The Lipidic and Volatile Components of Coffee Pods and Capsules Packaged in an Alternative Multilayer Film.

Coffee pods and capsules require packaging that guarantees the optimal coffee preservation. The chemical composition of coffee can undergo quality decay phenomena during storage, especially in terms of lipidic and volatile components. Amongst coffee packaging, aluminum multilayer materials are particularly widely diffused. However, aluminum is a negative component because it is not recoverable in a mixed plastic structure and its specific weight gives significant weight to packaging. In this study, a multilayer film with a reduced content of aluminum was used to package coffe pods and capsules and compared to a standard film with an aluminum layer. Their influence on the peroxides and volatile organic compounds of two coffee blends, 100% Coffea arabica L., 50% Coffea arabica L., and 50% Coffea canephora var. robusta L., were studied during their 180-day shelf life. The predominant volatile organic compounds detected belonged to the class of furans and pyrazines. Both packaging materials used for both coffee blends in the pods and capsules showed no significant differences during storage. Thus, the alternative packaging with less aluminum had the same performance as the standard with the advantage of being more sustainable, reducing the packaging weight, with benefits for transportation, and preserving the coffee aroma during the shelf life.

The Use of Carbon Dioxide as a Green Approach to Recover Bioactive Compounds from Spent Coffee Grounds.

Spent coffee grounds (SCG) contain bioactive compounds. In this work, given the increasing demand to valorize waste and use green technologies, SCG were submitted to extraction by carbon dioxide (CO2) in supercritical and liquid conditions. The extraction parameters were varied to obtain the maximum yield with the maximum antioxidant activity. The use of supercritical and liquid CO2 with 5% ethanol for 1 h provided yields (15 and 16%, respectively) comparable to those obtained by control methods for 5 h and extracts with high total polyphenolic contents (970 and 857 mg GAE/100 g oil, respectively). It also provided extracts with DPPH (3089 and 3136 µmol TE/100 g oil, respectively) and FRAP (4383 and 4324 µmol TE/100 g oil, respectively) antioxidant activity levels higher than those of hexane extracts (372 and 2758 µmol TE/100 g oil, respectively) and comparable to those of ethanol (3492 and 4408 µmol TE/100 g oil, respectively). The SCG extracts exhibited linoleic, palmitic, oleic, and stearic acids (predominant fatty acids) and furans and phenols (predominant volatile organic compounds). They were also characterized by caffeine and individual phenolic acids (chlorogenic, caffeic, ferulic, and 3,4-dihydroxybenzoic acids) with well-known antioxidant and antimicrobial properties; therefore, they could be used in the cosmetic, pharmaceutical, and food sectors.

Bioactive Compounds and Antioxidant Properties of Wild Rocket (Diplotaxis Tenuifolia L.) Grown under Different Plastic Films and with Different UV-B Radiation Postharvest Treatments.

Rocket species are rich in nutrients with well-known bioactive activity, but their content depends on several factors, such as plant-UV radiation interaction. In this work, we measured the production of nutritional elements in wild rocket (Diplotaxis tenuifolia L.) leaves as a function of exposure to UV-B radiation by adopting a combined approach. The wild rocket plants were grown under three greenhouse cover films (A, B, and C) having different transmittivity to UV-B and the fresh-cut leaves were exposed to UV-B in postharvest for 45, 150, 330, and 660 s. The content of chlorophyll, carotenoids, phenolic compounds, ascorbic acid, and the antioxidant activity were determined. Chlorophyll, carotenoids, and total phenolic content were significantly increased by the combination of Film C and treatment with UV-B for 45 s. The predominant phenolic compounds were kaempferol, isorhamnetin, and quercetin. Film C also elicited an increase in ascorbic acid (the most abundant antioxidant compound in the range 374-1199 per 100 g of dry matter) and antioxidant activity. These findings highlighted an increase in bioactive compound content in the wild rocket when it was cultivated under Film C (diffused light film with a tailored UV-B transmission dose) and treated with UV-B radiation for 45 s postharvest, corresponding to an energy dose of 0.2 KJ m-2.

Deep-frying purple potato Purple Majesty using sunflower oil: effect on the polyphenols, anthocyanins and antioxidant activity.

The potato is a root vegetable native to the Americas; it consists of the starchy tuber of the plant Solanum tuberosum. There are many varieties, and the flesh can have different colour ranging from yellow to red and purple. Coloured varieties have a denser texture and slightly nuttier, earthier flavour than other potatoes. The desirable quality characteristics of potatoes depends on the intended use, and the acceptability of raw potatoes is determined by size, shape, colour, and the quality of can be evaluated in terms of colour, flavour, and texture. Deep-frying is the century-old and it is among the most common cooking processes, still being used to prepare a variety of food products on both industrial and domestic scales. Frying the potatoes is among the tastiest and appreciated way to cook this vegetable. Purple fleshed potatoes are widely considered one of the best-tasting purple potatoes varieties, they have a nice taste and add colour to a meal. They are a source of beneficial health compounds which makes them interesting as functional food. The anthocyanins present in the Purple Majesty variety are interesting for their health promoting abilities, anti-oxidative activity, and even other health beneficial effects, e.g. anti-influenza virus activity, and anti-stomach cancer activity. The aim of this study has been to assess the effect of deep-frying of purple potato Purple Majesty using sunflower oil on the polyphenols, anthocyanins and to evaluate the antioxidant activity of the cooked matrix compared to the fresh one. The results seem to suggest that the healthy characteristics of this functional food are retained after the cooking by frying.

Basil (Ocimum basilicum L.) Leaves as a Source of Bioactive Compounds.

Basil (Ocimum basilicum L.) is an annual spicy plant generally utilized as a flavouring agent for food. Basil leaves also have pharmaceutical properties due to the presence of polyphenols, phenolic acids, and flavonoids. In this work, carbon dioxide was employed to extract bioactive compounds from basil leaves. Extraction with supercritical CO2 (p = 30 MPa; T = 50 °C) for 2 h using 10% ethanol as a cosolvent was the most efficient method, with a yield similar to that of the control (100% ethanol) and was applied to two basil cultivars: "Italiano Classico" and "Genovese". Antioxidant activity, phenolic acid content, and volatile organic compounds were determined in the extracts obtained by this method. In both cultivars, the supercritical CO2 extracts showed antiradical activity (ABTS●+ assay), caffeic acid (1.69-1.92 mg/g), linalool (35-27%), and bergamotene (11-14%) contents significantly higher than those of the control. The polyphenol content and antiradical activity measured by the three assays were higher in the "Genovese" cultivar than in the "Italiano Classico" cultivar, while the linalool content was higher (35.08%) in the "Italiano Classico" cultivar. Supercritical CO2 not only allowed us to obtain extracts rich in bioactive compounds in an environmentally friendly way but also reduced ethanol consumption.

"Sfogliatella Riccia Napoletana": Realization of a Lard-Free and Palm Oil-Free Pastry.

"Sfogliatella riccia napoletana" is a typical pastry from Naples (Italy), traditionally produced using lard. In the bakery industry, palm oil is widely used to replace lard in order to obtain products without cholesterol, but it is currently under discussion, which is mostly related to the sustainability of its cultivation. Therefore, in this work, lard was replaced with palm oil-free vegetable blends composed of sunflower oil, shea butter, and coconut oil in different percentages. Traditional pastries produced with lard and pastries produced with palm oil were used as controls. Moisture, aw, free acidity, peroxide value, fatty acids, total polar compounds, and global acceptability were determined in the obtained pastries. The results indicated that the use of a vegetable oil blend composed of 40% sunflower oil, 40% shea butter, and 20% coconut oil minimized the formation of oxidized compounds (peroxides and total polar compounds) during cooking and produced a product with a moisture content very similar to that of the traditional pastry that was appreciated by consumers.

Ontogenetic Variation in the Mineral, Phytochemical and Yield Attributes of Brassicaceous Microgreens.

Microgreens constitute novel gastronomic ingredients that combine visual, kinesthetic and bioactive qualities. The definition of the optimal developmental stage for harvesting microgreens remains fluid. Their superior phytochemical content against mature leaves underpins the current hypothesis of significant changes in compositional profile during the brief interval of ontogeny from the appearance of the first (S1) to the second true leaf (S2). Microgreens of four brassicaceous genotypes (Komatsuna, Mibuna, Mizuna and Pak Choi) grown under controlled conditions and harvested at S1 and S2 were appraised for fresh and dry yield traits. They were further analyzed for macro- and micromineral content using inductively coupled plasma optical emission spectrometry (ICP-OES), carotenoid content using high-performance liquid chromatography with a diode-array detector (HPLC-DAD), volatile organic compounds using solid-phase microextraction followed by gas chromatography-mass spectrometry (SPME-GC/MS), anthocyanins and polyphenols using liquid chromatography-high resolution-tandem mass spectrometry (LC-MS/MS) with Orbitrap technology and for chlorophyll and ascorbate concentrations, well as antioxidant capacity by spectrophotometry. Analysis of compositional profiles revealed genotype as the principal source of variation for all constituents. The response of mineral and phytochemical composition and of antioxidant capacity to the growth stage was limited and largely genotype-dependent. It is, therefore, questionable whether delaying harvest from S1 to S2 would significantly improve the bioactive value of microgreens while the cost-benefit analysis for this decision must be genotype-specific. Finally, the lower-yielding genotypes (Mizuna and Pak Choi) registered higher relative increase in fresh yield between S1 and S2, compared to the faster-growing and higher-yielding genotypes. Although the optimal harvest stage for specific genotypes must be determined considering the increase in yield against reduction in crop turnover, harvesting at S2 seems advisable for the lower-yielding genotypes.

Oxidative stability of high oleic sunflower oil during deep-frying process of purple potato Purple Majesty.

The behaviour of high oleic sunflower oil in deep frying process of purple potato Purple Majesty has been evaluated simulating a fast food cooking process. This oil was used for 8h/day for 6 days, filling up from the 2nd day. A discontinuous and prolonged procedure was tested. Free Fatty Acidity (FFA), Peroxide Value (PV), Total Polar Compounds (TPC), Fatty Acid (FA) composition, Volatile Organic Compounds (VOC) have been determined at different times in thermo-oxidized (T-OX) oil, and in frying oil. The FFA in T-OX oil samples showed values in the range 0.09%-0.24%, whereas in the frying oil values varied in a range between 0.09% and 0.16%. TPCs values varied from 1.76% to 38.24% in T-OX oils, whereas in frying oil used for frying purple potatoes (FOPP) showed values in the range from 1.76 to 29.13%. The peroxides values did not follow a regular pattern, both during thermo-oxidation and during frying. Among the Long Chain Fatty Acids (LCFAs), oleic acid was the most represented (84.13%). Short chain fatty acids (SCFAs) amount was 0.34% (octanoic acid). Medium chain fatty acids (MCFA) amount was 4.45% (palmitic acid). During the thermo-oxidation, the poly-unsaturated fatty acids (PUFA) amount decreased during the 48 h of heat treatment, reaching an amount of 6.21%. This determined the increase in short chain fatty acids (SCFA). Trans fatty acids increased with the frying time. Unsaturated fatty acids (UFA) reached the value of 90.19%; SFA was 9.79%, and octanoic acid was 0.20%. A correlation between TPC vs UFA/SFA and TPC vs C18:2/C16:0 was observed in the frying oil. The most abundant volatile compounds in frying oil (from 0 to 48 h) were the aldehydes produced by decomposition of hydroperoxides of oleic and linoleic acids.

Characterization of a new type of mead fermented with Cannabis sativa L. (hemp).

Mead, one of the oldest existing drinks, is a fermented product based on honey, water, and the possible addition of spices and selected yeasts. In this work, various parts (inflorescences, leaves, and steams) of Cannabis sativa L. at different concentrations and Saccharomyces cerevisiae biotype M3/5 were added during mead fermentation. The physicochemical parameters (pH, alcoholic content, sugar content, titratable acidity, and organic acids) of the mead were assessed at the beginning and end of fermentation. Moreover, polyphenols, cannabidiol and volatile organic compounds were identified at the end of fermentation and compared with the control sample prepared without hemp and with only indigenous yeasts. The mead fermented with hemp showed the highest quantity of polyphenols (227 to 256 mg GAE/L) and a level of cannabidiol ranging from 0.26 to 0.49 mg/kg. The volatile organic compounds found were mainly alcohols, esters and terpenes, which were present at higher concentrations in the mead prepared with C. sativa L. than in the control mead and conferred freshness and "hemp aroma" characteristics. PRACTICAL APPLICATION: Inflorescences, leaves, and steams of Cannabis sativa L. were added at different concentrations during mead fermentation. This type of mead showed high quantity of polyphenols (227 to 256 mg GAE/L) and a level of cannabidiol ranging from 0.26 to 0.49 mg/kg which have anxiolytic and neuro-protective properties. Moreover the volatile organic compounds found (mainly alcohols, esters, and terpenes) conferred freshness and "hemp aroma" characteristics.

Genotype and Successive Harvests Interaction Affects Phenolic Acids and Aroma Profile of Genovese Basil for Pesto Sauce Production.

Basil (Ocimum basilicum L.) is an essential ingredient of the Mediterranean cuisine due to its distinctive aroma. Genovese basil leaves are used to prepare "pesto", a condiment that has always caught the interest of consumers and producers. Usually, basil for industrial processing is harvested more than once to extract a higher yield. However, successive cuts can affect quality traits that play a crucial role in defining the product's final sensory profile. This research was aimed to evaluate the impact of cut on the quantitative and qualitative properties of three Genovese basil cultivars (Aroma 2, Eleonora and Italiano Classico) grown in an open field. Nitrate content, phenolic acids and aromatic profile were determined by ion chromatography (IC), high-performance liquid chromatography (HPLC), and gas chromatography coupled to a mass spectrometer (GC/MS) analysis, respectively. The second harvest increased fresh biomass and total phenolic acids content by 172% and 413%, respectively, with Italiano Classico recording the highest values. The combination of second-cut Aroma 2 yielded the lowest nitrate (473.8 mg kg-1 of fresh weight) and Eugenol (2.4%) levels. In the second harvest, Eleonora showed an increase in eugenol and trans-α-bergamotene of 75.3% and 48.2%, respectively; whereas, eucalyptol and ß-cis-ocimene decreased by 34.4% and 51.6%, respectively. Although successive harvests may increase basil yield and quality overall, the cultivar-dependent response to successive cuts needs to be accounted for in order to accomplish standardization of industrial "pesto" sauce.

Sweet Basil Functional Quality as Shaped by Genotype and Macronutrient Concentration Reciprocal Action.

Basil (Ocimum basilicum L.) is among the most widespread aromatic plants due to its versatility of use and its beneficial health properties. This aromatic plant thrives in hydroponics, which is a valid tool to improve the production and functional quality of crops, but nevertheless, it offers the possibility to de-seasonalize production. A floating raft system was adopted to test the production and quality potential during autumn season of three different genotypes of Genovese basil (Aroma 2, Eleonora and Italiano Classico) grown in three nutrient solutions with crescent electrical conductivity (EC: 1, 2 and 3 dS m-1). The aromatic and phenolic profiles were determined by GC/MS and HPLC analysis, respectively. The combination Aroma 2 and the EC 2 dS m-1 resulted in the highest production, both in terms of fresh weight and dry biomass. The 2 dS m-1 treatment determined the major phenolic content, 44%, compared to the other two EC. Italiano Classico showed a higher total polyphenolic content in addition to a different aromatic profile compared to the other cultivars, characterized by a higher percentage of Eucalyptol (+37%) and Eugenol (+107%) and a lower percentage of linalool (-44%). Correct management of the nutritional solution combined with adequate genetic material managed an improvement in the production and the obtainment of the desired aromatic and phenolic profiles.

A new type of tomato puree with high content of bioactive compounds from 100% whole fruit.

In this work, a new type of tomato puree was obtained from the whole fruit including seeds and skins without any waste. Currently, the food industry aims to develop products that both bring benefits to consumer health and are ecosustainable. Several efforts in fact have been carried out to revalue tomato by-products, such as seeds and skins, for human use. The physicochemical properties of the whole tomato puree immediately after production and after 6 months of storage were investigated and compared to the properties of a traditional tomato puree. Two tomato varieties were used in this study: Villa Literno and Piccadilly. pH, dry weight, water activity, titratable acidity, reducing sugars, soluble solid, lycopene, polyphenol, and volatile organic compound contents were evaluated. Tomato puree obtained from whole fruits mainly showed higher lycopene (90 to 150 mg/kg), flavonoid (39 to 41 mg/kg), and aldehyde (in particular hexanal) contents and a greater stability over time compared to those of the traditional puree. The proposed process allowed to obtain a tomato puree with a higher content of bioactive compounds, producing no waste. PRACTICAL APPLICATION: In this study, we proposed a new type of tomato puree made from the whole fruit. The whole tomato puree is richer in phenolic compounds and volatile organic compounds than the traditional puree that is made by removing skins and seeds. It can be an interesting product for the food industries because it is a source of bioactive compounds and reduces to zero the amount of waste.

Short-term effects of dietary bovine milk on fatty acid composition of human milk: A preliminary multi-analytical study.

The fatty acid (FA) composition of human milk (HM) from N = 9 Italian healthy donors following a free diet exhibited FA-dependent ranges of variability, as assessed by GC-FID. The possible short-term changes in the FA profile were monitored in the milk of lactating mothers (three) collected at five time points over a 6 h period, following an oral load (200 mL) of bovine milk. An array of techniques was exploited, including UHPLC-ESI-MS/MS of intact lipids and MALDI-TOF MS before and after chemical hydrogenation or bromination, in addition to MALDI-TOF MS analysis of FA after saponification, to monitor short-chain and odd-chain FA in HM as markers of bovine milk fat. A single administration of bovine milk did not appreciably modify the lipid pattern, suggesting that the maternal diet could induce not detectable short-term changes on the lipid composition of HM. Diet-induced increase of butyric acid was also excluded by 13C NMR. The functions that HM FA exert in infant physiology appear finely regulated through maternal metabolism.

Chemical Eustress Elicits Tailored Responses and Enhances the Functional Quality of Novel Food Perilla frutescens.

Consumer demand for fresh and functional horticultural products is on the rise. Perilla frutescens, L. Britt (Lamiaceae) is a potential specialty/niche crop for consumption and therapeutic uses with high contents of phenolic and volatile compounds. Plant growth, mineral composition, polyphenol profile and aroma volatile components of two perilla genotypes in response to salinity (non-salt control, 10, 20 or 30 mM NaCl) applied as chemical eustressor were assessed. Salinity suppressed growth and yield of both genotypes, although the red-pigmented genotype was less sensitive than the green-pigmented one. Mild (10 mM NaCl) and moderate (20 and 30 mM NaCl) salinity suppressed foliar potassium, magnesium, nitrate and chlorophyll a concentrations of both genotypes and increased the levels of rosmarinic acid, total polyphenols and target aroma volatile components. Green perilla showed higher yield and biomass production and higher content of protein, dry matter, calcium, magnesium, perilla ketone and cis-jasmone, whereas red perilla exhibited higher content of potassium, chlorophyll a, rosmarinic acid, total polyphenols, perilla aldehyde and benzaldehyde. Our findings support that chemical eustressors such as mild to moderate salinity offer valuable means to manipulate phytochemical and aroma profiles.

Degradation kinetic (D100) of lycopene during the thermal treatment of concentrated tomato paste.

Heat treatments can cause degradation in tomatoes of lycopene which has important antioxidant effects. No information about decimal reduction time (D100) of lycopene is available. D-value is the time required at a given temperature to reduce 90% of the molecule. This study for the first time determine the kinetic of lycopene thermal degradation. The content was measured at regular intervals of pasteurization using canned tomato paste to determinate D value. Microbiological analysis was carried out to verify product stability after packaging. Yeasts, molds and lactic acid bacteria were determined. The pasteurization time allowed to observe a loss of the red color. Lycopene content, after an increase at 8 min, decreased at 32 min of pasteurization. D100 value was calculated at 75 min; a diminution of 90% in lycopene content in the concentrated tomato paste was observed. Microbiological analysis confirmed the stability of products after 8 min of pasteurization.

Comparative Chemical Compositions of Fresh and Stored Vesuvian PDO "Pomodorino Del Piennolo" Tomato and the Ciliegino Variety.

The Vesuvian Piennolo cherry tomato (Lycopersicon esculentum Miller) (PdP) is an old and typical variety grown in the Campania region (Italy). PdP is referred to as a long-storage tomato due to its thick and coriaceous skin that allows long post-harvest storage and it has been granted Protected Designation of Origin (PDO) status since 2009. In this study, the chemical composition, focusing in particular on organic acids, antioxidant molecules and volatile compounds, were investigated in PdP and compared to another typical variety in Campania, the Ciliegino tomato (CIL). Chemical characterization was evaluated for both the CIL and PdP varieties during storage in the same environmental conditions until deterioration of 50% of the fruits; deterioration occurred in PdP after 6 months and in CIL tomatoes after 1 month. The results demonstrated variation in the chemical profiles of both varieties with storage length. Particularly, the PdP variety appears richer in antioxidants compounds (i.e., chlorogenic acids and lycopene) and organic acids (i.e., glutamic and malic acids) than does CIL. Additionally, both varieties display different profiles of volatile bioactive compounds and they are differently influenced by the storage time. The results indicate a typical chemical composition of this long-storage tomato closely linked to the geographic origin area.

Evaluation of microfiltration and heat treatment on the microbiological characteristics, phenolic composition and volatile compound profile of pomegranate (Punica granatum L.) juice.

BACKGROUND: Since processing technology and storage may influence the sensory and nutritional value as well as the shelf life of pomegranate juice (PJ), mild technologies based on microfiltration may be a promising alternative to heat treatments for fruit juice preservation. In this study, physicochemical and microbiological properties of raw (RPJ), microfiltered (MPJ) and cloudy pasteurized (PPJ) PJ were compared over a period of 4 weeks. RESULTS: Data demonstrated that microfiltration was comparable to pasteurization in guaranteeing microbiological stability of the juice, avoiding spoilage of the final product. After treatment, PPJ showed the highest amounts of gallic acid (GA) and ellagic acid derivatives (EAs). During storage, the amount of ellagitannins, EAs and GA similarly decreased in all types of juice. Trends towards variations of monomeric anthocyanins in MPJ and variations of polymeric and copigmented anthocyanins in both MPJ and PPJ were found over storage. CONCLUSION: The optimization of pretreatments and filtration parameters can lead to the industrial scale-up of microfiltration technology for the development of high-quality non-heat-treated PJ. © 2017 Society of Chemical Industry.

The effects of probiotics and prebiotics on the fatty acid profile and conjugated linoleic acid content of fermented cow milk.

The ability of probiotic bacteria (Lactobacillus acidophilus La5 and Bifidobacterium animalis Bb12), to produce conjugated linoleic acid (CLA) in association with Streptococcus thermophilus and Lb. bulgaricus during milk fermentation has been evaluated in this study. Pasteurized cow milk and infant formula were used. Infant formula was selected for its high linoleic acid content, for being a source of CLA and for its prebiotic compounds, e.g. galacto-oligosaccharides. The microorganisms were not able to increase the CLA content of the fermented products under the given experimental conditions. No statistically significant differences (p > 0.05) occurred between the CLA content in milk and the fermented samples. The CLA contents of 10 commercial fermented milk products were determined. The highest CLA content was observed in fermented milk containing only Str. thermophilus and Lb. bulgaricus.

Modulation of the pentose phosphate pathway induces endodermal differentiation in embryonic stem cells.

Embryonic stem (ES) cells can differentiate in vitro into a variety of cell types. Efforts to produce endodermal cell derivatives, including lung, liver and pancreas, have been met with modest success. Understanding how the endoderm originates from ES cells is the first step to generate specific cell types for therapeutic purposes. Recently, it has been demonstrated that inhibition of Myc or mTOR induces endodermal differentiation. Both Myc and mTOR are known to be activators of the Pentose Phosphate Pathway (PPP). We found that, differentely from wild type (wt), ES cells unable to produce pentose sugars through PPP differentiate into endodermal precursors in cell culture conditions generally non-permissive to generate them. The same effect was observed when wt ES cells were differentiated in presence of chemical inhibitors of the PPP. These data highlight a new role for metabolism. Indeed, to our knowledge, it is the first time that modulation of a metabolic pathway is described to be crucial in determining ES cell fate.

Primary structure of water buffalo alpha-lactalbumin variants A and B.

A novel electrophoretic alpha-lactalbumin (alpha-la) variant was detected in the Italian water buffalo breed. The isoelectric point of the variant, labelled A, was lower than the most frequent variant B. It presented an allelic frequency of 0.5% compared with the 97.1% of the BB allele. From Liquid Chromatography-Electrospray Ionization/Mass spectrometry, the molecular mass of the two alpha-la A and B variants were measured as 14,235.1+/-0.8 and 14,236.1+/-0.9 Da, respectively. The two proteins were sequenced and differentiated from one another by a single amino acid substitution, Asn45(B)-->Asp45(A). As this amino acid substitution altered the N-glycosylation sequence consensus Asn45-X-Ser46 it may be deduced that the protein glycosylation level of the alpha-la A would decrease.