultant AA profile is altered. For example, whole milk powder (WMP) and skim milk powder (SMP) contain both whey and casein while whey protein concentrate (WPC) derived products contain no intact casein, although they do contain variable amounts of casein derived peptides (e.g., caseinomacropeptides in sweet whey products). Higher fat products such as high fat whey protein concentrate (WPC-HF) contain higher levels of MFGM associated proteins while low-fat products such as whey protein isolate (WPI) contain little. Since each AA plays an important and often unique role in human health and wellbeing, knowledge of this variation is valuable for product design and formulation. For example, an accurate knowledge of the AA content of protein ingredients used for the production of infant formulas is essential as the AA requirements in such products are clearly defined via regulations. Previously this was not a major issue as manufacturers could simply add more protein and be confident the AA requirements would be met. However, there is now a movement towards lower protein formulations that more closely match the protein content of human breastmilk (Arnesen et al. 2022 ), meaning more consideration for AA content per gram of protein is required. In recent years there has also been considerable interest in determining the amino acid composition of a range of protein sources from non-animal sources (Gorissen et al. 2018 ). This paper aims to add to the knowledge of AA availability in our food supply by outlining the AA content of multiple dairy powders and provide some insight into the reasons for the variation found between different product types. Materials and methods Dairy powder samples and protein content Commercially available samples of WMP, SMP and a variety of WPCs were analysed for their AA composition. All samples were produced from NZ sourced milk, with the exception of the D90 samples, which were of European origin. Protein content was measured using near infrared spectroscopy (NIR) during production runs at the factories producing the powders. The NIR instruments are regularly calibrated via Kjeldahl testing of a range of representative samples. Protein analysis by Kjeldahl testing is based on the measurement of total nitrogen found in a sample (ISO 2014 ; Lynch and Barbano 1999 ). This inevitably leads to the measurement of not only nitrogen from protein, but nitrogen from non-protein sources as well. Due to this non-protein nitrogen (NPN) the Kjeldahl method utilises a protein conversion factor to convert total nitrogen to protein. This conversion factor varies based on the product type being assessed. For example, dairy products use a value of N = 6.38, while soy products use N = 6.25 due to its high NPN content (Tontisirin 2003 ). Amino acid analysis AA analysis of protein products is a two-step process comprising hydrolysis of proteins into free AAs followed by the separation and measurement of the level of the free AAs using HPLC. After hydrolysis in 6 n HCl for 24 h at approximately 110°C and further chemical stabilisation, samples were analysed using HPLC after pre-injection derivatisation. Prior to hydrolysis, the sulphur containing AAs cystine and methionine were oxidised to cysteic acid and methionine sulfone, respectively, and sodium metabisulphite was added to decompose the performic acid (AOAC 994.12; AOAC 2005a ). Additionally, under normal acid hydrolysis, glutamine and asparagine residues are converted to glutamic acid and aspartic acid, respectively, so the acids presented here include all residues. As tryptophan is destroyed by 6 n HCl hydrolysis, separate analysis using 4.2 m NaOH under vacuum to hydrolyse the protein was utilised (AOAC 988.15; AOAC 2005b ). Total nitrogen values were taken from the commercial grading process using near-infrared spectroscopy (NIR) analysis calibrated by reference Kjeldahl analysis. Total free AA contents of products are usually reported as "g 100 g − 1 powder"; however, this can be converted to "g 100 g − 1 protein" by dividing by the percentage protein content of the powder. Typically, the sum of the free AAs measured should ideally be around 114–116 g 100 g − 1 protein. The reason that this figure exceeds 100 g 100 g − 1 protein is that when a protein is hydrolysed by breaking the peptide bond between the AAs, one molecule of water is added to each AA, so increasing its weight. The individual AA levels in the profiles outlined in this paper have been presented in mg g − 1 protein to allow comparison of the AA content of the protein in each product. Results and discussion Variability within product groups One characteristic that is clearly demonstrated by our findings is the low variability in AA profiles within product groups (Table 1 ). This is expected as the expression of proteins in bovine milk is a tightly regulated biological process that will resist variability (Berry et al. 2020 ). If we accept that the AA profile of bovine milk is largely consistent, then the variability that is found within product groups can be attributed to variability either in the test methods used to establish the AA profiles or in the manufacturing process. It is possible to infer the variability of the testing based on the WPH results presented in this paper (Fig. 1 ) as all 15 of the samples were obtained from the same batch of powder and tested in the same laboratory. From this analysis it is apparent that the variability of the test is low, with the largest variations (as indicated by standard deviation, SD) being in isoleucine (SD = 1.68) valine (SD = 1.16), glutamic acid (SD = 1.09), serine (SD = 0.94) and tyrosine (SD = 0.82) with all others having a SD ≤ 0.6 (Table 1 ). Based on this, it is reasonable to assume that any variances appreciably larger than those found in these results are due to variability within the manufacturing process. With the exception of the WPH and the WMP, the samples analysed were manufactured across multiple years and across various months of those years. The low variability within the product types indicates that manufacturing process has limited impact on the variability of the AA profile. Table 1 Amino acid content of dried dairy powders a Amino acid Dairy powder WMP SMP WPC-C WPC-L WPI WPC-HF D90 WPH Alanine 33.6 ± 0.3 35.0 ± 0.4 58.9 ± 0.9 57.8 ± 1.1 58.3 ± 1.7 51.1 ± 1.25 50.7 ± 2.7 61.1 ± 0.5 Arginine 33.8 ± 0.9 34.8 ± 0.5 25.9 ± 0.7 28.7 ± 0.8 24.5 ± 1.0 27.4 ± 0.8 23.4 ± 1.3 30.1 ± 0.4 Aspartic acid 78.5 ± 1.3 80.9 ± 0.9 120.6 ± 1.7 121.4 ± 1.7 124.7 ± 2.9 115.4 ± 3.0 104.0 ± 3.4 120.7 ± 0.5 Cystine 7.3 ± 0.7 7.4 ± 0.3 28.3 ± 0.6 28.4 ± 0.7 35.5 ± 1.4 24.6 ± 1.0 23.4 ± 0.5 29.5 ± 0.4 Glutamic acid 208.5 ± 5.8 232.4 ± 2.7 189.2 ± 2.4 177.5 ± 3.3 188.1 ± 6.1 168.2 ± 3.1 166.6 ± 5.7 193.7 ± 1.1 Glycine 19.7 ± 0.4 20.0 ± 0.3 20.6 ± 0.3 20.7 ± 0.4 17.0 ± 0.6 21.8 ± 0.5 18.1 ± 0.8 20.2 ± 0.2 Histidine 27.2 ± 0.5 27.9 ± 0.4 19.0 ± 0.4 20.6 ± 0.4 19.3 ± 0.7 19.7 ± 0.4 18.0 ± 0.9 19.6 ± 0.1 Isoleucine 52.6 ± 1.3 48.8 ± 1.3 66.0 ± 2.7 51.3 ± 2.5 62.1 ± 2.2 61.5 ± 1.4 58.5 ± 2.5 56.9 ± 1.7 Leucine 96.5 ± 0.6 95.1 ± 1.3 114.1 ± 1.5 126.7 ± 2.4 141.5 ± 3.1 102.3 ± 2.9 103.0 ± 3.7 138.5 ± 0.6 Lysine 82.3 ± 0.7 84.2 ± 0.8 102.1 ± 1.5 106.4 ± 1.7 116.4 ± 2.2 92.1 ± 2.1 90.8 ± 3.4 110.3 ± 0.3 Methionine 23.8 ± 1.4 23.3 ± 0.7 25.0 ± 0.6 23.2 ± 0.5 25.8 ± 1.1 16.8 ± 0.8 20.7 ± 0.6 26.8 ± 0.4 Phenylalanine 48.1 ± 0.4 50.0 ± 0.6 33.5 ± 0.5 37.2 ± 0.9 37.1 ± 0.7 34.7 ± 0.5 31.4 ± 1.3 38.8 ± 0.2 Proline 96.0 ± 1.5 101.9 ± 1.2 65.5 ± 1.5 47.2 ± 1.1 45.5 ± 1.8 60.4 ± 3.7 59.5 ± 2.9 51.1 ± 0.6 Serine 53.9 ± 2.6 59.1 ± 1.2 58.1 ± 1.5 48.7 ± 0.9 38.3 ± 1.5 58.2 ± 1.0 48.4 ± 2.4 46.0 ± 0.9 Threonine 45.5 ± 1.0 46.6 ± 0.5 82.7 ± 1.5 59.8 ± 1.1 54.7 ± 1.5 79.2 ± 2.4 70.3 ± 2.8 57.3 ± 0.6 Tryptophan 15.6 ± 0.2 17.4 ± 0.5 23.8 ± 0.9 28.6 ± 0.6 30.2 ± 1.9 24.6 ± 1.1 20.1 ± 0.9 26.2 ± 0.3 Tyrosine 46.4 ± 1.0 43.7 ± 0.8 31.1 ± 0.6 34.8 ± 0.9 37.1 ± 0.7 31.6 ± 0.8 24.0 ± 1.3 39.9 ± 0.8 Valine 64.4 ± 2.4 63.2 ± 1.4 63.4 ± 2.3 51.9 ± 2.7 55.5 ± 1.8 62.7 ± 0.9 57.1 ± 2.1 59.5 ± 1.2 Total 1033.6 1071.8 1127.5 1070.6 1111.4 1052.3 988.0 1126.1 a Abbreviations are: WMP, whole milk powder; SMP, skim milk powder; WPC-C, cheese whey protein concentrate; WPC-L, lactic acid whey protein concentrate; WPI, whey protein isolate; WPC-HF, high fat whey protein concentrate; D90, demineralised whey protein; WPH, hydrolysed whey protein concentrate. Values (in mg g –1 protein, where protein = total nitrogen × 6.38) are means ± standard deviations (n = 15 for all powders except WMP and D90 where n = 4 and n = 11, respectively); values are rounded to one decimal place. Variability of whey protein hydrolysate Hydrolysed whey protein concentrate (WPH) is a concentrated whey stream that has undergone enzymatic hydrolysis to break down the proteins into smaller peptides. As commented above, the 15 WPH samples analysed all originated from the same batch and were tested in the same laboratory. Assuming homogeneity of the sample, the AA content of each sample should be the same. As can been seen in Fig. 1 , the variability within these samples is very low, indicating a high level of accuracy. The WPH analysed originates from an acid whey stream produced via acidification of milk (Blyund 2023 ), this means the caseinomacropeptide (CMP) protein fraction is retained in the casein fraction, in contrast to WPC-C that is enriched in CMP due to the cleavage of κ-casein with chymosin-like enzyme. Variability between WMP and SMP On a per gram of protein basis, both WMP and SMP exhibit similar AA profiles due to both products containing whey and casein proteins in a similar ratio to the raw milk (Fig. 2 ). The principal disparity between the two is that SMP undergoes processing to remove fat prior to drying (Blyund 2023 ). Our data do, however, show differences between the two products. Analysis shows that 12 of the 18 AAs are statistically different between the products ( p ≤ 0.05). However, the mean difference between them is low (less than 6 mg g − 1 protein) and so the differences will be of little consequence from a nutritional or functional point of view. There is, however, a clear variation in glutamic acid (this result represents both the glutamate and glutamine content of the product); WMP has a mean glutamic acid value of 217 mg g − 1 protein and SMP has a mean value of 237 mg g − 1 protein with the mean difference between the samples being 23.93 mg g − 1 protein. These observed differences may be explained by the presence of MFGM in the WMP. MFGM is a phospholipid tri-layer that surrounds the fat globules in milk and is perforated with a variety of glycosylated and non-glycosylated proteins such as mucin 1 (MUC 1), xanthine oxidase (XO), CD36 (PAS 4), mucin 15 (PAS 3), butyrophilin (BTN), PAS 6/7, adipophilin (ADPH), and fatty acid binding protein (FABP) (Fong and Norris 2009 ). The protein content of isolated MFGM has been reported to be between 22.3 and 28% (Fong et al. 2007 ; Kanno and Kim 1990 ) Variability between WPC-C and WPC-L The whey powders analysed here are produced in a variety of ways; this has a direct impact on their resulting AA profiles. A key difference is in how the whey is initially separated from casein. This can be done in several ways with two of the most common being (i) via the addition of rennet or chymosin to milk (in the cheese making and rennet casein process), and (ii) via the action of acid on milk (either via direct addition or from fermentation using lactic acid producing bacteria) (Blyund 2023 ). Both methods result in the coagulation of casein and its separation from whey, albeit via different mechanism. The addition of rennet or chymosin achieves aggregation of casein micelles by enzymatically cleaving the κ-casein off the exterior of the casein micelle, while the addition of acid nullifies the negative charge of the κ-casein (Lucey 2002 ). This disruption of the κ-casein peptide via these methods removes the steric repulsion of casein micelles provided by the negative charge on the κ-casein peptide (Vasbinder et al. 2003 ) allowing the casein micelles to aggregate and form a curd. When κ-casein is cleaved off the casein micelle by the addition of rennet or chymosin, insoluble para-κ-casein and water soluble caseinomacropeptide (CMP) are created (Lucey 2002 ). The soluble CMP is now associated with the whey protein fraction, while the para-κ-casein stays with the casein. On the other hand, when milk is acidified the κ-casein is not cleaved into CMP and para-κ-casein and is retained in the aggregated casein micelle curd instead of solubilising into the whey stream. CMP has its own unique AA profile that Neelima et al. (2013) describe as rich in threonine, serine, glutamine (converted to glutamic acid during analysis) and proline while being devoid of tyrosine, phenylalanine, cystine, isoleucine, valine, and tryptophan. Hence the presence or absence of CMP in a whey protein stream will result in a change in the overall AA composition. This is clearly observed when comparing WPC-C and WPC-L (Fig. 3 ). The WPC-C analysed here was produced via a renneting process and therefore contains CMP, while the WPC-L was produced via acidification and does not contain CMP (although other casein peptides may arise during fermentation). Threonine, serine, glutamic acid and proline are clearly present in higher levels in the CMP-containing WPC-C product compared with the WPC-L product, with mean differences of 22.92, 9.33, 11.73 and 18.27, respectively, while tyrosine, phenylalanine and tryptophan are lower in the WPC-C. The magnitude of this difference is smaller with the respective mean differences being − 3.67, − 3.71 and − 4.74. The magnitude of these differences is probably irrelevant for most applications. Cystine, on the other hand, shows no significant difference ( p = 0.753) between the two products. We do, however, see significant differences in the branch chain AAs valine, isoleucine and leucine with mean differences of 11.45, 14.65 and − 12.66, respectively. This is not predicted by the Neelima et al. (2013) analysis of CMP AA content, which is particularly notable as these branched chain AAs are involved in key metabolic roles such as glycogen synthesis (Monirujjaman and Ferdouse, 2014 , Peyrollier et al. 2000 ) and muscle protein synthesis. Leucine is considered the key signalling molecule for muscle protein synthesis (Monirujjaman and Ferdouse 2014 ) making it particularly important in products focused on muscle health and making these results particularly relevant to manufacturers of sports nutrition foods or supplements. Variability between WPI and WPC-HF The production of WPI from cheese whey typically uses either ion exchange (IX) or microfiltration (MF). Both processes produce a WPI stream devoid of fat and reduced in lactose as well as a co-product stream that has an elevated fat content, which is usually described as a high fat WPC (WPC-HF). The WPI and WPC-HF products analysed here show AA profiles substantially different from each other (Fig. 4 ); while they originate from similar sweet whey (WPC-C) streams the WPI was produced via an IX process and the WPC-HF was produced via a MF process. In the MF process the permeate (the portion that passes through the membrane filter) becomes the WPI stream, whilst the retentate (the portion that does not pass through the membrane) becomes the WPC-HF stream (Blyund 2023 ). As the WPC-HF retentate retains all the fat from the original sweet whey, all the proteins present in the MFGM are retained in this stream. The WPC-HF analysed here was produced via a MF process that included acidification and heating of the whey stream prior to the MF step resulting in the precipitation of minor proteins α-Lac, BSA and IgG, causing the WPC-HF to become enriched in these components, but depleted of minor proteins BLG and CMP. In the case of WPI produced via MF, the BLG and CMP would move into the accompanying WPI stream. However, the WPI analysed here was not produced from microfiltration of a sweet whey stream, but rather via an IX process. While the IX process still results in a WPI stream and a WPC-HF stream that parallels the MF process, the protein species are separated in such a way that the WPI rather than the WPC-HF is enriched in α-Lac and BSA. The WPI also retains an increased concentration of BLG. The WPC-HF stream resulting from the IX process also becomes enriched in CMP protein species. This results in this WPI having an AA profile somewhat similar to that of acid WPC, whereas a standard MF WPI process would be expected to result in a product more similar to a cheese WPC. The IX process also has the additional impact of separating the protein from the majority of non-protein nitrogen (NPN) in the stream (e.g., urea, nitrogen containing vitamins). Since (crude) protein content is determined by measuring total nitrogen content, the removal of NPN results in a lower total protein value than would be measured if the NPN was retained. This then impacts the calculation of AA content when expressed as mg g − 1 protein, as it will make the AA content per gram of protein slightly higher than it would be if the NPN was retained. These differences are clearly evidenced in Fig. 4 , where an obvious difference can be seen between almost every AA (with the exception of histidine). The mean difference in histidine between the two products is − 1.66 with no significant difference between the values found ( p = 0.113). If a comparison was to be made between a WPI and a WPC-HF that were both produced via the MF process, or both by the IX process, the AA profiles would be expected to be even more divergent as only one of the two would be enriched in α-Lac and BSA rather than both, which is the case here. Variability between D90 and WPC-C Demineralised whey (D90), as suggested by the name, has a mineral content (i.e., sodium and chlorine) lower than that of regular whey proteins (Blyund 2023 ), making it a useful ingredient for applications where mineral levels need to be carefully managed, e.g., infant formulas. The demineralisation process can be achieved via nanofiltration, electrolysis, ion exchange, or a combination of these processes. The D90 product analysed in this paper was produced via a combination of the nanofiltration and ion exchange processes and originates from a renneted sweet whey stream. The protein concentrating nanofiltration step causes the loss of some NPN resulting in a lower NPN content than that of the sweet whey stream from which it is derived. It will therefore have a higher total AA content per gram of crude protein than a standard non-nanofiltrated sweet whey powder. However, it will still contain more NPN than a standard WPC80 as the WPC80 undergoes ultrafiltration, which is much more effective at removing NPN. As can be seen in Fig. 5 , each AA in the D90 product is consistently lower than in the WPC-C product. However, if the AA contents of these products are compared as a percentage of total AAs, which effectively corrects for NPN content, the resulting profiles are essentially the same (Fig. 6 ). This is due to the fact that the D90 is just a demineralised sweet whey with some NPN removed and the WPC-C is an ultrafiltered sweet whey with more NPN removed. Conclusions The data outlined in this paper clearly describe the differences in AA profiles in a variety of dairy powders. These differences come down to two main factors: (i) changes in NPN distorting the apparent AA content when expressed on a per g of protein basis and (ii) the fractionation of the different classes of proteins into different product streams. When interpreting AA profiles both of these factors should be considered, it should be kept in mind that the distortion of AA profiles due to changes in NPN is just that, a distortion. The absolute AA content has not been changed, it only appears that way due to the fact protein testing is not a direct measurement of protein, but rather a calculation based on total nitrogen content. On the other hand, differences in AA profiles that are due to the fractionation of protein can result in real, and sometimes significant, changes to AA profiles. Categorisation of proteins can be done at multiple levels, as evidenced in milk, which can be defined at the most basic level as whey and casein. These classifications can then be broken down further into four primary caseins (α S1 -, α S2 -, β- and κ-caseins) and four primary whey proteins (α-Lac, BLG, BSA and immunoglobulins). Each of these proteins are made up of unique sequences of amino acids, meaning that when they eventuate in different product streams the resulting product will have an altered AA profile. While tools such as protein quality scores (e.g., DIAAS) provide useful insights into the overall quality of protein for consumers, understanding the specific differences in AA profiles of various powders is an important consideration when utilising them as ingredients into nutritional applications. For example, manufacturers of sports products may want to target ingredients high in leucine and other branched chain amino acids, while an infant formula manufacturer may need to know the levels of all the essential AAs to be able create a product that provides enough to support healthy infant growth and development while still maintaining appropriate protein levels. This paper provides a valuable insight into the nutritional differences between various dairy powders, as well as providing the context as to why these differences arise. Declarations Acknowledgements Funding This study was supported by Fonterra Co-operative Group Ltd Conflict of interest The authors have no relevant financial or non-financial interests to disclose. References Arnesen EK, Thorisdottir B, Lamberg-Allardt C, Bärebring L, Nwaru B, Dierkes J, Ramel A, Åkesson, A (2022) Protein intake in children and growth and risk of overweight or obesity: A systematic review and meta-analysis. Food Nut Res 66:8242. doi: 10.29219/fnr.v66.8242 AOAC (2005a) Official method 994.12-1997. Amino acids in feeds. Performic acid oxidation. In: Official methods of analysis of AOAC International 18 th edn. AOAC International, Gaithersburg AOAC (2005b) Official method 988.15-1988. Tryptophan in foods and food and feed ingredients - ion exchange chromatographic method. In: Official methods of analysis of AOAC International 18 th edn. AOAC International, Gaithersburg Berry S, Sheehy P, Williamson P, Sharp J, Menzies K, Lefèvre C, Snell, R (2020) Defining the origin and function of bovine milk proteins through genomics: the biological implications of manipulation and modification. In: Boland M, Singh H (eds) Milk proteins: From expression to food, 3rd edn. Academic Press, London, pp 143–171. https://doi.org/10.1016/B978-0-12-815251-5.00004-9 Blyund G (2023) Dairy processing handbook. Tetra Pak Processing Systems, Lund. https://dairyprocessinghandbook.tetrapak.com Fong BY, Norris CS (2009) Quantification of milk fat globule membrane proteins using selected reaction monitoring mass spectrometry. J Ag Food Chem 57:6021–6028. https://doi.org/10.1021/jf900511t Fong BY, Norris CS, MacGibbon AK (2007) Protein and lipid composition of bovine milk-fat-globule membrane. Int Dairy J 17:275–288. https://doi.org/10.1016/j.idairyj.2006.05.004 Gorissen SH, Crombag JJ, Senden JM, Waterval WH, Bierau J, Verdijk LB, van Loon LJ (2018) Protein content and amino acid composition of commercially available plant-based protein isolates. Amino Acids 50:1685–1695. https://doi.org/10.1007/s00726-018-2640-5 ISO (2014) Milk and milk products – Determination of nitrogen content – Part 1: Kjeldahl principle and crude protein calculation (ISO 8968-1:2014|IDF 20-12014). Geneva, International Standardisation Organisation. https://wwwisoorg/standard/61020html Kanno C, Kim DH (1990) A simple procedure for the preparation of bovine milk fat globule membrane and a comparison of its composition, enzymatic activities, and electrophoretic properties with those prepared by other methods. Ag Biol Chem 54:2845–2854. https://doi.org/10.1080/00021369.1990.10870405 Lucey JA (2002) Formation and physical properties of milk protein gels. J Dairy Sci 85:281–294. https://doi.org/10.3168/jds.S0022-0302(02)74078-2 Lynch JM, Barbano DM (1999) Kjeldahl nitrogen analysis as a reference method for protein determination in dairy products J AOAC Int 82:1389–1398. https://doi.org/10.1093/jaoac/82.6.1389 Mathai JK, Liu Y, Stein HH (2017) Values for digestible indispensable amino acid scores (DIAAS) for some dairy and plant proteins may better describe protein quality than values calculated using the concept for protein digestibility-corrected amino acid scores (PDCAAS) Brit J Nut 117:490–499. https://doi.org/10.1017/S0007114517000125 Monirujjaman MD, Ferdouse A (2014) Metabolic and physiological roles of branched-chain amino acids. Adv. Mol. Biol. 2014: 364976. https://doi.org/10.1155/2014/364976 Neelima, Sharm R, Rajpu YS, Mann B (2013) Chemical and functional properties of glycomacropeptide (CMP) and its role in the detection of cheese whey adulteration in milk: a review. Dairy Sci Tech 93:21–43. https://doi.org/10.1007/s13594-012-0095-0 OECD/FAO (2022) OECD-FAO agricultural outlook 2022–2031. OECD Publishing, Paris https://doiorg/101787/f1b0b29c-en Peyrollier K, Hajduch E, Blair AS, Hyde R, Hundal HS (2000) l-Leucine availability regulates phosphatidylinositol 3-kinase, p70 S6 kinase and glycogen synthase kinase-3 activity in L6 muscle cells: evidence for the involvement of the mammalian target of rapamycin (mTOR) pathway in the l-leucine-induced up-regulation of system amino acid transport. Biochem J 350:361–368. https://doi.org/10.1042/bj3500361 Teodoro M (2023) The future of nutrition: Health and wellness: 2023 [Industry Report] Mintel, London. https://wwwmintelcom/ Tontisirin K (2003) Food energy: Methods of analysis and conversion factors: Report of a technical workshop, Rome, 3–6 December 2002 (Publication No ISSN0254-4725). Food and Agriculture Organisation of the United Nations, Rome. https://wwwfaoorg Vasbinder AJ, Rollema HS, Bot A, De Kruif CG (2003) Gelation mechanism of milk as influenced by temperature and pH; studied by the use of transglutaminase cross-linked casein micelles. J Dairy Sci 86:1556–1563. https://doi.org/10.3168/jds.S0022-0302(03)73741-2 Additional Declarations Competing interest reported. Authors of the paper are employees of Fonterra Co-operative Ltd. The paper outlines the AA profiles of bovine dairy powders produced by Fonterra and the differences between the products. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4356289","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":299512533,"identity":"848c064d-8ffc-4a3a-a808-5bb4ce69efb0","order_by":0,"name":"Simon Robert Gilmour","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYDACZgjFwwemKuRI0MIGps4YMzCwEWsbWCFjGxFa5Nu5Ex8XMNTKsIkdPiZdOc9Azly++djnihoGeX6xA1i1GBzm3Ww8g+E4D5t0Wprk2W0GxpZtbMkzzxxjMJw5OwG7FmbebdI8DMeAWnLMJBu3/UnccIzHmLGBjSHB4DZ2LfLNKFrmGAC18H9mbPiHWwvDYbCWGqiWBpAWHmbGxjbcWsB+4TE4APJLsmXDMQNjg2NpxoyNfRI4/SLff3bjY56KOnt+6eSDNxtqDOQMDh9+zNjwzUaeXxqHw6B2YQhJ4FEOBnWEFIyCUTAKRsFIBgCtqU4WXGTiBwAAAABJRU5ErkJggg==","orcid":"","institution":"Fonterra (New Zealand)","correspondingAuthor":true,"prefix":"","firstName":"Simon","middleName":"Robert","lastName":"Gilmour","suffix":""},{"id":299512534,"identity":"8d79ebb1-6618-4677-87e0-b166b6c6ce05","order_by":1,"name":"Stephen E. Holroyd","email":"","orcid":"","institution":"Fonterra (New Zealand)","correspondingAuthor":false,"prefix":"","firstName":"Stephen","middleName":"E.","lastName":"Holroyd","suffix":""},{"id":299512535,"identity":"15ff6b37-38fe-45e8-b3d0-a93a6008cef3","order_by":2,"name":"Maher D. Fuad","email":"","orcid":"","institution":"Fonterra (New Zealand)","correspondingAuthor":false,"prefix":"","firstName":"Maher","middleName":"D.","lastName":"Fuad","suffix":""},{"id":299512536,"identity":"d17451ca-2fcf-413a-bd4f-322316c43b1d","order_by":3,"name":"Dave Elgar","email":"","orcid":"","institution":"Fonterra (New Zealand)","correspondingAuthor":false,"prefix":"","firstName":"Dave","middleName":"","lastName":"Elgar","suffix":""},{"id":299512537,"identity":"e9991ba3-bfde-4144-a275-318ba5a0c1ec","order_by":4,"name":"Aaron C. Fanning","email":"","orcid":"","institution":"Fonterra (New Zealand)","correspondingAuthor":false,"prefix":"","firstName":"Aaron","middleName":"C.","lastName":"Fanning","suffix":""}],"badges":[],"createdAt":"2024-05-02 01:16:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4356289/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4356289/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56067808,"identity":"bd92b3a4-bd20-4a98-99ca-8d7cf8cd1c46","added_by":"auto","created_at":"2024-05-08 06:42:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":33545,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4356289/v1/570a121e711a9177426baa80.png"},{"id":56067809,"identity":"215e9723-dd67-4fc8-8c02-92d608cff9d1","added_by":"auto","created_at":"2024-05-08 06:42:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":37849,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4356289/v1/0d866a47f434e2a074b54c76.png"},{"id":56068618,"identity":"385049b7-2d98-4b24-bb26-40655dc7ad09","added_by":"auto","created_at":"2024-05-08 06:50:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":38373,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4356289/v1/75c63544782f25da5d7659fe.png"},{"id":56067814,"identity":"cadb1aeb-ea03-4fc2-9cbe-8aa8fcc0943b","added_by":"auto","created_at":"2024-05-08 06:42:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":215909,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4356289/v1/a76efbe6aaa3c494a54f323c.png"},{"id":56067813,"identity":"1170988b-8076-40b5-bd11-8bba86d7eac7","added_by":"auto","created_at":"2024-05-08 06:42:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":25432,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4356289/v1/19173f45cc75202ce2536b7a.png"},{"id":56067810,"identity":"93a9ccd7-d55a-47e5-9ffb-a6d4a8577640","added_by":"auto","created_at":"2024-05-08 06:42:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":50889,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4356289/v1/545fa32af946fb35621ac3fb.png"},{"id":56957074,"identity":"7644c8f0-65ea-4e97-a8a4-67c946d87994","added_by":"auto","created_at":"2024-05-22 16:19:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":899496,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4356289/v1/7c80e3f8-2ae6-4f37-9afe-9baa3190de67.pdf"}],"financialInterests":"Competing interest reported. Authors of the paper are employees of Fonterra Co-operative Ltd. The paper outlines the AA profiles of bovine dairy powders produced by Fonterra and the differences between the products.","formattedTitle":"Variation of Amino acid composition in dried bovine dairy powders from a range of product streams","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDairy powders are a nutrient dense source of high-quality nutrition that are used as ingredients in a wide range of foods consumed by a variety of people across all age ranges. As public awareness of the importance of good nutrition in maintaining health and wellness continues to grow globally (Teodoro \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), so too does demand for dairy worldwide, and this growing demand is expected to continue over the next decade (OECD/FAO \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Dairy proteins are amongst the highest quality available in the food supply due to a combination of their high digestibility and essential amino acid content (Mathai et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Moreover, there is a wide variety of dairy powders that consist of different protein fractions, produced by a variety of processes, resulting in different amino acid (AA) profiles across these products.\u003c/p\u003e \u003cp\u003eDairy proteins are often categorised into two main types based solely on relative solubility, i.e., casein (contained in colloidal micelles) that makes up approximately 80% of the total protein, and whey that makes up the remaining 20%. However, there is more diversity in dairy proteins than suggested by this high-level categorisation. There are four principal caseins, α\u003csub\u003eS1\u003c/sub\u003e-, α\u003csub\u003eS2\u003c/sub\u003e-, β- and κ-casein, and four principal whey proteins, α-lactalbumin (α-Lac), β-lactoglobulin (BLG), bovine serum albumin (BSA) and immunoglobulins. In addition to these main proteins there are numerous minor proteins, including those associated with the milk fat globular membrane (MFGM), a phospholipid tri-layer that encapsulates milk fat (Fong and Norris \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEvery one of these proteins has a unique AA profile that contributes to the overall AA profile of total milk protein; when these proteins are separated by the various processes utilised in the manufacture of dairy products the resultant AA profile is altered. For example, whole milk powder (WMP) and skim milk powder (SMP) contain both whey and casein while whey protein concentrate (WPC) derived products contain no intact casein, although they do contain variable amounts of casein derived peptides (e.g., caseinomacropeptides in sweet whey products). Higher fat products such as high fat whey protein concentrate (WPC-HF) contain higher levels of MFGM associated proteins while low-fat products such as whey protein isolate (WPI) contain little.\u003c/p\u003e \u003cp\u003eSince each AA plays an important and often unique role in human health and wellbeing, knowledge of this variation is valuable for product design and formulation. For example, an accurate knowledge of the AA content of protein ingredients used for the production of infant formulas is essential as the AA requirements in such products are clearly defined via regulations. Previously this was not a major issue as manufacturers could simply add more protein and be confident the AA requirements would be met. However, there is now a movement towards lower protein formulations that more closely match the protein content of human breastmilk (Arnesen et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), meaning more consideration for AA content per gram of protein is required.\u003c/p\u003e \u003cp\u003eIn recent years there has also been considerable interest in determining the amino acid composition of a range of protein sources from non-animal sources (Gorissen et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This paper aims to add to the knowledge of AA availability in our food supply by outlining the AA content of multiple dairy powders and provide some insight into the reasons for the variation found between different product types.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDairy powder samples and protein content\u003c/h2\u003e \u003cp\u003eCommercially available samples of WMP, SMP and a variety of WPCs were analysed for their AA composition. All samples were produced from NZ sourced milk, with the exception of the D90 samples, which were of European origin.\u003c/p\u003e \u003cp\u003eProtein content was measured using near infrared spectroscopy (NIR) during production runs at the factories producing the powders. The NIR instruments are regularly calibrated via Kjeldahl testing of a range of representative samples. Protein analysis by Kjeldahl testing is based on the measurement of total nitrogen found in a sample (ISO \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lynch and Barbano \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). This inevitably leads to the measurement of not only nitrogen from protein, but nitrogen from non-protein sources as well. Due to this non-protein nitrogen (NPN) the Kjeldahl method utilises a protein conversion factor to convert total nitrogen to protein. This conversion factor varies based on the product type being assessed. For example, dairy products use a value of N\u0026thinsp;=\u0026thinsp;6.38, while soy products use N\u0026thinsp;=\u0026thinsp;6.25 due to its high NPN content (Tontisirin \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAmino acid analysis\u003c/h2\u003e \u003cp\u003eAA analysis of protein products is a two-step process comprising hydrolysis of proteins into free AAs followed by the separation and measurement of the level of the free AAs using HPLC.\u003c/p\u003e \u003cp\u003eAfter hydrolysis in 6 \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003en\u003c/span\u003e HCl for 24 h at approximately 110\u0026deg;C and further chemical stabilisation, samples were analysed using HPLC after pre-injection derivatisation. Prior to hydrolysis, the sulphur containing AAs cystine and methionine were oxidised to cysteic acid and methionine sulfone, respectively, and sodium metabisulphite was added to decompose the performic acid (AOAC 994.12; AOAC \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005a\u003c/span\u003e). Additionally, under normal acid hydrolysis, glutamine and asparagine residues are converted to glutamic acid and aspartic acid, respectively, so the acids presented here include all residues. As tryptophan is destroyed by 6 \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003en\u003c/span\u003e HCl hydrolysis, separate analysis using 4.2 \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003em\u003c/span\u003e NaOH under vacuum to hydrolyse the protein was utilised (AOAC 988.15; AOAC \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2005b\u003c/span\u003e). Total nitrogen values were taken from the commercial grading process using near-infrared spectroscopy (NIR) analysis calibrated by reference Kjeldahl analysis.\u003c/p\u003e \u003cp\u003eTotal free AA contents of products are usually reported as \"g 100 g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e powder\"; however, this can be converted to \"g 100 g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e protein\" by dividing by the percentage protein content of the powder. Typically, the sum of the free AAs measured should ideally be around 114\u0026ndash;116 g 100 g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e protein. The reason that this figure exceeds 100 g 100 g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e protein is that when a protein is hydrolysed by breaking the peptide bond between the AAs, one molecule of water is added to each AA, so increasing its weight. The individual AA levels in the profiles outlined in this paper have been presented in mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e protein to allow comparison of the AA content of the protein in each product.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eVariability within product groups\u003c/h2\u003e \u003cp\u003eOne characteristic that is clearly demonstrated by our findings is the low variability in AA profiles within product groups (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This is expected as the expression of proteins in bovine milk is a tightly regulated biological process that will resist variability (Berry et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). If we accept that the AA profile of bovine milk is largely consistent, then the variability that is found within product groups can be attributed to variability either in the test methods used to establish the AA profiles or in the manufacturing process. It is possible to infer the variability of the testing based on the WPH results presented in this paper (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) as all 15 of the samples were obtained from the same batch of powder and tested in the same laboratory. From this analysis it is apparent that the variability of the test is low, with the largest variations (as indicated by standard deviation, SD) being in isoleucine (SD\u0026thinsp;=\u0026thinsp;1.68) valine (SD\u0026thinsp;=\u0026thinsp;1.16), glutamic acid (SD\u0026thinsp;=\u0026thinsp;1.09), serine (SD\u0026thinsp;=\u0026thinsp;0.94) and tyrosine (SD\u0026thinsp;=\u0026thinsp;0.82) with all others having a SD\u0026thinsp;\u0026le;\u0026thinsp;0.6 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Based on this, it is reasonable to assume that any variances appreciably larger than those found in these results are due to variability within the manufacturing process. With the exception of the WPH and the WMP, the samples analysed were manufactured across multiple years and across various months of those years. The low variability within the product types indicates that manufacturing process has limited impact on the variability of the AA profile.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAmino acid content of dried dairy powders \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAmino acid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e \u003cp\u003eDairy powder\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWMP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSMP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWPC-C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWPC-L\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWPI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWPC-HF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eD90\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWPH\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlanine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e57.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e58.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e51.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e50.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e61.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArginine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e28.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e27.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e23.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e30.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAspartic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e78.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e80.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e120.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e121.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e124.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e115.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e104.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e120.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCystine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e28.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e35.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e24.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e23.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e29.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlutamic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e208.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e232.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e189.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e177.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e188.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e168.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e166.6\u0026thinsp;\u0026plusmn;\u0026thinsp;5.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e193.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlycine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e17.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e18.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e20.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHistidine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e19.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e19.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e18.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e19.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsoleucine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e51.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e62.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e61.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e58.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e56.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeucine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e96.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e114.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e126.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e141.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e102.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e103.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e138.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLysine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e82.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e84.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e102.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e106.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e116.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e92.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e90.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e110.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethionine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e20.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e26.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhenylalanine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e37.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e37.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e34.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e31.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e38.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e96.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e101.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e65.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e47.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e45.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e60.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e59.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e51.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSerine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e59.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e48.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e38.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e58.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e48.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e46.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThreonine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e59.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e54.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e79.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e70.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e57.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTryptophan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e28.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e24.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e20.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e26.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTyrosine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e46.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e34.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e37.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e31.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e24.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e39.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eValine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e63.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e63.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e51.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e55.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e62.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e57.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e59.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1033.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1071.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1127.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1070.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1111.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1052.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e988.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1126.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003e Abbreviations are: WMP, whole milk powder; SMP, skim milk powder; WPC-C, cheese whey protein concentrate; WPC-L, lactic acid whey protein concentrate; WPI, whey protein isolate; WPC-HF, high fat whey protein concentrate; D90, demineralised whey protein; WPH, hydrolysed whey protein concentrate. Values (in mg g\u003csup\u003e\u0026ndash;1\u003c/sup\u003e protein, where protein\u0026thinsp;=\u0026thinsp;total nitrogen \u0026times; 6.38) are means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (n\u0026thinsp;=\u0026thinsp;15 for all powders except WMP and D90 where n\u0026thinsp;=\u0026thinsp;4 and n\u0026thinsp;=\u0026thinsp;11, respectively); values are rounded to one decimal place.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eVariability of whey protein hydrolysate\u003c/h2\u003e \u003cp\u003eHydrolysed whey protein concentrate (WPH) is a concentrated whey stream that has undergone enzymatic hydrolysis to break down the proteins into smaller peptides. As commented above, the 15 WPH samples analysed all originated from the same batch and were tested in the same laboratory. Assuming homogeneity of the sample, the AA content of each sample should be the same. As can been seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the variability within these samples is very low, indicating a high level of accuracy.\u003c/p\u003e \u003cp\u003eThe WPH analysed originates from an acid whey stream produced via acidification of milk (Blyund \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), this means the caseinomacropeptide (CMP) protein fraction is retained in the casein fraction, in contrast to WPC-C that is enriched in CMP due to the cleavage of κ-casein with chymosin-like enzyme.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eVariability between WMP and SMP\u003c/h2\u003e \u003cp\u003eOn a per gram of protein basis, both WMP and SMP exhibit similar AA profiles due to both products containing whey and casein proteins in a similar ratio to the raw milk (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The principal disparity between the two is that SMP undergoes processing to remove fat prior to drying (Blyund \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Our data do, however, show differences between the two products. Analysis shows that 12 of the 18 AAs are statistically different between the products (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05). However, the mean difference between them is low (less than 6 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e protein) and so the differences will be of little consequence from a nutritional or functional point of view.\u003c/p\u003e \u003cp\u003eThere is, however, a clear variation in glutamic acid (this result represents both the glutamate and glutamine content of the product); WMP has a mean glutamic acid value of 217 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e protein and SMP has a mean value of 237 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e protein with the mean difference between the samples being 23.93 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e protein. These observed differences may be explained by the presence of MFGM in the WMP. MFGM is a phospholipid tri-layer that surrounds the fat globules in milk and is perforated with a variety of glycosylated and non-glycosylated proteins such as mucin 1 (MUC 1), xanthine oxidase (XO), CD36 (PAS 4), mucin 15 (PAS 3), butyrophilin (BTN), PAS 6/7, adipophilin (ADPH), and fatty acid binding protein (FABP) (Fong and Norris \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The protein content of isolated MFGM has been reported to be between 22.3 and 28% (Fong et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Kanno and Kim \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1990\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eVariability between WPC-C and WPC-L\u003c/h2\u003e \u003cp\u003eThe whey powders analysed here are produced in a variety of ways; this has a direct impact on their resulting AA profiles. A key difference is in how the whey is initially separated from casein. This can be done in several ways with two of the most common being (i) via the addition of rennet or chymosin to milk (in the cheese making and rennet casein process), and (ii) via the action of acid on milk (either via direct addition or from fermentation using lactic acid producing bacteria) (Blyund \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Both methods result in the coagulation of casein and its separation from whey, albeit via different mechanism. The addition of rennet or chymosin achieves aggregation of casein micelles by enzymatically cleaving the κ-casein off the exterior of the casein micelle, while the addition of acid nullifies the negative charge of the κ-casein (Lucey \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). This disruption of the κ-casein peptide via these methods removes the steric repulsion of casein micelles provided by the negative charge on the κ-casein peptide (Vasbinder et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) allowing the casein micelles to aggregate and form a curd.\u003c/p\u003e \u003cp\u003eWhen κ-casein is cleaved off the casein micelle by the addition of rennet or chymosin, insoluble para-κ-casein and water soluble caseinomacropeptide (CMP) are created (Lucey \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The soluble CMP is now associated with the whey protein fraction, while the para-κ-casein stays with the casein. On the other hand, when milk is acidified the κ-casein is not cleaved into CMP and para-κ-casein and is retained in the aggregated casein micelle curd instead of solubilising into the whey stream.\u003c/p\u003e \u003cp\u003eCMP has its own unique AA profile that Neelima et al. (2013) describe as rich in threonine, serine, glutamine (converted to glutamic acid during analysis) and proline while being devoid of tyrosine, phenylalanine, cystine, isoleucine, valine, and tryptophan. Hence the presence or absence of CMP in a whey protein stream will result in a change in the overall AA composition. This is clearly observed when comparing WPC-C and WPC-L (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The WPC-C analysed here was produced via a renneting process and therefore contains CMP, while the WPC-L was produced via acidification and does not contain CMP (although other casein peptides may arise during fermentation).\u003c/p\u003e \u003cp\u003eThreonine, serine, glutamic acid and proline are clearly present in higher levels in the CMP-containing WPC-C product compared with the WPC-L product, with mean differences of 22.92, 9.33, 11.73 and 18.27, respectively, while tyrosine, phenylalanine and tryptophan are lower in the WPC-C. The magnitude of this difference is smaller with the respective mean differences being \u0026minus;\u0026thinsp;3.67, \u0026minus;\u0026thinsp;3.71 and \u0026minus;\u0026thinsp;4.74. The magnitude of these differences is probably irrelevant for most applications. Cystine, on the other hand, shows no significant difference (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.753) between the two products. We do, however, see significant differences in the branch chain AAs valine, isoleucine and leucine with mean differences of 11.45, 14.65 and \u0026minus;\u0026thinsp;12.66, respectively. This is not predicted by the Neelima et al. (2013) analysis of CMP AA content, which is particularly notable as these branched chain AAs are involved in key metabolic roles such as glycogen synthesis (Monirujjaman and Ferdouse, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Peyrollier et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) and muscle protein synthesis. Leucine is considered the key signalling molecule for muscle protein synthesis (Monirujjaman and Ferdouse \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) making it particularly important in products focused on muscle health and making these results particularly relevant to manufacturers of sports nutrition foods or supplements.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eVariability between WPI and WPC-HF\u003c/h2\u003e \u003cp\u003eThe production of WPI from cheese whey typically uses either ion exchange (IX) or microfiltration (MF). Both processes produce a WPI stream devoid of fat and reduced in lactose as well as a co-product stream that has an elevated fat content, which is usually described as a high fat WPC (WPC-HF).\u003c/p\u003e \u003cp\u003eThe WPI and WPC-HF products analysed here show AA profiles substantially different from each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e); while they originate from similar sweet whey (WPC-C) streams the WPI was produced via an IX process and the WPC-HF was produced via a MF process.\u003c/p\u003e \u003cp\u003eIn the MF process the permeate (the portion that passes through the membrane filter) becomes the WPI stream, whilst the retentate (the portion that does not pass through the membrane) becomes the WPC-HF stream (Blyund \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). As the WPC-HF retentate retains all the fat from the original sweet whey, all the proteins present in the MFGM are retained in this stream. The WPC-HF analysed here was produced via a MF process that included acidification and heating of the whey stream prior to the MF step resulting in the precipitation of minor proteins α-Lac, BSA and IgG, causing the WPC-HF to become enriched in these components, but depleted of minor proteins BLG and CMP. In the case of WPI produced via MF, the BLG and CMP would move into the accompanying WPI stream.\u003c/p\u003e \u003cp\u003eHowever, the WPI analysed here was not produced from microfiltration of a sweet whey stream, but rather via an IX process. While the IX process still results in a WPI stream and a WPC-HF stream that parallels the MF process, the protein species are separated in such a way that the WPI rather than the WPC-HF is enriched in α-Lac and BSA. The WPI also retains an increased concentration of BLG. The WPC-HF stream resulting from the IX process also becomes enriched in CMP protein species. This results in this WPI having an AA profile somewhat similar to that of acid WPC, whereas a standard MF WPI process would be expected to result in a product more similar to a cheese WPC. The IX process also has the additional impact of separating the protein from the majority of non-protein nitrogen (NPN) in the stream (e.g., urea, nitrogen containing vitamins). Since (crude) protein content is determined by measuring total nitrogen content, the removal of NPN results in a lower total protein value than would be measured if the NPN was retained. This then impacts the calculation of AA content when expressed as mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e protein, as it will make the AA content per gram of protein slightly higher than it would be if the NPN was retained.\u003c/p\u003e \u003cp\u003eThese differences are clearly evidenced in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, where an obvious difference can be seen between almost every AA (with the exception of histidine). The mean difference in histidine between the two products is \u0026minus;\u0026thinsp;1.66 with no significant difference between the values found (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.113). If a comparison was to be made between a WPI and a WPC-HF that were both produced via the MF process, or both by the IX process, the AA profiles would be expected to be even more divergent as only one of the two would be enriched in α-Lac and BSA rather than both, which is the case here.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eVariability between D90 and WPC-C\u003c/h2\u003e \u003cp\u003eDemineralised whey (D90), as suggested by the name, has a mineral content (i.e., sodium and chlorine) lower than that of regular whey proteins (Blyund \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), making it a useful ingredient for applications where mineral levels need to be carefully managed, e.g., infant formulas. The demineralisation process can be achieved via nanofiltration, electrolysis, ion exchange, or a combination of these processes. The D90 product analysed in this paper was produced via a combination of the nanofiltration and ion exchange processes and originates from a renneted sweet whey stream. The protein concentrating nanofiltration step causes the loss of some NPN resulting in a lower NPN content than that of the sweet whey stream from which it is derived. It will therefore have a higher total AA content per gram of crude protein than a standard non-nanofiltrated sweet whey powder. However, it will still contain more NPN than a standard WPC80 as the WPC80 undergoes ultrafiltration, which is much more effective at removing NPN.\u003c/p\u003e \u003cp\u003eAs can be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, each AA in the D90 product is consistently lower than in the WPC-C product. However, if the AA contents of these products are compared as a percentage of total AAs, which effectively corrects for NPN content, the resulting profiles are essentially the same (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This is due to the fact that the D90 is just a demineralised sweet whey with some NPN removed and the WPC-C is an ultrafiltered sweet whey with more NPN removed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe data outlined in this paper clearly describe the differences in AA profiles in a variety of dairy powders. These differences come down to two main factors: (i) changes in NPN distorting the apparent AA content when expressed on a per g of protein basis and (ii) the fractionation of the different classes of proteins into different product streams.\u003c/p\u003e \u003cp\u003eWhen interpreting AA profiles both of these factors should be considered, it should be kept in mind that the distortion of AA profiles due to changes in NPN is just that, a distortion. The absolute AA content has not been changed, it only appears that way due to the fact protein testing is not a direct measurement of protein, but rather a calculation based on total nitrogen content. On the other hand, differences in AA profiles that are due to the fractionation of protein can result in real, and sometimes significant, changes to AA profiles. Categorisation of proteins can be done at multiple levels, as evidenced in milk, which can be defined at the most basic level as whey and casein. These classifications can then be broken down further into four primary caseins (α\u003csub\u003eS1\u003c/sub\u003e-, α\u003csub\u003eS2\u003c/sub\u003e-, β- and κ-caseins) and four primary whey proteins (α-Lac, BLG, BSA and immunoglobulins). Each of these proteins are made up of unique sequences of amino acids, meaning that when they eventuate in different product streams the resulting product will have an altered AA profile.\u003c/p\u003e \u003cp\u003eWhile tools such as protein quality scores (e.g., DIAAS) provide useful insights into the overall quality of protein for consumers, understanding the specific differences in AA profiles of various powders is an important consideration when utilising them as ingredients into nutritional applications. For example, manufacturers of sports products may want to target ingredients high in leucine and other branched chain amino acids, while an infant formula manufacturer may need to know the levels of all the essential AAs to be able create a product that provides enough to support healthy infant growth and development while still maintaining appropriate protein levels.\u003c/p\u003e \u003cp\u003eThis paper provides a valuable insight into the nutritional differences between various dairy powders, as well as providing the context as to why these differences arise.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis study was supported by Fonterra Co-operative Group Ltd\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eArnesen EK, Thorisdottir B, Lamberg-Allardt C, B\u0026auml;rebring L, Nwaru B, Dierkes J, Ramel A, \u0026Aring;kesson, A (2022) Protein intake in children and growth and risk of overweight or obesity: A systematic review and meta-analysis. Food Nut Res 66:8242. doi: 10.29219/fnr.v66.8242\u003c/li\u003e\n\u003cli\u003eAOAC (2005a) Official method 994.12-1997. Amino acids in feeds. Performic acid oxidation. In: Official methods of analysis of AOAC International 18\u003csup\u003eth\u003c/sup\u003e edn. AOAC International, Gaithersburg\u003c/li\u003e\n\u003cli\u003eAOAC (2005b) Official method 988.15-1988. Tryptophan in foods and food and feed ingredients - ion exchange chromatographic method. In: Official methods of analysis of AOAC International 18\u003csup\u003eth\u003c/sup\u003e edn. AOAC International, Gaithersburg\u003c/li\u003e\n\u003cli\u003eBerry S, Sheehy P, Williamson P, Sharp J, Menzies K, Lef\u0026egrave;vre C, Snell, R (2020) Defining the origin and function of bovine milk proteins through genomics: the biological implications of manipulation and modification. In: Boland M, Singh H (eds) Milk proteins: From expression to food, 3rd edn. Academic Press, London, pp 143\u0026ndash;171. https://doi.org/10.1016/B978-0-12-815251-5.00004-9\u003c/li\u003e\n\u003cli\u003eBlyund G (2023) Dairy processing handbook. Tetra Pak Processing Systems, Lund. https://dairyprocessinghandbook.tetrapak.com \u003c/li\u003e\n\u003cli\u003eFong BY, Norris CS (2009) Quantification of milk fat globule membrane proteins using selected reaction monitoring mass spectrometry. J Ag Food Chem 57:6021\u0026ndash;6028. https://doi.org/10.1021/jf900511t\u003c/li\u003e\n\u003cli\u003eFong BY, Norris CS, MacGibbon AK (2007) Protein and lipid composition of bovine milk-fat-globule membrane. Int Dairy J 17:275\u0026ndash;288. \u003cbr\u003e https://doi.org/10.1016/j.idairyj.2006.05.004\u003c/li\u003e\n\u003cli\u003eGorissen SH, Crombag JJ, Senden JM, Waterval WH, Bierau J, Verdijk LB, van Loon LJ (2018) Protein content and amino acid composition of commercially available plant-based protein isolates. Amino Acids 50:1685\u0026ndash;1695. https://doi.org/10.1007/s00726-018-2640-5\u003c/li\u003e\n\u003cli\u003eISO (2014) Milk and milk products \u0026ndash; Determination of nitrogen content \u0026ndash; Part 1: Kjeldahl principle and crude protein calculation (ISO 8968-1:2014|IDF 20-12014). Geneva, International Standardisation Organisation. https://wwwisoorg/standard/61020html\u003c/li\u003e\n\u003cli\u003eKanno C, Kim DH (1990) A simple procedure for the preparation of bovine milk fat globule membrane and a comparison of its composition, enzymatic activities, and electrophoretic properties with those prepared by other methods. Ag Biol Chem 54:2845\u0026ndash;2854. https://doi.org/10.1080/00021369.1990.10870405 \u003c/li\u003e\n\u003cli\u003eLucey JA (2002) Formation and physical properties of milk protein gels. J Dairy Sci 85:281\u0026ndash;294. https://doi.org/10.3168/jds.S0022-0302(02)74078-2\u003c/li\u003e\n\u003cli\u003eLynch JM, Barbano DM (1999) Kjeldahl nitrogen analysis as a reference method for protein determination in dairy products J AOAC Int 82:1389\u0026ndash;1398. https://doi.org/10.1093/jaoac/82.6.1389 \u003c/li\u003e\n\u003cli\u003eMathai JK, Liu Y, Stein HH (2017) Values for digestible indispensable amino acid scores (DIAAS) for some dairy and plant proteins may better describe protein quality than values calculated using the concept for protein digestibility-corrected amino acid scores (PDCAAS) Brit J Nut 117:490\u0026ndash;499. https://doi.org/10.1017/S0007114517000125\u003c/li\u003e\n\u003cli\u003eMonirujjaman MD, Ferdouse A (2014) Metabolic and physiological roles of branched-chain amino acids. Adv. Mol. Biol. 2014: 364976. https://doi.org/10.1155/2014/364976\u003c/li\u003e\n\u003cli\u003eNeelima, Sharm R, Rajpu YS, Mann B (2013) Chemical and functional properties of glycomacropeptide (CMP) and its role in the detection of cheese whey adulteration in milk: a review. Dairy Sci Tech 93:21\u0026ndash;43. https://doi.org/10.1007/s13594-012-0095-0\u003c/li\u003e\n\u003cli\u003eOECD/FAO (2022) OECD-FAO agricultural outlook 2022\u0026ndash;2031. OECD Publishing, Paris https://doiorg/101787/f1b0b29c-en\u003c/li\u003e\n\u003cli\u003ePeyrollier K, Hajduch E, Blair AS, Hyde R, Hundal HS (2000) l-Leucine availability regulates phosphatidylinositol 3-kinase, p70 S6 kinase and glycogen synthase kinase-3 activity in L6 muscle cells: evidence for the involvement of the mammalian target of rapamycin (mTOR) pathway in the l-leucine-induced up-regulation of system amino acid transport. Biochem J 350:361\u0026ndash;368. https://doi.org/10.1042/bj3500361\u003c/li\u003e\n\u003cli\u003eTeodoro M (2023) The future of nutrition: Health and wellness: 2023 [Industry Report] Mintel, London. https://wwwmintelcom/\u003c/li\u003e\n\u003cli\u003eTontisirin K (2003) Food energy: Methods of analysis and conversion factors: Report of a technical workshop, Rome, 3\u0026ndash;6 December 2002 (Publication No ISSN0254-4725). Food and Agriculture Organisation of the United Nations, Rome. https://wwwfaoorg\u003c/li\u003e\n\u003cli\u003eVasbinder AJ, Rollema HS, Bot A, De Kruif CG (2003) Gelation mechanism of milk as influenced by temperature and pH; studied by the use of transglutaminase cross-linked casein micelles. J Dairy Sci 86:1556\u0026ndash;1563. https://doi.org/10.3168/jds.S0022-0302(03)73741-2 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Dairy Protein, Amino Acid, Whey Protein Concentrate, Milk Powder Variability, Protein, Fractionation, Nutritional Composition","lastPublishedDoi":"10.21203/rs.3.rs-4356289/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4356289/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMultiple samples of a range of dairy powders were analysed for their amino acid (AA) content, allowing an in-depth analysis of the differences between their AA profiles and how various manufacturing processes give rise to the differences between product types. The products analysed were whole milk powder (WMP), skim milk powder (SMP), cheese whey protein concentrate (WPC-C), lactic acid whey protein concentrate (WPC-L), high fat whey protein concentrate (WPC-HF), hydrolysed whey protein concentrate (WPH) and demineralised whey protein (D90). Analysis demonstrated that WMP and SMP share broadly similar AA profiles with minor differences that were most probably due to the small levels of protein in milk fat, which is close to absent in SMP. When comparing WPC-C and WPC-L, there were higher levels of threonine, serine, glutamic acid, and proline in the former, but lower levels of tyrosine, phenylalanine and tryptophan. This is due to these products being separated from casein via different methods. WPI and WPC-HF show differences in the levels of every AA with the exception of histidine; they originate from similar sweet whey streams, but then processing diverges, resulting in the AA variation. D90 was consistently lower in every AA when compared with WPC-C; while both originate from sweet whey streams, D90 has a nanofiltration step in its manufacture that increases its non-protein nitrogen content, impacting its AA levels.\u003c/p\u003e","manuscriptTitle":"Variation of Amino acid composition in dried bovine dairy powders from a range of product streams","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-08 06:42:16","doi":"10.21203/rs.3.rs-4356289/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"63909946-1bec-4e00-92db-65efbd77b504","owner":[],"postedDate":"May 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-22T16:11:38+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-08 06:42:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4356289","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4356289","identity":"rs-4356289","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}