Location: Dairy and Functional Foods Research
2024 Annual Report
Objectives
Objective 1: Characterize bioactive peptides released from dairy proteins by enzymatic digestion and investigate the effect of protein-carbohydrate interactions on the stability and bioavailability of these peptides in dairy foods. [NP306, C1, PS1C]
Sub-objective 1.A: Production and characterization of bioactive peptides from dairy proteins through enzymatic digestion.
Sub-objective 1.B: Develop encapsulation-based delivery systems to protect and enhance the bioactivity and effectiveness of these peptides.
Objective 2: Identify novel bioactive prebiotic oligosaccharides from plant (fruit and vegetable) and animal (milk) sources in food processing low-value by-products. [NP306, C1, PS1B]
Approach
New bioactive compounds will be developed from dairy proteins, oligosaccharides and plant dietary fiber. Bioactive compounds will include antioxidative peptides from milk and dairy products. We will identify these bioactive peptides released through the enzymatic digestion of dairy proteins and products, and investigate the effect of milk fatty acids and oligosaccharides on the production of these peptides. Novel prebiotics will be characterized and compared with galacto-oligosaccharides derived from lactose and milk oligosaccharides. Pectic oligosaccharide prebiotic activity will be evaluated using animal models, mixed culture fecal fermentation and determining their effects as human bioactive food ingredients. We will develop synbiotics in live microbial systems such as yogurt and with tea products. Oligosaccharide anti-adhesive activity against pathogens will be determined with tissue culture cells. We will determine dietary fiber characteristics in fruit and vegetable processing byproducts.
Progress Report
Sub-objective 1a. Consumers' growing interest in fermented dairy foods necessitates research on a wide array of lactic acid bacterial strains to be explored and used. This study aimed to investigate the differences in the proteolytic capacity of Lactobacillus helveticus strains B1929 and ATCC 15009 on the fermentation of commercial ultra-pasteurized (UHT) skim milk and reconstituted nonfat dried milk powder (at a comparable protein concentration, 4%). The antihypertensive properties of the fermented milk, measured by angiotensin-I-converting enzyme inhibitory (ACE-I) activity, were compared. The B1929 strain lowered the pH of the milk to 4.13 ± 0.09 at 37°C after 24 h, whereas ATCC 15009 needed 48 h to drop the pH to 4.70 ± 0.18 at 37°C. Two soluble protein fractions, one (CFS1) obtained after fermentation (acidic conditions) and the other (CFS2) after the neutralization (pH 6.70) of the pellet from CFS1 separation, were analyzed for d-/l-lactic acid production, protein concentration, the degree of protein hydrolysis, and ACE-I activity. The CFS1 fractions, dominated by whey proteins, demonstrated a greater degree of protein hydrolysis (7.9%) than CFS2. On the other hand, CFS2, mainly casein proteins, showed a higher level of ACE-I activity (33.8%) than CFS1. Significant differences were also found in the d- and l-lactic acid produced by the UHT milk between the 2 strains. These results attest that milk casein proteins possessed more detectable ACE-I activity than whey fractions, even without a measurable degree of hydrolysis. Findings from this study suggest that careful consideration must be given when selecting the bacterial strain and milk substrate for fermentation.
Sub-objective 1b. Arabinoxylans (AX) and protease treated AX (AXP) were subjected to enzymatic hydrolysis with endoxylanase and arabinofuranosidase to obtain hydrolyzed AX (HAX) and AXP (HAXP), whose ability to promote Bifidobacterium infantis and Bifidobacterium longum growth was investigated. Further, the effect of cross-linked AX on the growth of bifidobacteria was also explored. Bifidobacteria showed the highest growth on AX and AXP, while HAX and HAXP did not have a significant impact on bacterial growth. The laccase cross-linking of AX stimulated the growth of bifidobacteria, possibly due to its gel-like structure which favored the bacteria-substrate (AX) interaction. The AX and alginate (SA) were used to prepare synbiotic matrices. The ability of AX-SA matrices to encapsulate and protect probiotic bacteria (Lactobacillus rhamnosus GG, Streptococcus thermophilus and B. longum) viability under storage conditions was investigated. The AX synbiotic matrices presented the highest encapsulation efficiencies (55-77%) for all three strains, when AX-SA and SA matrices were compared. Significantly higher levels (~7 logs) of L. rhamnosus GG were recovered from AX and AX-SA synbiotic matrices after 28 days of storage under aerobic conditions at 4 °C compared to SA matrices (~4 logs). The results indicated that the incorporation of AX into synbiotic matrices played a significant role on the survival of encapsulated bacteria during storage, which could be attributed to the stability of the covalent cross-linked network formed during AX gelation that protected bacterial viability. Thus the synbiotic matrices based on AX could be promising materials to encapsulate and protect probiotic bacteria for targeted-delivery to the colon.
Objective 2. ß-galactosidase is currently applied in foods for reduction of lactose but can also be utilized for its transgalactosylation activity to synthesize galacto-oligosaccharides (GOS) as prebiotics. The ability of GRAS-status Lactobacillus delbrueckii strains to exhibit such activities would benefit consumers given their extensive history with dairy products. The objective of this study was to characterize the production of GOS in six L. delbueckii strains for their ability to synthesize GOS in 50 mM sodium phosphate (pH 6.5) with high lactose concentrations at 50 °C. L. delbrueckii subsp. bulgaricus strains B548, LB11, and YB1 lysates released the most glucose at 112.8 ± 6.2 mM, 150.4 ± 11.7 mM, and 190.2 ± 12.2 mM, respectively. However, the ratio of free glucose to free galactose released by B548 lysate (1.4 ± 0.3) was significantly lower than that of LB11 (2.8 ± 0.6) and YB1 (2.5 ± 0.6) lysates, so the latter strains were screened further at different initial lactose concentrations. GOS yield from YB1 was not dependent on initial lactose concentration, averaging 54.3 ± 0.6% across all starting lactose concentrations. However, optimal LB11 transgalatosylation had an initial concentration of 394.4 mM lactose instead of 788.8 mM, resulting in a GOS yield of 56.8% instead of 47.3%. In all cases for LB11, the lysates had greater free glucose, galactose, and GOS yield than the whole cells. The ability of two Lactobacillus delbrueckii subsp. bulgaricus strains to produce GOS from common lactose-containing ingredients can have a large range of applications in the dairy industry. Indeed, oligosaccharides were produced with ß-galactosidase from Lactobacillus bulgaricus or Kluyveromyces lactis in reconstituted nonfat dry milk powder sweetened with either sucrose or corn syrup solids. These oligosaccharides have prebiotic activity since they can be extracted and used to increase the growth of probiotic Bifidobacterium breve 2141 and Limosilactobacillus reuteri 1428 compared to nonfat dry milk without ß-galactosidase treatment.
Objective 2. Strawberries are a nutrient dense food rich in vitamins, minerals, non-nutrient antioxidant phenolics, and fibers. Strawberry fiber bioactive structures are not well characterized and limited information is available about the interaction between strawberry fiber and phenolics. Therefore, we analyzed commercial strawberry pomace in order to provide a detailed carbohydrate structural characterization, and to associate structures with functions. The pomace fraction, which remained after strawberry commercial juice extraction, contained mostly insoluble (49.1% vs. 5.6% soluble dietary fiber) dietary fiber, with pectin, xyloglucan, xylan, ß-glucan and glucomannan polysaccharides; glucose, fructose, xylose, arabinose, galactose, fucose and galacturonic acid free carbohydrates; protein (15.6%), fat (8.34%), and pelargonidin 3-glucoside (562 µg/g). Oligosaccharides from fucogalacto-xyloglucan, methyl-esterified rhamnogalacturonan I with branched arabinogalacto-side chains, rhamnogalacturonan II, homogalacturonan and ß-glucan were detected by MALDI-TOF MS, NMR and glycosyl-linkage analysis. Previous reports suggest that these oligosaccharide and polysaccharide structures have prebiotic, bacterial pathogen anti-adhesion, and cholesterol-lowering activity, while anthocyanins are well-known antioxidants. A strawberry pomace microwave acid-extracted (10 min, 80°C) fraction had high molar mass (2376 kDa) and viscosity (3.75 dL/g), with an extended rod shape. A random coil shape, that was reported previously to bind to phenolic compounds, was observed for other strawberry microwave-extracted fractions. These strawberry fiber structural details suggest that they can thicken foods, while the polysaccharide and polyphenol interaction indicates great potential as a multiple-function bioactive food ingredient important for gut and metabolic health.
Accomplishments
1. Strawberry pomace source of healthy dietary fiber. Strawberries are well known healthy fruit containing bioactive compounds, yet little is known about the pomace remaining after commercial juice extraction. ARS scientists in Wyndmoor, Pennsylvania, characterized commercial strawberry pomace by as mostly insoluble dietary fiber containing pectin and other polysaccharides, protein, fat and red pigments. These compounds are known to have gut health, cholesterol-lowering, antioxidant and anti-pathogen activity. The binding between pectin and red pigment compounds suggests that strawberry pomace bioactive compounds may have synergistic properties to promote health. This information will help food processors develop better functional food ingredients and will provide more value for this specialty crop.
Review Publications
Hotchkiss, A.T., Chau, H.K., Strahan, G.D., Nunez, A., Harron, A.F., Simon, S., White, A.K., Dieng, S., Heuberger, E., Black, I., Yadav, M.P., Welchoff, M., Hirsch, J. 2024. Structural characterization of strawberry pomace. Heliyon. 10:e29787.
Cai Shi, D., Hotchkiss, A.T., Lawton, M.A., Di, R. 2023. Modified citrus pectin confers a preventative effect on cancer-related pathways in CdCl2-treated C. elegans. Food Hydrocolloid for Health. 4:100161. https://doi.org/10.1016/j.fhfh.2023.100161.
Guron, G.P., Qi, P.X., Mcanulty, M.J., Renye Jr, J.A., Miller, A.L., Oest, A.M., Wickham, E.D., Harron, A.F. 2023. Differential behavior of Lactobacillus helveticus B1929 and ATCC 15009 on the hydrolysis and angiotensin-I-converting enzyme inhibition activity of fermented ultra-high-temperature milk and nonfat dried milk powder. Journal of Dairy Science. 106(7):4502-4515. https://doi.org/10.3168/jds.2022-22842.