Determining the safety of microbial cultures for consumption by humans and animals.

Fermented foods and feeds have been consumed for millennia, and microorganisms isolated from traditional fermentations have been used as probiotics. There is interest in developing new microbial cultures for these uses, but to date safety evaluation procedures have only been discussed in general terms. We propose a comprehensive approach for determining the safety of microbial cultures that lack an established history of safe use for their intended new applications. Three scenarios are considered: (1) substantially increased exposure to a culture that has an established record of safety in a more limited application; (2) a new strain without a history of safe use that was isolated from a food or feed that has a history of safe use; and (3) a new strain isolated from a non-food or non-feed source. Our safety evaluation process is based on scientific procedures and is in the form of a decision tree composed of 13 questions. Our decision tree for determining the safety of microbial cultures for consumption by humans or animals is modeled on previous decision trees that are used worldwide to evaluate the safety of microbial enzymes for use in human food or animal feed.

Total antioxidant capacity is significantly lower in cocaine-dependent and methamphetamine-dependent patients relative to normal controls: results from a preliminary study.

BACKGROUND: Oxidative stress can result in damage to the brain and other organs. To protect from oxidative damage, the human body possesses molecular defense systems, based on the activity of antioxidants, and enzymatic defense systems, including the enzymes catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Although pre-clinical research has shown that stimulant use is associated with oxidative damage, oxidative stress and the antioxidant defense systems have not been evaluated in clinical samples of stimulant-dependent patients. OBJECTIVES: This study aimed to investigate the link between stimulant dependence and oxidative stress. METHODS: Peripheral blood samples from 174 methamphetamine (n = 48) and/or cocaine-dependent (n = 126) participants as well as 30 normal control participants were analyzed for the enzyme activities of CAT, SOD, and GSH-Px in the erythrocytes and the total antioxidant capacity and the malondialdehyde concentration in the plasma. RESULTS: We could show an association of stimulant dependence with a depletion of total antioxidant capacity to 54.6 ± 4.7%, which correlates with a reduced activity of the SOD to 71.3 ± 0.03% compared with healthy control participants (100%). CONCLUSION: Stimulant-dependent patients had significantly lower antioxidant capacity relative to controls, suggesting that they may be at greater risk for oxidative damage to the brain and other organs.

Characteristics and safety assessment of intractable proteins in genetically modified crops.

Genetically modified (GM) crops may contain newly expressed proteins that are described as "intractable". Safety assessment of these proteins may require some adaptations to the current assessment procedures. Intractable proteins are defined here as those proteins with properties that make it extremely difficult or impossible with current methods to express in heterologous systems; isolate, purify, or concentrate; quantify (due to low levels); demonstrate biological activity; or prove equivalency with plant proteins. Five classes of intractable proteins are discussed here: (1) membrane proteins, (2) signaling proteins, (3) transcription factors, (4) N-glycosylated proteins, and (5) resistance proteins (R-proteins, plant pathogen recognition proteins that activate innate immune responses). While the basic tiered weight-of-evidence approach for assessing the safety of GM crops proposed by the International Life Sciences Institute (ILSI) in 2008 is applicable to intractable proteins, new or modified methods may be required. For example, the first two steps in Tier I (hazard identification) analysis, gathering of applicable history of safe use (HOSU) information and bioinformatics analysis, do not require protein isolation. The extremely low level of expression of most intractable proteins should be taken into account while assessing safety of the intractable protein in GM crops. If Tier II (hazard characterization) analyses requiring animal feeding are judged to be necessary, alternatives to feeding high doses of pure protein may be needed. These alternatives are discussed here.

Determining the safety of enzymes used in animal feed.

The purpose of this paper is to provide guidance for evaluating the safety of enzyme preparations used in animal feed. Feed enzymes are typically added to animal feed to increase nutrient bioavailability by acting on feed components prior to or after consumption, i.e., within the gastrointestinal tract. In contrast, food processing enzymes are generally used during processing and then inactivated or removed prior to consumption. The enzymes used in both applications are almost always impure mixtures of active enzyme and other metabolites from the production strain, hence similar safety evaluation procedures for both are warranted. We propose that the primary consideration should be the safety of the production strain and that the decision tree mechanism developed previously for food processing enzymes (Pariza and Johnson, 2001) is appropriate for determining the safety of feed enzymes. Thoroughly characterized non-pathogenic, non-toxigenic microbial strains with a history of safe use in enzyme manufacture are also logical candidates for generating safe strain lineages, from which additional strains may be derived via genetic modification by traditional and non-traditional strategies. For new feed enzyme products derived from a safe strain lineage, it is important to ensure a sufficiently high safety margin for the intended use, and that the product complies with appropriate specifications for chemical and microbial contamination.

NF-kappaB independent inhibition of lipopolysaccharide-induced cyclooxygenase by a conjugated linoleic acid cognate, conjugated nonadecadienoic acid.

10t, 12c-CLA was shown to inhibit COX-2 expression through the NF-kappaB pathway. In the current study, conjugated nonadecadienoic acid (CNA) was shown to decrease inducible COX-2 protein and mRNA and PGE(2) release to the similar extent as 10t, 12c-CLA in Raw264.7 macrophage. However, unlike 10t, 12c-CLA, inhibition of COX-2 mRNA/protein by CNA was independent of the NF-kappaB pathway. The data indicate the regulation of COX-2 by select conjugated fatty acids and hence their anti-inflammatory actions could operate through different signal transduction pathways.

Selective conjugated fatty acids inhibit guinea pig platelet aggregation.

Conjugated linoleic acids have been shown to reduce eicosanoid release from select tissues and/or cells. To elucidate effects of conjugated linoleic acid isomers on cyclooxygenase-1 (COX-1) activity and their application as platelet aggregation inhibitors, conjugated linoleic acid isomers and conjugated nonadecadienoic acid were incubated with ovine COX-1 and Raw264.7 macrophage to examine their effects on COX-1 activity. The effects were further examined in collagen and ADP-induced guinea pig whole blood platelet aggregation. Fatty acids tested were shown to inhibit COX-1 enzymatic activity. However, only 10t, 12c-conjugated linoleic acid, 9t, 11t-conjugated linoleic acid and conjugated nonadecadienoic acid inhibited collagen and ADP-induced platelet aggregation with IC(50) 125.9 microM (74.2 microM to 213.4 microM, 95% confidence interval), 99.3 microM (52.8 microM to 187.2 microM, 95% confidence interval) and 124.3 microM (85.1 microM to 181.5 microM, 95% confidence interval) respectively in collagen-induced aggregation. TxB(2) release was also appreciably inhibited by 10t, 12c-conjugated linoleic acid, 9t, 11t-conjugated linoleic acid and conjugated nonadecadienoic acid. Based on these data, we conclude 10t, 12c-conjugated linoleic acid, 9t, 11t-conjugated linoleic acid and conjugated nonadecadienoic acid are platelet aggregation inhibitors while 9c, 11t-conjugated linoleic acid is a moderate inhibitor and linoleic acid, and 9c, 11c-conjugated linoleic acid have no effect on whole blood platelet aggregation.

Effects of conjugated linoleic acid on long term feeding in Fischer 344 rats.

Weanling male Fischer 344 rats were fed either control or diet containing 1% CLA for 18 months. Weight gain and survival rate were not different between treatments, but CLA-fed animals ate slightly less food. CLA feeding did not significantly reduce body fat compared to that of control. Clinical chemistry and hematology analyses were performed on blood samples at week 69-72. CLA had no effects except on blood glucose, which was reduced in CLA-fed animals compared to control. All animals had chronic renal failure at the end of the study; however, CLA decreased the amount of protein in urine at week 70 of feeding. Necropsy and histo-pathology results indicated that there was no difference between treatment groups. Although this study used a limited number of animals and a single dose of CLA, our results suggest that long term CLA feeding did not cause any adverse effects in rats.

Influence of feeding soybean oil on conjugated linoleic acid content in beef.

Forty-eight steers were used to study the influence of feeding soybean oil (SO) on the conjugated linoleic acid (CLA) content of beef. Steers were fed either a control diet containing 954 g/kg of dry matter (DM) corn-based concentrate (CTL) or a control diet supplemented with SO at 20 (SO2) or 40 g/kg (SO4) of diet DM for 105 days. Adipose tissue samples were collected from the M. longissimus dorsi (LD) and from the M. semitendinosus (ST) on days 0 and 63 of the experiment. Adipose and muscle tissue samples were collected from the LD and ST immediately after slaughter. Feeding 40 g/kg of DM as SO increased the proportions of trans-C(18:1) in beef lipid as compared to CTL and SO2 treatments. The C(18:2) cis-9, trans-11 isomer of CLA as a proportion of total fat was not different in adipose and muscle across treatments. Supplementing SO increased C(18:2) trans-10, cis-12 CLA in adipose tissue of the LD. Supplementing high-grain finishing diets with SO is not an effective strategy to enhance the C(18:2) cis-9, trans-11 isomer of CLA in beef.

Biological activities of conjugated fatty acids: conjugated eicosadienoic (conj. 20:2delta(c11,t13/t12,c14)), eicosatrienoic (conj. 20:3delta(c8,t12,c14)), and heneicosadienoic (conj. 21:2delta(c12,t14/c13,t15)) acids and other metabolites of conjugated linoleic acid.

The elongated form of conjugated linoleic acid (CLA), conjugated eicosadienoic acid (CEA, conj. 20:2delta(c11,t13/t12,c14)), was generated from CLA by liver microsomal fractions. Subsequent testing showed that dietary CEA significantly reduced body fat, and increased lean mass similar to CLA when compared to controls. CEA also decreased lipoprotein lipase activity and triacylglyceride, and increased glycerol release in 3T3-L1 adipocytes, correlated with the trans-12,cis-14 isomer, but CEA required a longer incubation period than cells treated with CLA. Based on the fact that CEA fed animals had CLA in tissue, we suggest that the effect of CEA is due to the CLA converted from CEA in the system. The delta-6 desaturated and elongated form of trans-10,cis-12 CLA (conjugated eicosatrienoic acid, CETA, conj. 20:3delta(c8,t12,c14)) inhibited LPL activity and increased glycerol release but was less active than trans-10,cis-12 CLA or CEA. The 21-carbon conjugated fatty acid, conjugated heneicosadienoic acid (CHDA, conj. 21:2delta(c12,t14/c13,t15)), was not active on LPL inhibition, triacylglyceride, or glycerol release in 3T3-L1 adipocytes. We also provide evidence that CLA was metabolized to conjugated dodecadienoic acid (conj. 12:2delta(c3,t5/t4,c6)). In addition, there were indications of the presence of conjugated tetradecadienoic acid (conj. 14:2delta(c5,t7/t6,c8)), suggesting that CLA can be metabolized through fatty acid beta-oxidation. This is the first work to report the presence of conjugated 12 and 14 carbon fatty acids, originated from CLA, and the biological activities of CEA, CETA and CHDA.

Controlling acrylamide in French fry and potato chip models and a mathematical model of acrylamide formation: acrylamide: acidulants, phytate and calcium.

We previously reported that in potato chip and French fry models, the formation of acrylamide can be reduced by controlling pH during processing steps, either by organic (acidulants) or inorganic acids. Use of phytate, a naturally occurring chelator, with or without Ca++ (or divalent ions), can reduce acrylamide formation in both models. However, since phytate itself is acidic, the question remains as to whether the effect of phytate is due to pH alone or to additional effects. In the French fry model, the effects on acrylamide formation of pH, phytate, and/or Ca++ in various combinations were tested in either blanching or soaking (after blanching) steps. All treatments significantly reduced acrylamide levels compared to control. Among variables tested, pH may be the single most important factor for reducing acrylamide levels, while there were independent effects of phytate and/or Ca++ in this French fry model. We also developed a mathematical formula to estimate the final concentration of acrylamide in a potato chip model, using variables that can affect acrylamide formation: glucose and asparagine concentrations, cut potato surface area and shape, cooking temperature and time, and other processing conditions.

Conjugated linoleic acid preserves gastrocnemius muscle mass in mice bearing the colon-26 adenocarcinoma.

Cancer cachexia is a syndrome of weight loss, muscle wasting, fatigue, and anorexia that occurs in patients with advanced or recurrent solid tumor disease. Tumor necrosis factor-alpha (TNFalpha) and prostaglandin E2 (PGE2) have been implicated in the biology of cachexia and serve as possible targets for treatment of this condition. Conjugated linoleic acid (CLA) is a polyunsaturated fatty acid that alters the synthesis of PGE2 and reduces the negative effects of TNF on body weight of healthy mice. We hypothesized that a diet supplemented with .5% CLA might reduce muscle wasting in mice bearing the colon-26 adenocarcinoma, an animal model of cancer cachexia. CLA preserved gastrocnemius muscle mass and reduced TNF receptors in muscle of tumor-bearing mice. These data suggest that CLA may preserve muscle mass by reducing the catabolic effects of TNF on skeletal muscle.

Structure-activity relationship of conjugated linoleic acid and its cognates in inhibiting heparin-releasable lipoprotein lipase and glycerol release from fully differentiated 3T3-L1 adipocytes.

Conjugated linoleic acid (CLA) reduces body fat in part by inhibiting the activity of heparin-releasable lipoprotein lipase (HR-LPL) activity in adipocytes, an effect that is induced by the trans-10,cis-12 CLA isomer. In this study we used a series of compounds that are structurally related to CLA (i.e., CLA cognates) to investigate the structural basis for this phenomenon. None of the 18:1 CLA cognates that were tested, nor trans-9,cis-12 18:2, cis-12-octadecen-10-ynoic acid (10y,cis-12) or 11-(2'-(n-pentyl)phenyl)-10-undecylenic acid (designated P-t10), exhibited any significant effect on HR-LPL activity. Among the CLA derivatives (alcohol, amide, and chloride) that were tested, only the alcohol form inhibited HR-LPL activity, although to a lesser extent than CLA itself. In addition, intracellular TG was reduced only by trans-10,cis-12 CLA and the alcohol form of CLA. Hence it appears that the trans-10,cis-12 conjugated double bond in conjunction with a carboxyl group at C-1 is required for inhibition of HR-LPL activity, and that an alcohol group can partially substitute for the carboxyl group. We also studied glycerol release from the cells, observing that this was enhanced by trans-10 18:1, trans-13 18:1, cis-12 18:1, cis-13 18:1, P-t10 but was reduced by cis-9 18:1, the alcohol and amide forms of CLA or 10y,cis-12. Accordingly the structural feature or features involved in regulating lipolysis appear to be more complex. Despite enhancing lipolysis in cultured 3T3-L1 adipocytes, trans-10 18:1 did not reduce body fat gain when fed to mice.

Perspective on the safety and effectiveness of conjugated linoleic acid.

The amount of scientific literature on conjugated linoleic acid (CLA) is growing at a phenomenal rate. Animal studies and clinical trials indicate the possibility that CLA could be useful in improving human health in a number of areas, eg, controlling body fat gain and enhancing immunity while also reducing inflammation and other adverse effects typically associated with immune enhancement. The background of this growing research field and mechanistic insights from animal and cell culture experiments are briefly reviewed. Experimental and clinical data relating to the safety and effectiveness of CLA in humans are presented and discussed.

Evidence that the anti-obesity effect of conjugated linoleic acid is independent of effects on stearoyl-CoA desaturase1 expression and enzyme activity.

The trans-10,cis-12 isomer of conjugated linoleic acid (CLA) reduces body fat gain in animals and inhibits stearoyl-CoA desaturase (SCD) activity in 3T3-L1 adipocytes. To test whether CLA's body fat reduction is mediated by SCD1, wild-type and SCD1-null mice were fed diet supplemented with 0.2% trans-10,cis-12 (t10c12) CLA for 4 weeks. The t10c12 CLA-supplemented diet significantly reduced body fat mass in both wild type and SCD1-null mice. Similarly, t10c12 CLA diet decreased blood triglyceride and free fatty acid levels regardless of SCD1 genotypes. Mice fed t10c12 CLA exhibited increased mRNA expression of fatty acid synthase and uncoupling protein 2 in both genotypes. Taken together, the effects of t10c12 CLA on reduction of body fat gain, blood parameters, and mRNA expression in both SCD1-null mice and wild-type mice were similar, indicating that the anti-obesity effect of t10c12 CLA may be independent of the effects of this CLA isomer on SCD1 gene expression and enzyme activity.

Isomer-specific regulation of metabolism and PPARgamma signaling by CLA in human preadipocytes.

Trans-10,cis-12 conjugated linoleic acid (CLA) has previously been shown to be the CLA isomer responsible for CLA-induced reductions in body fat in animal models, and we have shown that this isomer, but not the cis-9,trans-11 CLA isomer, specifically decreased triglyceride (TG) accumulation in primary human adipocytes in vitro. Here we investigated the mechanism behind the isomer-specific, CLA-mediated reduction in TG accumulation in differentiating human preadipocytes. Trans-10,cis-12 CLA decreased insulin-stimulated glucose uptake and oxidation, and reduced insulin-dependent glucose transporter 4 gene expression. Furthermore, trans-10,cis-12 CLA reduced oleic acid uptake and oxidation when compared with all other treatments. In parallel to CLA's effects on metabolism, trans-10,cis-12 CLA decreased, whereas cis-9,trans-11 CLA increased, the expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and several of its downstream target genes when compared with vehicle controls. Transient transfections demonstrated that both CLA isomers antagonized ligand-dependent activation of PPARgamma. Collectively, trans-10,cis-12, but not cis-9, trans-11, CLA decreased glucose and lipid uptake and oxidation and preadipocyte differentiation by altering preadipocyte gene transcription in a manner that appeared to be due, in part, to decreased PPARgamma expression.

trans-10,cis-12 CLA inhibits differentiation of 3T3-L1 adipocytes and decreases PPAR gamma expression.

The trans-10,cis-12 isomer of conjugated linoleic acid (CLA) has been shown to reduce body fat gain in mice. However, the underlying molecular mechanism is not well characterized. Here we report evidence that trans-10,cis-12 (t10c12) CLA inhibits preadipocyte differentiation. Treating differentiating 3T3-L1 preadipocytes with t10c12 CLA and conjugated nonadecadienoic acid (CNA, a 19-carbon CLA cognate) resulted in decreased intracellular triglyceride accumulation and mRNA levels of the adipogenic gene fatty acid synthase and adipocyte lipid binding protein. T10c12 CLA and CNA also reduced protein levels of adipocyte transcription factors, peroxisome proliferator-activated receptor gamma and CCAAT/enhancer binding protein alpha. Similarly, CLA reduced body fat gain and significantly inhibited the expression of PPAR gamma and its downstream target lipoprotein lipase in mouse adipose tissue. These observations indicate that CLA decreases body fat gain in part by inhibiting the differentiation of preadipocytes.

Short-term intake of conjugated linoleic acid inhibits lipoprotein lipase and glucose metabolism but does not enhance lipolysis in mouse adipose tissue.

Feeding diets supplemented with t10c12 conjugated linoleic acid (CLA) to growing mice reduces body fat mass. The effects are evident after 1 wk and maximal by 3 wk and are accompanied by reductions in fat cell size. This may complicate direct comparisons with adipocytes from control mice. Accordingly, we investigated the early biochemical events that occur within adipocytes during the first week of CLA feeding, before changes in the size of adipocytes have occurred. Female ICR mice were fed a control diet or a diet supplemented with 0.5 g/100 g of CLA for 4 d, at which time there were no differences in body weight, fat mass or adipocyte size (except that CLA-fed mice had fewer adipocytes >90 micro m in diameter). Parametrial adipose tissue from the CLA-fed mice had significantly reduced heparin-releasable lipoprotein lipase (LPL) and intracellular LPL activities and significantly reduced glucose incorporation into CO(2), fatty acid and glycerol. There were no differences between adipose tissues from CLA-fed or control mice in the ratios of 16:0 to 16:1 and 18:0 to 18:1 fatty acids or in norepinephrine-stimulated lipolysis. Serum insulin levels in food-deprived mice, measured at 4 d and 7 wk, did not differ between groups nor did the concentration of free fatty acids in serum of food-deprived or fed mice measured at the same time points. In mice, CLA-induced inhibition of heparin-releasable LPL and glucose metabolism may be the most important early steps leading to subsequent body fat reduction. In addition, CLA does not appear to enhance lipolysis in mouse adipose tissue in vivo.

Effects of conjugated linoleic acid (CLA) on immune responses, body composition and stearoyl-CoA desaturase.

Conjugated linoleic acid (CLA) has shown a wide range of biologically beneficial effects; reduction of incidence and severity of animal carcinogenesis, reduction of the adverse effects of immune stimulation, reduction of severity of atherosclerosis, growth promotion in young rats, and modulation of stearoyl-CoA desaturase (SCD). One of the most interesting aspects of CLA is its ability to reduce body fat while enhancing lean body mass which is associated with the trans-10,cis-12 isomer of CLA. The effects of CLA are unique characteristics that have not been observed with other polyunsaturated fatty acids. In this review, we will focus on the effects of CLA on immune responses, body compositional changes and stearoyl-CoA desaturase.

Inhibition of stearoyl-CoA desaturase activity by the cis-9,trans-11 isomer and the trans-10,cis-12 isomer of conjugated linoleic acid in MDA-MB-231 and MCF-7 human breast cancer cells.

Conjugated linoleic acid (CLA) is a collective term for a group of positional and geometric conjugated dienoic isomers of linoleic acid. CLA has been shown to have strong inhibitory effects on mammary carcinogenesis both in vitro and in vivo. In this study, we investigated the regulation of human stearoyl-CoA desaturase (SCD, EC 1.14.99.5) expression by CLA in human breast cancer cell lines, MDA-MB-231 and MCF-7. Treatment of the cells with the cis-9,trans-11 and trans-10,cis-12 CLA isomers (45 microM) did not repress SCD mRNA in both MDA-MB-231 and MCF-7 cells. However, the cis-9,trans-11 and trans-10,cis-12 CLA isomers significantly decreased SCD protein levels and SCD activity in MDA-MB-231 cells. In MCF-7 cells, both isomers did not affect protein levels, but they inhibited SCD activity. These results suggest that in MDA-MB-231 cells the cis-9,trans-11 and trans-10,cis-12 CLA isomers regulate human SCD by reducing SCD protein levels, while in MCF-7 cells both isomers have a direct inhibitory effect on SCD enzyme activity.