Normal-Phase HPLC-ELSD to Compare Lipid Profiles of Different Wheat Flours.

Normal-phase high-performance liquid chromatography (HPLC) is widely used in combination with evaporative light scattering detection (ELSD) for separating and detecting lipids in various food samples. ELSD responses of different lipids were evaluated to elucidate the possibilities and challenges associated with quantification by means of HPLC-ELSD. Not only the number and type of polar functional groups but also the chain length and degree of unsaturation of (free or esterified) fatty acids (FAs) had a significant effect on ELSD responses. Tripalmitin and trilinolein yielded notably different ELSD responses, even if their constituting free FAs produced identical responses. How FA structure impacts ELSD responses of free FAs is thus not predictive for those of triacylglycerols and presumably other lipids containing esterified FAs. Because ELSD responses of lipids depend on the identity of the (esterified) FA(s) which they contain, fully accurate lipid quantification with HPLC-ELSD is challenging and time-consuming. Nonetheless, HPLC-ELSD is a good and fast technique to semi-quantitatively compare the levels of different lipid classes between samples of comparable FA composition. In this way, lipid profiles of different flours from near-isogenic wheat lines could be compared.

Impact of wheat endogenous lipids on the quality of fresh bread: Key terms, concepts, and underlying mechanisms.

While wheat (Triticum aestivum L.) flour contains only low levels of lipids (2.0% to 3.0%), they tremendously affect fresh bread quality. They are either starch (30% to 40%) or nonstarch (60% to 70%) lipids. While the former are important in bread staling, they affect neither bread loaf volume nor crumb structure as they are only set free at the very end of the baking process and prior to that they are not available because of their location inside starch granules. This review mainly focuses on wheat nonstarch lipids and how they impact on bread quality. Traditional approaches for investigating their role in bread making have been to use flours varying in bread making quality, to defat and reconstitute flour with (fractions of) the extracted lipids and/or to supplement flour with lipids from wheat or other sources. More recently, lipases have been successfully applied to investigate how wheat lipids affect bread making. It is generally accepted that their impact on bread loaf volume and crumb structure largely if not entirely relates to that on bread dough gas cells stability. However, today there are still different views and hypotheses on the mechanism(s) whereby they impact fresh bread quality. This review first defines and introduces the key terms, concepts, and theories related to lipids, lipases, and bread making. Next, the effects that wheat endogenous lipids and their enzymatically released hydrolysis products have on fresh bread properties and the mechanisms whereby they exert these effects are reviewed.

Do puroindolines affect the impact of enzymatic lipid hydrolysis on loaf volume in bread making?

This paper is the first to study whether and how interactions between puroindolines (PINs) and lipids affect bread loaf volume (LV). Flour from near-isogenic wheat lines differing in PIN haplotype and lipases were used in bread making. That lipase impact on LV strongly depended on the flour used supported the hypothesis that PINs modify the impact of enzymatic lipid hydrolysis on LV. In dough prepared from gluten-starch blends (GSB) differing in PIN levels, PINs did not affect enzymatic lipid hydrolysis itself. Gas cells in these GSB doughs were apparently not surrounded by surface-active compounds so that the impact of PIN-lipid interactions on LV could not be evaluated. This allowed concluding that lipase impact on LV is exclusively related to stabilization of gas cell interfaces in dough since lipase application did not change GSB LVs. Our results advance knowledge on PIN-lipid interactions and the impact of lipases in bread making.

Lipases in wheat flour bread making: Importance of an appropriate balance between wheat endogenous lipids and their enzymatically released hydrolysis products.

Lipids are only minor wheat flour constituents but play major roles in bread making (BM). Here, the importance of a well-balanced lipid population in BM was studied by applying a lipase from Fusarium oxysporum in the process. Monogalactosyldiacylglycerols and N-acyl phosphatidylethanolamines were the most accessible lipase substrates. Hydrolysis thereof into their corresponding lysolipids was largely if not entirely responsible for loaf volume increases upon lipase application. Degradation of endogenously present lipids and enzymatically released lysolipids caused loaf volume to decrease, confirming that an appropriate balance between different types of lipids is crucial in BM. For optimal dough gas cell stability, the level of lipids promoting lamellar mesophases and, thus, liquid condensed monolayers needs to be maximal while maintaining an appropriate balance between lipids promoting hexagonal I phases, non-polar lipids and lipids promoting hexagonal II or cubic phases.

Lipases as Processing Aids in the Separation of Wheat Flour into Gluten and Starch: Impact on the Lipid Population, Gluten Agglomeration, and Yield.

Three lipases with different hydrolysis specificities were tested in a laboratory-scale dough-batter wheat flour separation process in two concentrations. Lipolase specifically hydrolyzed nonpolar flour lipids. At the highest concentration tested, it significantly improved gluten agglomeration and yield, also when combined with a xylanase with hydrolysis specificity toward water-extractable arabinoxylan. We hypothesize that its action is due to the release of adequate levels of free fatty acids, which, because at least a part of them is dissociated, act as anionic surfactants. Lipolase at the lowest concentration, Lecitase Ultra, hydrolyzing both nonpolar and polar lipids, and YieldMAX, which specifically hydrolyzed phospholipids, had no or a negative impact on gluten agglomeration and yield. In conclusion, this study demonstrated that lipases with hydrolysis specificity toward nonpolar lipids can be used as processing aids in wheat flour separation in the absence or presence of added xylanases to maximize gluten agglomeration and yield.

ESX1 mRNA expression in seminal fluid is an indicator of residual spermatogenesis in non-obstructive azoospermic men.

STUDY QUESTION: Is the presence of ESX1 mRNA in seminal fluid (SF) an indicator of residual spermatogenesis in men with non-obstructive azoospermic (NOA)? SUMMARY ANSWER: ESX1 mRNA in SF is a suitable molecular marker for predicting the presence of residual spermatogenesis in testis. WHAT IS KNOWN ALREADY: ESX1 is an X-linked homeobox gene whose expression in testis is restricted to germ cells. We previously reported, in the testicular biopsies from azoospermic men, a positive correlation between the presence of ESX1 mRNA and residual spermatogenesis. STUDY DESIGN, SIZE, DURATION: We investigated ESX1 mRNA expression in 70 testicular fragments (TF) and 56 (SF) of 70 NOA men. As controls, we analyzed 8 TF from men with obstructive azoospermic (OA) and 9 SF from normozoospermic men. For all patients we considered the histological classification of testis biopsies and the recovery of spermatozoa by surgical procedures. PARTICIPANTS/MATERIALS, SETTING, METHODS: Relative ESX1 mRNA expression was evaluated by quantitative RT-PCR using the ΔΔCt method. The results were compared with the recovery of spermatozoa at surgery. MAIN RESULTS AND THE ROLE OF CHANCE: In TF from NOA patients we found that: (i) ESX1 mRNA level was significantly decreased as the severity of spermatogenic defects increased (P < 0.0001, one-way analysis of variance); (ii) the presence of ESX1 mRNA can predict the success of sperm retrieval (sensitivity: 80%). In SF from NOA patients we found that: (i) ESX1 mRNA was present in 78.5% of NOA men; (ii) the presence of ESX1 mRNA could predict the success of sperm retrieval (sensitivity: 84%). LIMITATIONS, REASONS FOR CAUTION: Spermatozoa were recovered at surgery in 5 out of 12 patients whose SF was negative for ESX1 mRNA expression. We think that discrepancies between molecular and clinical results could be reduced by analyzing more than one ejaculate from each man. WIDER IMPLICATIONS OF THE FINDINGS: The data confirm that the ESX1 transcript in the semen of men with NOA is a suitable molecular marker for predicting the presence of residual foci of spermatogenesis in the testis. The implication of these results is that some patients 'with azoospermia', although having a severe impairment of spermatogenesis, could still maintain residual foci of spermatogenesis in limited areas of the testes, not always recovered by surgery. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico: Ricerca Corrente [grant number RC2014/519-02] to M.M. and from ASM onlus 2010-2011 to M.M. The authors declare that they have no conflict of interest.