Fabrication of Human Milk Fat Substitute: Based on the Similarity Evaluation Model and Computer Software.

We aimed to obtain the optimal formula for human milk fat substitute (HMFS) through a combination of software and an evaluation model and further verify its practicability through an animal experiment. The results showed that a total of 33 fatty acid (FA) and 63 triglyceride (TAG) molecular species were detected in vegetable oils. Palmitic acid, oleic acid, linoleic acid, 18:1/16:0/18:1, 18:2/16:0/18:2, 18:1/18:1/18:1 and 18:1/18:2/18:1, were the main molecular species among the FAs and TAGs in the vegetable oils. Based on the HMFS evaluation model, the optimal mixed vegetable oil formula was blended with 21.3% palm oil, 2.8% linseed oil, 2.6% soybean oil, 29.9% rapeseed oil and 43.4% maize oil, with the highest score of 83.146. Moreover, there was no difference in the weight, blood routine indices or calcium and magnesium concentrations in the feces of the mice between the homemade mixed vegetable oil (HMVO) group and the commercial mixed vegetable oil (CMVO) group, while nervonic acid (C24:1) and octanoic acid (C8:0) were absorbed easily in the HMVO group. Therefore, these results demonstrate that the mixing of the different vegetable oils was feasible via a combination of computer software and an evaluation model and provided a new way to produce HMFS.

Fatty acid, triglyceride, and kinetic properties of milk fat fractions made by the combination of dry fractionation and short-path molecular distillation.

In this study, we aimed to detect the physicochemical properties of distilled products (residue and distillate) obtained from anhydrous milk fat (AMF) and its dry fractionation products (liquid and solid fractions at 25°C [25 L and 25 S]). The results showed that the saturated fatty acids and low- and medium molecular-weight triglycerides were easily accumulated in the distillate, and the percentage of unsaturated fatty acid and high molecular-weight triglycerides in the residue were higher, and these components in 25 S and 25 L were influenced more significantly than those in the AMF. In addition, the distillate had larger melting ranges in comparison with the distilled substrate, while the melting ranges of residue was smaller. The triglycerides were presented as the mixture crystal forms (α, ß', and ß crystal) in 25 S, AMF, and their distilling products, and it was transformed gradually to a single form as the increasing of distilling temperature. Moreover, the accumulated pattern of triglycerides was double chain length in 25 S, AMF, and their distilling products. These results provide a new approach to obtain the milk fat fractions with different properties, and the findings of this study enrich the theoretical basis of milk fat separation in practical production.

Insight into the effects and biotechnological production of kestoses, the smallest fructooligosaccharides.

Kestoses, the smallest fructooligosaccharides, are trisaccharides composed of a fructose molecule and a sucrose molecule linked by either ß-(2,1) or ß-(2,6) linkage. 1-kestose, 6-kestose and neokestose are the three types of kestoses occurring in nature. As the main kind of fructooligosaccharide, kestoses share similar physiological effects with other fructooligosaccharides, and they have recently been determined to show more notable effects in promoting the growth of probiotics including Faecalibacterium prausnitzii and Bifidobacterium than those of other fructooligosaccharides. Kestoses exist in many plants, but the relatively low content and the isolation and purification are the main barriers limiting their industrial application. The production of kestoses by enzymatic biosynthesis and microbial fermentation has the potential to facilitate its production and industrial use. In this article, the recent advances in the research of kestoses were overviewed, including those studying their functions and production. Kestose-producing enzymes were introduced in detail, and microbial production and fermentation optimization techniques for enhancing the yield of kestoses were addressed. ß-Fructofuranosidase is the main one used to produce kestoses because of the extensive range of microbial sources. Therefore, the production of kestoses by microorganisms containing ß-fructofuranosidase has also been reviewed. However, few molecular modification studies have attempted to change the production profile of some enzymes and improve the yield of kestoses, which is a topic that should garner more attention. Additionally, the production of kestoses using food-grade microorganisms may be beneficial to their application in the food industry.

Metabolite secretions of Lactobacillus plantarum YYC-3 may inhibit colon cancer cell metastasis by suppressing the VEGF-MMP2/9 signaling pathway.

BACKGROUND: Colorectal cancer (CRC) is a major clinical challenge, and the gut microbiome plays important roles in the occurrence and metastasis of CRC. Lactobacillus and their metabolites are thought to be able to suppress the growth of CRC cells. However, the antimetastatic mechanism of Lactobacillus or their metabolites toward CRC cells is not clear. Therefore, the aim of this study was to assess the inhibitory mechanism of cell-free supernatants (CFSs) of L. rhamnosus GG, L. casei M3, and L. plantarum YYC-3 on metastasis of CRC cells. RESULTS: YYC-3 CFS showed the highest inhibitory effect on CRC cell growth, invasion and migration, and inhibited MMP2, MMP9, and VEGFA gene and protein expression, and protein secretion. Furthermore, it suppressed the activities of MMPs by gelatin zymography. Moreover, the effective compounds in these CFSs were analyzed by Q Exactive Focus liquid chromatography-mass spectrometry. CONCLUSIONS: Our results showed that metabolite secretions of YYC-3 may inhibited cell metastasis by downregulating the VEGF/MMPs signaling pathway. These data suggest that treatment of CRC cells with metabolites from L. plantarum YYC-3 may reduce colon cancer metastasis.

Whole-genome sequencing and genomic-based acid tolerance mechanisms of Lactobacillus delbrueckii subsp. bulgaricus LJJ.

The probiotic efficacy and fermentative ability of Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus), a widely used probiotic, is majorly affected by its acid tolerance. Here, we conducted whole-genome sequencing of the high acid-tolerant L. bulgaricus LJJ stored in the laboratory. Compared with the whole genome of low acid-tolerant strain L. bulgaricus ATCC11842, the results show that 16 candidate acid-tolerant genes may be involved in the regulation of the acid tolerance of L. bulgaricus LJJ. Association analysis of candidate acid-tolerant genes and acid-tolerant traits of different L. bulgaricus strains revealed that the three genes dapA, dapH, and lysC are the main reasons for the strong acid tolerance of L. bulgaricus LJJ. The results of real-time quantitative PCR (RT-qPCR) supported this conclusion. KEGG pathway analysis showed that these three acid-tolerant genes are involved in the synthesis of lysine; the synthesis of lysine may confer L. bulgaricus LJJ strong acid tolerance. This study successfully revealed the acid tolerance mechanism of L. bulgaricus LJJ and provides a theoretical basis for the subsequent selection of strains with high acid tolerance for improved probiotic functions. KEY POINTS: • Three genes are identified as acid-tolerant genes, respectively, lysC, dapA, and dapH. • LysC and dapA are the major key genes in the synthesis of lysine. • The synthesis of lysine may confer L. bulgaricus LJJ strong acid tolerance.

A new method for quantitative detection of Lactobacillus casei based on casx gene and its application.

BACKGROUND: The traditional method of bacterial identification based on 16S rRNA is a widely used and very effective detection method, but this method still has some deficiencies, especially in the identification of closely related strains. A high homology with little differences is mostly observed in the 16S sequence of closely related bacteria, which results in difficulty to distinguish them by 16S rRNA-based detection method. In order to develop a rapid and accurate method of bacterial identification, we studied the possibility of identifying bacteria with other characteristic fragments without the use of 16S rRNA as detection targets. RESULTS: We analyzed the potential of using cas (CRISPR-associated proteins) gene as a target for bacteria detection. We found that certain fragment located in the casx gene was species-specific and could be used as a specific target gene. Based on these fragments, we established a TaqMan MGB Real-time PCR method for detecting bacteria. We found that the method used in this study had the advantages of high sensitivity and good specificity. CONCLUSIONS: The casx gene-based method of bacterial identification could be used as a supplement to the conventional 16 s rRNA-based detection method. This method has an advantage over the 16 s rRNA-based detection method in distinguishing the genetic relationship between closely-related bacteria, such as subgroup bacteria, and can be used as a supplement to the 16 s rRNA-based detection method.

Screening for Cholesterol-Lowering Probiotics from Lactic Acid Bacteria Isolated from Corn Silage Based on Three Hypothesized Pathways.

A total of 85 strains of lactic acid bacteria were isolated from corn silage in this study and analyzed in vitro for their cholesterol removal, NPC1L1 protein down-regulation and bile salt deconjugation ability, respectively. Nineteen strains were selected for further analysis for their probiotic potential. Finally, 3 strains showing better probiotic potential were evaluated for their cholesterol-lowering activity in hamsters. The strains showing the greater cholesterol removal and NPC1L1 protein down-regulation activity had no significant effects on serum and hepatic cholesterol levels in hamsters (p > 0.05). However, Lactobacillus plantarum CAAS 18008 (1 × 109 CFU/d) showing the greater bile salt deconjugation ability significantly reduced serum low-density lipoprotein cholesterol, total cholesterol, and hepatic total cholesterol levels by 28.8%, 21.7%, and 30.9%, respectively (p < 0.05). The cholesterol-lowering mechanism was attributed to its bile salt hydrolase activity, which enhanced daily fecal bile acid excretion levels and thereby accelerated new bile acid synthesis from cholesterol in liver. This study demonstrated that the strains showing greater cholesterol removal and NPC1L1 protein down-regulation activity in vitro hardly reveal cholesterol-lowering activity in vivo, whereas the strains showing greater bile salt deconjugation ability in vitro has large potential to decrease serum cholesterol levels in vivo.

Development an effective system to expression recombinant protein in E. coli via comparison and optimization of signal peptides: Expression of Pseudomonas fluorescens BJ-10 thermostable lipase as case study.

BACKGROUND: Thermostable lipases from microbial sources have been substantially overexpressed in E. coli, however, these enzymes are often produced with low-level enzymatic activity and mainly in the form of inclusion bodies. Several studies have reported that the secretory production of recombinant proteins fused their N-terminus to a signal peptide has been employed to resolve the problem. In general, the feasibility of this approach largely depends on the secretory pathway of signal peptide and the type of target protein to be secreted. This study was performed to compare and optimize signal peptides for efficient secretion of thermostable lipase lipBJ10 from Pseudomonas fluorescens BJ-10. Meanwhile, a comparative study between this method and cytoplasmic secretion was implemented in secreting soluble and active lipases. RESULTS: Fusion expression using six signal peptides, i.e., PelB and five native E. coli signal peptides, as fusion partners produced more soluble and functional recombinant lipBJ10 than non-fusion expression. Recombinant lipBJ10, fused to these six diverse signal peptides, was secreted into the periplasm in E. coli. The total lipase activity in all cases of fusion expression was higher than those in non-fusion expression. The relative activity peaked when lipBJ10 was fused to DsbA, yielding a value 73.3 times greater than that of the non-fusion protein. When DsbA was used as the fusion partner, the highest activity (265.41 U/ml) was achieved with the least formation of inclusion bodies; the other four E. coli signal peptides, to some extent, led to low activity and insoluble inclusion bodies. Therefore, DsbA is the optimal signal peptide partner to fuse with lipBJ10 to efficiently produce soluble and functional protein. CONCLUSION: We found that fusing to these signal peptides, especially that of DsbA, can significantly decrease the formation of inclusion bodies and enhance the function and solubility of lipBJ10 compared to non-fusion lipBJ10. Our results reported here can provide a reference for the high-level expression of other lipases with respect to a possible industrial application.

Functional Characteristics of Milk Protein Concentrates and Their Modification.

A major deterrent to the usage of milk protein concentrate (MPC), a high-protein milk product with increasing demand as a food and sports drink ingredient, has been its poor functional characteristics when compared with other milk protein products such as whey protein concentrate and sodium caseinates. This review discusses the recent research on functional properties of MPC, focusing on factors that may contribute to the poor functional characteristics before, during, and after production. Current research, methods employed, and new understanding on the causes of poor solubility of MPC at mild temperatures (about 20°C) has been presented, including loss of solubility during storage as these areas have received unprecedented attention over the past decade, and also affects other useful functional properties of MPC, such as emulsifying properties, gelation, and foaming. Processing methods, which include heat treatment, high-pressure application, microwave heating, ultrasound application, and enzyme and salts modification, have been used or have potential to modify or improve the functional properties of MPCs. Future research on the effects of these processing methods on the functional properties, including effects of enzyme hydrolysis on bitterness and bioactivity, has also been discussed.

Effect of power ultrasound pretreatment on peptidic profiles and angiotensin converting enzyme inhibition of milk protein concentrate hydrolysates.

BACKGROUND: The use of power ultrasound as a pretreatment to enhance the hydrolysis of milk protein concentrate (MPC) and subsequent angiotensin converting enzyme (ACE) inhibitory activity has been studied. Liquid chromatography was used to analyse peptide profiles of Neutrase-derived MPC hydrolysates after pretreatment at 0, 1, 3, 5 and 8 min at an ultrasound power level of 800 W. RESULTS: The peptide profiles indicated an increase in number of peptides when ultrasound pretreatment was applied. There was also an increase in the degree of hydrolysis of MPC hydrolysates. The profiles indicated that new small peptides in ultrasound pretreated samples (1-5 min) which were not present in the control samples and 8 min pretreated samples, could be responsible for increased ACE inhibitory activity. These small peptides were digested in the 8 min pretreated samples. CONCLUSION: Ultrasound pretreatment of MPC increases the ACE inhibitory activity of the hydrolysates because of the production of new small peptides. This can be used as a means to derive potent ACE inhibitory peptides at industrial scale in complex protein sources.

Purification and properties of heat-stable extracellular protease from Pseudomonads fluorescens BJ-10.

Pseudomonas fluorescens BJ-10, a kind of psychrotrophic bacteria, was isolated from raw milk. It produced an extracellular protease of 47 kDa by SDS-PAGE. The crude proteases were purified by ammonium sulfate fractionation, ion-exchange and gel filtration chromatography. The specific activity of purified protease increased 61.38-fold. The optimum pH and temperature were pH 7.0 and 30 °C, respectively. The purified protease was partially inhibited by DL-dithiothreitol, and the activity increased a little upon Fe(2+) addition. The protease showed typical heat-stable behavior. After treatment at 100 °C for 3 min, more than 94% activity remained. This work might lay the foundation for possible relationship between the heat stable protease and gelation of UHT milk.

Optimizing Akkermansia muciniphila Isolation and Cultivation: Insights into Gut Microbiota Composition and Potential Growth Promoters in a Chinese Cohort.

The study aims to analyze the composition of the gut microbiota in Chinese individuals using metagenomic sequencing technology, with a particular focus on the abundance of Akkermansia muciniphila (Akk). To improve the efficiency of Akk isolation and identification accuracy, modifications were made to the enrichment culture medium and 16S rRNA universal primers. Additionally, potential growth-promoting factors that stimulate Akk growth were explored through in vitro screening. The research results revealed that the abundance of Akk in Chinese fecal samples ranged from 0.004% to 0.4%. During optimization, a type of animal protein peptide significantly enhanced the enrichment efficiency of Akk, resulting in the isolation of three Akk strains from 14 fecal samples. Furthermore, 17 different growth-promoting factors were compared, and four factors, including galactose, sialic acid, lactose, and chitosan, were identified as significantly promoting Akk growth. Through orthogonal experiments, the optimal ratio of these four growth-promoting factors was determined to be 1:1:2:1. After adding 1.25% of this growth-promoting factor combination to the standard culture medium, Akk was cultivated at 37° for 36 h, achieving an OD600nm value of 1.169, thus realizing efficient proliferation and optimized cultivation of Akk. This study provides important clues for a deeper understanding of the gut microbiota composition in Chinese individuals, while also offering effective methods for the isolation and cultivation of Akk, laying the groundwork for its functional and application research in the human body.

Characteristics of casein phosphopeptides in Chinese human milk and its correlation with infant growth: A cross-sectional study.

This research aimed to investigate the characteristics of casein phosphopeptides in Chinese human milk, and their potential relationship to infant growth. Using the liquid chromatography-Orbitrap-mass spectrometry technique, a total of 15 casein phosphopeptides were identified from 200 human milk samples. Also, our results indicate that casein phosphopeptides were phosphorylated with only one phosphate. The relative concentrations of casein phosphopeptides at 6 months postpartum were increased compared with milk at 2 months (FDR < 0.05). Significantly positive correlations were observed between casein phosphopeptides and infant growth, as shown by four casein phosphopeptides were positively correlated with the infants' weight-for-age Z-scores (rs range from 0.20 to 0.29), and three casein phosphopeptides were positively correlated with the infants' length-for-age Z-scores (rs range from 0.19 to 0.27). This study is the first to reveal the phosphorylated level and composition of casein phosphopeptides in Chinese human milk, and their potential relationship with infant growth.

Effects of endogenous DHA milk and exogenous DHA milk on oxidative stress and cognition in SAMP8 mice.

In this study, Senescence Accelerated Mice (SAMP8) were supplemented with exogenous DHA milk, endogenous DHA milk, normal milk, or 0.9 % saline solution. Enzyme-linked immunosorbent assay (ELISA), gas chromatography (GC), ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI MS/MS), and Morris water maze were used to characterize the effects of diet on oxidative stress and cognition in SAMP8 mice. Supplementation endogenous DHA milk or exogenous DHA milk can enhance the antioxidant capacity of mice organs. Endogenous DHA milk increased the superoxide dismutase (SOD) activity of mice brain and serum than normal milk and 0.9 % saline solution (P ≤ 0.05), as well as increased SOD activity of mice liver and glutathione peroxidase (GSH-Px) activity of mice brain than normal milk (P ≤ 0.05). Exogenous DHA milk increased SOD activity of mice brain than normal milk and 0.9 % saline solution, as well as increased SOD activity of mice serum than 0.9 % saline solution (P ≤ 0.05). Several polar lipid relative content, such as 18:0/18:2 PS, 17:0 Ceramide, and 20:4 LPC in mice brain was affected by dietary supplementation with DHA-containing milk. Lipid oxidation metabolites in mice brain were not affected by DHA-containing milk. Endogenous DHA milk increased the number of platform location crossing times of mice in the Morris water maze test, compared with Exogenous DHA milk, normal milk, and 0.9 % saline solution (P ≤ 0.05).

Determination and Risk Assessment of Flavor Components in Flavored Milk.

This study aimed to determine chemical composition and assess exposure in flavored milk among Chinese residents, based on risk assessment methodologies of acceptable daily intake (ADI) and toxicological concern threshold (TTC). Esters (32.17%), alcohols (11.19%), olefins (9.09%), aldehydes (8.39%), and ketones (7.34%) comprised the majority of the flavoring samples. Methyl palmitate (90.91%), ethyl butyrate (81.82%), and dipentene (81.82%) had the highest detection rates in flavor samples. This study screened fifteen flavor components of concern and discovered that 2,3,5-trimethylpyrazine, furfural, benzaldehyde, and benzenemethanol were detected in 100% of flavored milk samples. Benzenemethanol was found in the highest concentration (14,995.44 µg kg-1). The risk assessment results revealed that there was no risk for Chinese residents in consuming flavored milk, and the maximum per capita daily consumption of 2,3,5-trimethylpyrazine, furfural, and benzenemethanol were 226.208 g, 140.610 g, and 120.036 g, respectively. This study could provide guidelines for amounts of flavor additive ingredients in milk.

Advances in the Metabolic Mechanism and Functional Characteristics of Equol.

Equol is the most potent soy isoflavone metabolite and is produced by specific intestinal microorganisms of mammals. It has promising application possibilities for preventing chronic diseases such as cardiovascular disease, breast cancer, and prostate cancer due to its high antioxidant activity and hormone-like activity. Thus, it is of great significance to systematically study the efficient preparation method of equol and its functional activity. This paper elaborates on the metabolic mechanism of equol in humans; focuses on the biological characteristics, synthesis methods, and the currently isolated equol-producing bacteria; and looks forward to its future development and application direction, aiming to provide guidance for the application and promotion of equol in the field of food and health products.

Effects of different heat treatments on Maillard reaction products and volatile substances of camel milk.

Camel milk has unique compositional, functional and therapeutic properties compared to cow's milk and also contains many protective proteins with anti-cancer, anti-diabetic and anti-bacterial properties. In this experiment, fresh camel milk was heat-treated at different temperatures and times, and the changes in Millard reaction products were analyzed. Meanwhile, headspace-gas chromatography-ion migration spectrometry (HS-GC-IMS), electronic nose and electronic tongue were used to analyze the changes of volatile components in camel milk after different heat treatments. The results showed that the Maillard reaction was more severe with the increase of heat treatment, and the contents of furosine and 5-hydroxymethylfurfural increased significantly when the heat treatment temperature was higher than 120°C. HS-GC-IMS results showed that the contents of aldehydes and ketones increased obviously with the increase of heat treatment degree. The study clarifies the effects of different heat treatment degrees on Maillard reaction degree and flavor of camel milk, which has practical production guidance significance for the research and industrialization of liquid camel milk products.

Changes in glycosylated proteins in colostrum and mature milk and their implication.

Introduction: Glycosylation is one of the essential post-translational modifications that influences the function of milk proteins. Methods: In the present study, 998 proteins and 764 glycosylated sites from 402 glycoproteins were identified in human milk by TMT labeling proteomics. Compared to human milk proteins, the glycoproteins were mainly enriched in cell adhesion, proteolysis, and defense/immune process. Results: The abundance of 353 glycosylated sites and their 179 parent proteins was quantified. After normalization to their parent protein's abundance, 78 glycosylated sites in 56 glycoproteins and 10 glycosylated sites in 10 glycoproteins were significantly higher in colostrum and mature milk, respectively. These changed glycoproteins were mainly related to host defense. Intriguingly, one glycosylated site (Asp144) in IgA and two glycosylated sites (Asp38 and Asp1079) in tenascin are significantly upregulated even though their protein abundance was downregulated during lactation. Discussion: This study helps us figure out the critical glycosylated sites in proteins that might influence their biological function in an unbiased way.

Comparison of glycerophospholipid and sphingolipid in mature milk from different sampled regions in the Chinese human milk project (CHMP) study.

Milk phospholipids (PLs) are critical components of infant growth. This study aimed to discover PL in mature human milk (HM) from China (n = 201) and mainly assessed the effect caused by sampled regions. The average total PL concentration was quantified from 3.65 to 11.25 mg per g of lipid, and the major PL class identified was sphingomyelin (SM, 38.06-47.62 %), followed by phosphatidylcholine (PC, 29.61-34.39 %), and phosphatidylethanolamine (PE, 10.54-24.46 %). In addition, the 36:2 (18:0/18:2), 38:6 (16:0/22:6), 40:1 (d18:1/22:0), and 42:2 (d18:1/24:1) were the most abundant molecular species identified in glycerophospholipid and SM molecular species respectively. Some PL molecular species were strongly related with region of sampling, like lysophosphatidylinositol 18:1 was only detected in Beijing. In conclusion, those findings showed that the PL molecular species and concentration of HM had significant regional diversity, and it will give the Chinese human milk database more accurate PL data.

Effects of Pre-Emulsification with Thermal-Denatured Whey Protein on Texture and Microstructure of Reduced-Sodium Processed Cheese.

Thermal-denatured whey protein-milk fat emulsion gels with different degrees of pre-emulsification were prepared by pre-emulsifying milk fat with thermal-denatured whey protein and used in the preparation of reduced-sodium processed cheeses. The effect of the thermal-denatured whey protein pre-emulsification process on the texture and microstructure of reduced-sodium processed cheeses was evaluated by studying the composition, color, texture, functional properties, microstructure and sensory analysis of the processed cheeses. The results showed that compared with cheese without pre-emulsified fat (1.5% ES control), the moisture content of cheese with pre-emulsified 100% fat (1.5% ES100) increased by 5.81%, the L* values increased by 7.61%, the hardness increased by 43.24%, and the free oil release decreased by 38%. The microstructure showed that the particle size of fat was significantly reduced, and the distribution was more uniform. In addition, compared with the cheese added with 3% emulsifying salt (3% ES control), the amount of emulsifying salt in the 1.5% ES100 decreased by 50%, but the fat distribution of the two kinds of cheese tended to be consistent, and there was no obvious change in texture characteristics and meltability. Sensory scores increased with the increase in pre-emulsification degree. Overall, the pre-emulsification of milk fat with thermal-denatured whey protein can reduce the sodium content of processed cheese and improve its quality.