Vicki Schlegel

Resistant Starches Types 2 and 4 Have Differential Effects on the Composition of the Fecal Microbiota in Human Subjects

Background To systematically develop dietary strategies based on resistant starch (RS) that modulate the human gut microbiome, detailed in vivo studies that evaluate the effects of different forms of RS on the community structure and population dynamics of the gut microbiota are necessary. The aim of the present study was to gain a community wide perspective of the effects of RS types 2 (RS2) and 4 (RS4) on the fecal microbiota in human individuals. Methods and Findings Ten human subjects consumed crackers for three weeks each containing either RS2, RS4, or native starch in a double-blind, crossover design. Multiplex sequencing of 16S rRNA tags revealed that both types of RS induced several significant compositional alterations in the fecal microbial populations, with differential effects on community structure. RS4 but not RS2 induced phylum-level changes, significantly increasing Actinobacteria and Bacteroidetes while decreasing Firmicutes. At the species level, the changes evoked by RS4 were increases in Bifidobacterium adolescentis and Parabacteroides distasonis, while RS2 significantly raised the proportions of Ruminococcus bromii and Eubacterium rectale when compared to RS4. The population shifts caused by RS4 were numerically substantial for several taxa, leading for example, to a ten-fold increase in bifidobacteria in three of the subjects, enriching them to 18–30% of the fecal microbial community. The responses to RS and their magnitudes varied between individuals, and they were reversible and tightly associated with the consumption of RS. Conclusion Our results demonstrate that RS2 and RS4 show functional differences in their effect on human fecal microbiota composition, indicating that the chemical structure of RS determines its accessibility by groups of colonic bacteria. The findings imply that specific bacterial populations could be selectively targeted by well designed functional carbohydrates, but the inter-subject variations in the response to RS indicates that such strategies might benefit from more personalized approaches.

Improved production of recombinant ovine interferon-τ by Mut+ strain of Pichia pastoris using an optimized methanol feed profile

Recombinant ovine interferon‐τ (r‐oIFN‐τ) production by Pichia pastoris was studied using methanol as the sole carbon source during induction. The cells were grown on glycerol up to a certain cell density before induction of the AOX1 promoter by methanol for expression of the recombinant protein. Cell growth on methanol has been modeled using a substrate‐feed equation, which served as the basis for an effective computer control of the process. The r‐oIFN‐τ concentration in the culture began to decline despite continued cell growth after 50 (± 6) h of induction, which was associated with an increase in proteolytic activity of the fermentation broth. A specific growth rate of 0.025 h‐1 was found to be optimal for r‐oIFN‐τ production. No significant improvement in r‐oIFN‐τ production was observed when the specific growth rate was stepped up before the critical point when r‐oIFN‐τ concentration started decreasing during fermentation. However, best results were obtained when the specific growth rate was stepped down from 0.025 to 0.02 h‐1 at 38 h of induction, whereby the active production period was prolonged until 70 h of induction and the broth protease activity was correspondingly reduced. The corresponding maximum protein yield was 391.7 mg·L‐1 after 70 h of fermentation. The proteolytic activity could be reduced by performing fermentations at specific growth rates of 0.025 h‐1 or below. The recombinant protein production can be performed at an optimal yield by directly controlling the methanol feed rate by a computer‐controlled model. The production profile of r‐oIFN‐τ was found to be significantly different from other secreted and intracellular recombinant protein processes, which is an indication that recombinant protein production in Pichia pastoris needs to be optimized as individual processes following established principles.

Identification of a Putative Operon Involved in Fructooligosaccharide Utilization by Lactobacillus paracasei

ABSTRACT The growth and activity of some Lactobacillus and Bifidobacterium strains are stimulated by the presence of nondigestible fructooligosaccharides (FOS), which are selectively fermented by specific intestinal bacteria. Consumption of FOS, therefore, enriches for those bacteria that possess metabolic pathways necessary for FOS metabolism. In this study, a DNA microarray consisting of 7,680 random genomic library fragments of Lactobacillus paracasei 1195 was used to examine genes involved in the utilization of FOS in this organism. Differential expression profiles between cells grown on FOS and those grown on glucose provided a basis for identifying genes specifically induced by FOS. Several of the FOS-induced genes shared sequence identity with genes encoding β-fructosidases and components of phosphoenolpyruvate-dependent phosphotransferase systems (PTS). These genes were organized in a putative operon, designated the fos operon, that may play an essential role in FOS utilization. The complete 7,631-bp nucleotide sequence of the putative fos operon was determined and consists of fosABCDXE genes, which encode a putative fructose/mannose PTS (FosABCDX) and a β-fructosidase precursor (FosE). The latter contains an N-terminal signal peptide sequence and cell wall sorting signals at the C-terminal region, suggesting its localization at the cell wall. Inactivation of the fosE gene led to impaired growth on FOS and other β-fructose-linked carbohydrates. Transcriptional analysis by reverse transcriptase PCR suggested that fosABCDXE was cotranscribed as a single mRNA during growth on FOS. Expression array analysis revealed that when glucose was added to FOS-grown cells, transcription of the FOS-induced genes was repressed, indicating that FOS metabolism is subject to catabolite regulation.

Factors Influencing the Freeze‐Thaw Stability of Emulsion‐Based Foods

Many of the sauces used in frozen meals are oil-in-water emulsions that consist of fat droplets dispersed within an aqueous medium. This type of emulsion must remain physically and chemically stable throughout processing, freezing, storage, and defrosting conditions. Knowledge of the fundamental physicochemical mechanisms responsible for the stability of emulsion-based sauces is needed to design and fabricate high-quality sauces with the desired sensory attributes. This review provides an overview of the current understanding of the influence of freezing and thawing on the stability of oil-in-water emulsions. In particular, it focuses on the influence of product composition (such as emulsifiers, biopolymers, salts, and cryoprotectants), homogenization conditions, and freezing/thawing conditions on the stability of emulsions. The information contained in this review may be useful for optimizing the design of emulsion-based sauces for utilization in commercial food products.

Design of methanol feed control in Pichia pastoris fermentations based upon a growth model

The methylotrophic yeast Pichia pastoris is an effective system for recombinant protein productions that utilizes methanol as an inducer, and also as carbon and energy source for a Mut+ (methanol utilization plus) strain. Pichia fermentation is conducted in a fed‐batch mode to obtain a high cell density for a high productivity. An accurate methanol control is required in the methanol fed‐batch phase (induction phase) in the fermentation. A simple “on‐off” control strategy is inadequate for precise control of methanol concentrations in the fermentor. In this paper we employed a PID (proportional, integral and derivative) control system for the methanol concentration control and designed the PID controller settings on the basis of a Pichia growth model. The closed‐loop system was built with four components: PID controller, methanol feed pump, fermentation process, and methanol sensor. First, modeling and transfer functions for all components were derived, followed by frequency response analysis, a powerful method for calculating the optimal PID parameters Kc (controller gain), θI (controller integral time constant), and θD (controller derivative time constant). Bode stability criteria were used to develop the stability diagram for evaluating the designed settings during the entire methanol fed‐batch phase. Fermentations were conducted using four Pichia strains , each expressing a different protein, to verify the control performance with optimal PID settings. The results showed that the methanol concentration matched the set point very well with only small overshoot when the set point was switched, which indicated that a very good control performance was achieved. The method developed in this paper is robust and can serve as a framework for the design of other PID feedback control systems in biological processes.

Stearate-Enriched Plant Sterol Esters Lower Serum LDL Cholesterol Concentration in Normo- and Hypercholesterolemic Adults

Studies in our laboratory have previously demonstrated in hamsters a superior cholesterol-lowering ability of plant sterol (PS) esters enriched in stearate compared with linoleate. We therefore conducted a randomized, double-blind, 2-group parallel, placebo-controlled study to test the cholesterol-lowering properties of stearate-enriched PS esters in normo- and hypercholesterolemic adults. Thirty-two adults, 16 per group with equal number of males and females in each group, participated in the 4-wk study. Participants consumed 3 g/d (1 g three times per day with meals) of either PS esters or placebo delivered in capsules. Serum LDL cholesterol concentration significantly decreased 0.42 mmol/L (11%) and the LDL:HDL cholesterol ratio decreased 10% with PS ester supplementation, whereas LDL particle size and lipoprotein subclass particle concentrations (as measured by NMR) were not affected. The percent change in LDL cholesterol was positively correlated with baseline lathosterol concentration (r = 0.729; P = 0.0014), indicating an association between the magnitude of LDL change and the rate of whole-body cholesterol synthesis. Serum campesterol (but not sitosterol) concentration significantly increased in the PS ester group. Serum tocopherol, retinol, and beta-carotene concentrations were not affected by PS ester supplementation. Thus, our findings demonstrate the usefulness of a novel stearate-enriched PS ester compound in decreasing LDL cholesterol in both normo- and hypercholesterolemic adults. The extent to which PS ester fatty acid composition affects intestinal micelle formation and cholesterol absorption in humans requires further study.

A small variation in diet influences the Lactobacillus strain composition in the crop of broiler chickens

Feed composition has the potential to influence the activities of bacteria that colonize the digestive tract of broiler chickens with important consequences for animal health, well being, and food safety. In this study, the gut microbiota of two groups of broiler chickens raised in immediate vicinity but fed either a standard corn/soybean meal ration (corn-soy, CS) or a ration high in wheat middlings (high wheat, HW) was characterized. The findings revealed that this small variation in feed composition did not influence the distribution of microbial species present in the microbial community throughout the digestive tract. However, diet variation markedly influenced the Lactobacillus strain composition in the crop. Most striking, the dominant type in birds on the CS diet (Lactobacillus agilis type R5), which comprised 25% of the isolates, was not detected in birds fed the HW diet. The latter birds harbored a different strain of L. agilis (type R1) in a significantly higher ratio than birds on the CS diet. Several other strains were also specific to the particular diet. In conclusion, this study showed that a small variation in the composition of chicken feed that does not result in detectable differences in species composition can still have an impact on which microbial strains become dominant in the digestive tract. This finding has relevance in the application of probiotics and other direct-fed microbials in poultry husbandry.

Identification and assessment of markers of biotin status in healthy adults

Human biotin requirements are unknown and the identification of reliable markers of biotin status is necessary to fill this knowledge gap. Here, we used an outpatient feeding protocol to create states of biotin deficiency, sufficiency and supplementation in sixteen healthy men and women. A total of twenty possible markers of biotin status were assessed, including the abundance of biotinylated carboxylases in lymphocytes, the expression of genes from biotin metabolism and the urinary excretion of biotin and organic acids. Only the abundance of biotinylated 3-methylcrotonyl-CoA carboxylase (holo-MCC) and propionyl-CoA carboxylase (holo-PCC) allowed for distinguishing biotin-deficient and biotin-sufficient individuals. The urinary excretion of biotin reliably identified biotin-supplemented subjects, but did not distinguish between biotin-depleted and biotin-sufficient individuals. The urinary excretion of 3-hydroxyisovaleric acid detected some biotin-deficient subjects, but produced a meaningful number of false-negative results and did not distinguish between biotin-sufficient and biotin-supplemented individuals. None of the other organic acids that were tested were useful markers of biotin status. Likewise, the abundance of mRNA coding for biotin transporters, holocarboxylase synthetase and biotin-dependent carboxylases in lymphocytes were not different among the treatment groups. Generally, datasets were characterised by variations that exceeded those seen in studies in cell cultures. We conclude that holo-MCC and holo-PCC are the most reliable, single markers of biotin status tested in the present study.

Distillation time alters essential oil yield, composition and antioxidant activity of female Juniperus scopulorum trees

The objective of this study was to evaluate the effect of distillation time (DT) (1.25, 2.5, 5, 10, 20, 40, 80, 160, 240, 360, 480 minutes) on yield, oil profile and antioxidant capacity of female Juniperus scopulorum trees. Analysis of the data revealed that essential oil yields reached a maximum of 0.77% at 240 minutes DT; the concentrations of alpha-thujene, alpha-pinene, camphene, myrcene and para-cymene decreased with longer DT; and the concentrations of cis-sabinene hydrate and linalool/trans-sabinene hydrate reached maximum at 40 minutes DT, whereas that of pregeijerene-B, delta-carene reached maximum at 240 minutes DT. The concentrations of alpha-terpinene, limonene, gamma-terpinene and 4-terpineol reached their maximum at 360 minutes DT, whereas terpinolene, alpha-eudesmol/beta-eudesmol and 8-alpha acetoxyelemol reached maximum at 480 minutes DT. The yield of various essential oil constituents increased with increasing DT and reached maximum at 240 minutes or longer. The antioxidant capacity of J. scopulorum leaf essential oil increased with longer DT and was highest at 480 minutes DT. In conclusion, DT can be used as a tool to obtain an essential oil with differential composition and antioxidant activity. This paper can be used as a reference for comparing reports where different DTs were applied to extract essential oil from the leaves of female J. scopulorum.

Antiproliferation Properties of Grain Sorghum Dry Distiller's Grain Lipids in Caco-2 Cells

Antiproliferative properties of lipids extracted from grain sorghum (GS) dry distillers grain (DDG) were analyzed to determine the feasibility of developing GS coproducts as a source for human health dietary ingredients. The lipid extract of GS-DDG was delivered to human colon carcinoma (Caco-2) cells by solubilizing 0-1000 microg/mL of GS-DDG lipids in 100 microg/mL increments with micelles. A significant reduction in cell viability (25-50%) resulted at treatment levels of 400-1000 microg/mL GS-DDG lipids (p < 0.05). Alternatively, total protein levels of cells treated with 400, 500, and 600 microg/mL of GS-DDG lipid were not significantly different from the control, indicating cell growth during the treatment period. Total cell counts for the control were not significantly different from the GS-DDG lipid treated cells, but dead cell counts increased by approximately 10% for the latter sample with a concomitant increase of the intercellular protein lactate dehydrogenase leakage (30-40%) in the medium. Preliminary analysis by the fluorescence-activated cell method (FACs) demonstrated that nonviable cells were in either the early apoptotic, late apoptotic, or necrotic stage post-treatment with 400, 500, and 600 microg/mL GS-DDG lipids. Physiochemical characterization of the GS-DDG lipids used for the antiproliferation study showed the presence of vitamin E (predominantly gamma-tocopherol), triacylglycerides (predominantly linoleic acid), policosanols, aldehydes, and sterols (predominantly campesterol and stigmasterol), each of which or as synergistic/additive group of constituents may be responsible for the antiproliferative effect.