The bovine genetic variant A2 beta-casein is associated with fewer digestive and absorption issues compared to A1 beta-casein, leading to increased global demand for A2 milk. However, contamination with the A1 variant during collection, transportation, or sterilization of A2 milk poses a risk, necessitating a verification test to ensure A2 milk does not contain A1 beta-casein. We developed an A1-specific monoclonal antibody (mAb) and a general mAb that reacts with both A1 and A2 variants, using the iliac lymph node method. A sandwich ELISA was created using the general mAb as the capture antibody and the A1-specific mAb as the detection antibody to identify A1 beta-casein in milk. This ELISA successfully detected A1 beta-casein in raw and pasteurized A2 milk, including ultra-high temperature treated milk. The test identified A1 beta-casein when the A1 spike in A2 milk exceeded 1% in volume, indicating its capability to detect contamination from one A1A1 cow in a herd of one hundred A2A2 cows. The developed A1 beta-casein ELISA is suitable for high-throughput analysis and valuable for monitoring A1 beta-casein contamination in commercially produced A2 milk.

Reconstituted low-heat skim milk powder (SMP) exhibits rennet-induced gelation. Although it is known that the gelling properties of reconstituted SMP are poorer than those of preheated milk, the cause has not yet been identified. In this study, we analysed the denatured proteins, soluble calcium, rennet-induced gelation, and syneresis properties of skim milk subjected to preheating, concentration, and spray drying. Furthermore, the effects of the denatured proteins and decrease in soluble calcium caused by concentration and spray drying on gelling and syneresis properties were investigated using ultrafiltrate or simulated milk ultrafiltrate. The reduction in gelling and syneresis properties was found to be caused by a decrease in soluble calcium following the concentration. Our results also indicate that slight denaturation of whey proteins caused by concentration and spray drying had little effect on the gelling and syneresis properties. These findings provide important insights for the use of SMP in the dairy industry.

This study focused on the factors that soften the crumb texture of vacuum-cooled bread by examining the effect of residual heat applied to bread during the vacuum cooling process, focusing on how suppressing residual heat affects the starch and protein content of the bread. The results showed that in vacuum cooling breads, the removal of residual heat preserved the grain shape and crystallinity of the starch and inhibited the progress of gelatinization. In addition, residual heat also enhanced the insolubilization of proteins caused by baking, indicating that protein insolubilization was suppressed in vacuum cooled breads with less residual heat. To further elucidate the effect on protein properties, the isoforms of gliadin that were soluble in the EtOH fraction, whose hydrophobicity was reduced by residual heat, were identified. The results showed that alpha-/beta- and gamma-gliadins, which exhibit higher hydrophobicity among gliadin isoforms, demonstrated increased solubility in vacuum-cooled bread where residual heat was suppressed.

The use of low-heat skim milk powder (SMP) in natural cheese production offers signi ficant industrial advantages. However, SMP takes a long time to undergo rennet-induced gelation and forms a soft gel, which makes it dif ficult to use in cheese production. In this study, we investigated whether dry -heat treatment of low-heat SMP contributes to improving rennet-induced gel properties. We found that dry -heat treatment (65 degrees C; relative humidity, 40%; 2-6 h) accelerated rennet coagulation time (RCT), increased gel strength, and improved the water -holding capacity of the gel. Dry -heat treatment resulted in lactosylation, an increase in hydrophobicity, and a decrease in the zeta potential of casein micelles. These changes possibly contributed to the improvement in rennet-induced gelation. Additionally, the dry -heat treatment likely affected the surface properties of casein micelles by altering their secondary structure. This study offers valuable insights into SMP applications, particularly in the production of natural cheese in the dairy industry. (c) 2024 Elsevier Ltd. All rights reserved.

Abstract In this study, the effects of the degree of thermal denaturation of whey protein (WP) added to milk on the dissociation of κ-casein from casein micelles were investigated, since they are related to the strength of acid milk gel and its factors. Acid milk gels were prepared by heating thermally denatured WP isolate (WPI) and undenatured milk mixtures and treating them with glucono-δ-lactone as a coagulant. The strength of these gels was negatively correlated with the WPI denaturation degree and strongly positively correlated with the extent of κ-casein dissociation from casein micelles. This behavior was ascribed to the fact that α-lactalbumin (α-La) and β-lactoglobulin (β-Lg) contained in WPI denatured after heating and engaged in disulfide bond formation with each other. With an increase in the degree of denaturation and disulfide bond formation, the bonding between β-lactoglobulin and κ-casein was suppressed to decrease the amount of κ-casein–WPI complexes. When β-Lg forms SS bonds with α-La, the number of highly reactive, free SH groups decreases, which complicates the formation of SS bridges between β-Lg and κ-casein. Thus, the denaturation degree of WPI largely determined the degree of κ-casein dissociation from casein micelles and, consequently, the strength of acid milk gels. Adding WP to milk increases the strength of acid milk gel, and it can be controlled by changing the degree of thermal denaturation of the WP. Furthermore, it was clarified for the first time that the dissociation of κ-casein from casein micelles influences this effect. Further studies are needed to elucidate the structural features of κ-casein-dissociated micelles.

Wheat flour is added to and mixed with water to hydrate and associate gliadin and glutenin to form gluten. In addition to various minor interactions such as hydrogen bonding and hydrophobic interaction for proper gluten formation, it is acknowledged that disulfide bonds (SS bonds) between cysteine residues are important. Further, in terms of breeding, genes encoding proteins having excellent SS bond-forming ability, represented by Glu-Dld, are critical. There has been much research on endogenous redox substances and enzymes in the mechanism of SS bond formation in the manufacturing process; however, consensus regarding the relationship has not been obtained. Protein disulfide isomerase (PDI, EC 5.3.4.1) is present in the endoplasmic reticulum, and is a molecular chaperone that forms intramolecular (interstitial) SS bonds during protein expression. When PDI forms SS bonds in gluten, it is reduced (PDIred) and loses its oxidizing ability. It is known that oxidative regeneration to oxidized PDI (PDIoxi) by endoplasmic reticulum oxide reductase 1 (ERO1) occurs in vivo, and ascorbic acid (AA) as a dough-improving agent is involved in this regeneration in wheat flour dough. AA is converted to dehydroascorbic acid (DHA) by ascorbate oxidase in the dough. When examining the effect of DHA on PDIred, conversion to PDIoxi progresses efficiently, and since it catalyzes SS bond formation in substrate protein, it is inferred that the dough-improving effect by AA is brought about in cooperation with PDI.

The purpose of this study was to analyze the properties of wheat flour and rice flour that are used in the production of wheat flour and rice flour blended bread with the addition of acid-soluble wheat protein (ASP), and to examine the optimal additional amounts of sugars and enzymes that can improve the quality of the wheat flour and rice flour blended bread. In the sponge and dough method of bread-making, rice flour was used to replace 40 % of the wheat flour, and 10 % wheat protein (gluten and ASP) was added to the wheat flour and rice flour. Results of flour analysis confirmed that the rice flour (rice flour for bread and high amylose rice flour) had less damaged starch content than the wheat flour. Also, rice flour particles were smaller than wheat flour particles, and each displayed different viscosity characteristics. In wheat flour and rice flour blended bread, the addition of rice flour, with its low-damaged starch content, decreased the amount of maltose produced during the fermentation of the sponge dough when compared to bread made with wheat flour only. Accordingly, the measured amount of gas generated during sponge dough fermentation also decreased. Therefore, when rice flour was used to replace 40 % of the wheat flour, 2 % maltose was added to the sponge dough to increase the gas amount and improve fermentability. When wheat flour and rice flour blended bread was made in which 40 % of the wheat flour was replaced with rice flour, in comparison to the use of rice flour for bread, the use of high amylose rice flour resulted in a higher specific volume, and caving did not occur. When using two alpha-amylases with different optimum temperatures (50 ppm each), it was possible to improve the specific volume of the bread and suppress the degradation of crumb firmness over time. Furthermore, the addition of 150 ppm of lipase was effective at preventing the bread from caving, indicating that the addition of appropriate amounts of these enzymes improved the quality of wheat flour and rice flour blended bread.

Backgrounds and objectives: The structure of molded frozen dough cannot be reconstructed before baking; therefore, molding directly affects bread quality. The effect of the addition of acid-soluble wheat protein (ASP), which consists mainly of gliadin, to molded frozen dough on molding conditions was examined. Findings: Molded (unfermented) frozen dough samples were prepared from the same dough batch at different floor times (assumed potential time differences during dividing). Scanning electron microscopy revealed the presence of cracks in the microstructure of the molded dough. The cracks were caused by gluten damage owing to changes in rheological properties and appeared when the floor time was extended from 5 to 20 min. The addition of ASP maintained the uniformity of the gluten sheet regardless of floor time. Conclusions: The added ASP increased shape uniformity of bread rolls prepared from molded frozen dough and also crumb grain structure and crust appearance uniformity. Significance and novelty: The addition of ASP to molded frozen dough improved bread quality and prevented manufacturing problems that can occur during mechanized bread-making. Therefore, we anticipate that ASP could also be added to pre-fermented and par-baked frozen doughs.

The purpose of this study was to investigate the effects of two types of rice flour, with different starch damage degrees, on bread-making properties and temporal changes in bread crumb rheological properties. The subject of this study was wheat flour and rice flour blended bread, in which rice flour was used to replace 40 % of the wheat flour. It was found that rice flour with a high degree of starch damage had high water retention capacity. Also, differences in the shape and surface microstructure of rice flour particles were observed. When producing the subject bread on a laboratory scale, it was found that replacing 25 % or 50 % of low starch damage rice flour with high starch damage rice flour reduced the temporal changes in bread crumb rheological Properties compared to the product made using only rice flour with a low degree of starch damage (2.7 %). With the aim of simulating mass production, the subject bread was produced on an experimental factory scale with each rice flour type at a ratio of 50 % (starch damage degree 8.2 %). Results showed that when the starch damage degree increased, dough hardening was suppressed and temporal changes in the rheological properties of the subject bread crumb (firmness and cohesiveness) decreased. It was speculated that as the starch damage degree increased, alpha-amylase, which was added during the dough mixing stage, acted more easily.

In this study, we analyzed the molecular properties and thermal behavior of dried egg white (DEW) proteins to understand the mechanisms underlying DEW gel hardening via dry-heat treatment. Dry-heattreated DEW proteins displayed a higher surface negative charge and more isopeptide bonds, such as those in lanthionine and lysinoalanine. In addition, secondary structure and surface hydrophobicity measurements suggested that structural changes that occur during heating in solution were suppressed in dry-heat-treated DEW. The size of the protein aggregates did not change during heating in the diluted solution, and was almost the same as that of the gel structure unit. These results indicated that the structural changes of the protein in solution were restricted due to isopeptide bonds, and that the surface negative charge of the protein caused intermolecular repulsion. Consequently, protein interactions were limited, and the proteins formed a finer and more homogeneous network, which is believed to harden the gel.

Backgrounds and objectives Time differences in processing with mechanized bread-making cause changes in the rheological properties of dough. The effect of the addition of acid-soluble wheat protein (ASP), mainly composed of gliadin, on these changes was examined. Findings Bread rolls were prepared from the same batch of dough in two different floor times. The shape of bread rolls became wider when the floor time was extended to 40 min compared with the normal time of 20 min. The addition of ASP to dough (0.5% or 0.75%) led to a reduction in the influence of the time difference. The stress applied to the sheeting roller in the molding process increased with extended floor times, although this was suppressed by the addition of ASP. Farinograph and extensigraph results as well as the results of the microstructure analysis performed using a scanning electron microscope showed that the addition of ASP to dough favorably changed the gluten network structure. Conclusions In mechanized bread-making, the addition of ASP contributed to the uniformity of the product quality by suppressing changes in the rheological properties of dough during production. Significance and novelty The results revealed that the addition of ASP could potentially provide industrial uniformity to various bread products in mechanized bread-making.

In this study, the relationship between the dissociation of κ-casein from casein micelles due to heat-induced denaturation and the strength of acid milk gel was investigated. The κ-casein-dissociated micelles were fractionated by gel filtration chromatography and two-dimensional polyacrylamide gel electrophoresis, and their zeta potential and surface hydrophobicity were measured. The negative charge of the κ-casein-dissociated micelles was lower than that of native micelles, and micellar surface hydrophobicity was higher. For confirmation, the isoelectric point of the casein micelles was measured. The κ-casein-dissociated micelles were found to cohere at an earlier stage of acidification than the native micelles. These results demonstrated that the heat-induced increase in the strength of acid milk gel was partly due to the decrease in micellar surface charge and partly to the increase in surface hydrophobicity caused by the dissociation of κ-casein.

To clarify the processing properties of “ Sendango ”， a traditional food from Tsushima Island, obtained from fermented sweet potato, and the factors that show the characteristic physical properties of “ Rokubee ”， a Sendango processed product, we examined and compared the physicochemical and viscosity properties, and chain lengths of amylopectin in Sendango starch and its raw material, the sweet potato starch. The surface of Sendango starch granules was rough and with holes, confirming that the granules were degraded by fermentation. Amylose ratio in Sendango starch was high, suggesting that amylopectin was degraded. In addition, the properties of Sendango starch indicated that it hard to gelatinization and easy to retrogradation ; starch gel was hard, elastic, and viscous. These properties were associated with specific degradation of the short (A and Bl) chains in the outmost shell of the amylopectin chain-length.

The effect adding acid-soluble wheat protein (ASP), mainly composed of gliadin, to wheat flour and rice flour blend bread in mechanized bread-making was investigated using the following method. Rice flour was used to replace 40 % of the wheat flour, and wheat protein (gluten and ASP) was added to constitute 10 % of the total mixture, thus replacing 20 % to 40 % of the gluten with ASP. In the sponge and dough method, the addition of ASP improved the gas holding capacity of the sponge, and the dough development time shortened during the mixing process. In addition, molding conditions for mass production were improved due the suppression of changes in dough rheological properties caused by the time lag during the dividing process. Microstructural observation of sheeted dough showed that the gluten network of the dough extended in a uniform direction. Consequently, it was shown that the addition of ASP resulted in increased extensibility of the dough. As a result, the addition of ASP improved uneven crumb structure after baking and decreased the rate of crumb firmness during storage (20 degrees C, 24-96 hours). In conclusion, the addition of ASP improved the stabilization of the bread-making process and increased the product quality of wheat flour and rice flour blend bread in mechanized bread-making.

We examined the effects of components of starch surface granule (included lipids and proteins) on the gelatinization and viscosity properties of rice starch. In water absorbing and solubility test, lipids and proteins inhibited the water absorption power and solubility of starch granule. There effect was stronger for proteins than lipids. The viscosity profile showed that lipids and proteins in the starch granule increased pasting temperature of starch granule. In addition, proteins decreased swelling power of starch granule. The viscosity behavior of gelatinized starch paste showed lipids and proteins promoted retrogradation of starch. These data were suggested promotion of retrogradation of starch was due to interaction with hydrophobicity protein such as glutelin and prolamiin in the rice starch granule. These data were suggested component of starch granule such as lipids and proteins are also significant in determining the characteristics of starch granules. It can be valuable in developing novel technologies for starch processing.

To improve quality and production efficiency of yuba films, this study aimed to clarify the mechanism underlying the formation of yuba films, by investigating the effects of lipids and elucidating the molecular interaction between proteins and lipids. A thin film with dense structure was produced after heating soybean protein isolate (SPI) solution, whereas a thick film with a porous structure was formed upon heating soybean milk, and SPI solution with added soybean oil, indicating that lipids are indispensable to the formation of yuba films. FT-IR imaging demonstrated the distribution of lipids as oil bodies in the protein phase of the yuba films. Analysis of structural proteins by diagonal two-dimensional electrophoresis (D-2DE) identified a specific 17-kDa protein in the yuba films, which was identified to be oleosin 1-like by LC/MS/MS. This lipophilic protein is known to have a cysteine residue. The existence of oleosin in the yuba films suggested the presence of disulfide (SS) bonds in polymers formed by oleosin with β-conglycinin (β-GCα), β-conglycinin (β-GCα′) and glycinin acidic (GA), and with GA and glycinin basic (GB) protein subunits. These proteins and oil bodies polymerize to form an aggregate and form the structural framework of yuba films.

We studied the effects of K-casein (K-CN) dissociation from casein micelles upon heating on cheese curd formation. Cheese curd was formed by the addition of chymosin to unheated and heated (80 degrees C, 30 min) defatted milk. In heated defatted milk, cheese curd was not formed; however, the amount of free glycomacropeptides (GMPs) and the degree of formation of para-K-CN were the same in both the unheated and heated defatted milk. In addition, because calcium ions insolubilize upon heating, the calcium ion concentration in the heated defatted milk was adjusted to its equivalent amount in the unheated defatted milk; however, cheese curd was not formed in this case either. Therefore, chymosin was added to the casein micelles, which dissociated K-CN. As a result, the amount of free GMP was significantly less (p < 0.05) than that in the native casein micelles. The results suggest that the quantity of formation of para-K-CN on the micelle surface decreases due to K-CN dissociation from the casein micelles.

デンプン粒は，起源によって粒子径，形状，アミロース含量およびデンプン分解酵素の作用性が異なることで性状の違いがある。さらにデンプン粒の性状にデンプン粒表層のタンパク質（SSP）の関与が推察されており，SSPを減少させたデンプンは糊化特性に変化が生じることが報告されている。しかし，SSPを構成するタンパク質に関する研究例は少ない。そこで私たちは，コメ，トウモロコシ，コムギ，サツマイモおよびジャガイモのSSPを同定することを試みた。デンプン粒のタンパク質含量は起源によって異なり，その値はデンプン粒のクマシーブリリアントブルー（CCB）による染色度（b＊値）と高い相関がみられた。0.1％水酸化ナトリウムによるデンプン粒中のタンパク質の減少率はデンプンの起源によって異なり，コメ，サツマイモ，ジャガイモ，トウモロコシ，コムギの順で高かった。SDS-PAGEにより，各起源のSSPが19のタンパク質バンドに分離され，そのうち16のタンパク質を同定した。その結果，各起源のSSPからデンプン合成酵素が同定されたほか，コムギSSPからはαアミラーゼインヒビター，ジャガイモからはアスパラギン酸およびシステインプロテアーゼインヒビターといった酵素阻害剤が同定された。また，コメからはグルテリンおよびプロラミン，トウモロコシからはゼイン，コムギからはグロブリン，サツマイモからはスポラミンといった貯蔵タンパク質が同定された。一般的に貯蔵タンパク質は疎水性の高いタンパク質であり，これら貯蔵タンパク質が各起源のデンプン粒の性状に影響を与えている可能性が示唆された。