Antagonistic properties against Fusarium sporotrichioides and glycosylation of HT-2 and T-2 toxins by selected Trichoderma strains.

The present study assessed the ability of Trichoderma to combat F. sporotrichioides, focusing on their antagonistic properties. Tests showed that Trichoderma effectively inhibited F. sporotrichioides mycelial growth, particularly with T. atroviride strains. In co-cultures on rice grains, Trichoderma almost completely reduced the biosynthesis of T-2 and HT-2 toxins by Fusarium. T-2 toxin-α-glucoside (T-2-3α-G), HT-2 toxin-α-glucoside (HT-2-3α-G), and HT-2 toxin-ß-glucoside (HT-2-3ß-G) were observed in the common culture medium, while these substances were not present in the control medium. The study also revealed unique metabolites and varying metabolomic profiles in joint cultures of Trichoderma and Fusarium, suggesting complex interactions. This research offers insights into the processes of biocontrol by Trichoderma, highlighting its potential as a sustainable solution for managing cereal plant pathogens and ensuring food safety.

Adsorption of Zearalenone by Aureobasidium pullulans Autolyzed Biomass Preparation and Its Detoxification Properties in Cultures of Saccharomyces cerevisiae Yeast.

Different preventive strategies are needed to minimize the intake risks of mycotoxins, including zearalenone (ZEN). The aim of this study was to determine the ZEN adsorption ability of an autolyzed biomass preparation of polymorphic yeast Aureobasidium pullulans A.p.-3. The evaluation of the antitoxic properties of the preparation was also performed in relation to Saccharomyces cerevisiae yeast (ATCC 2366, ATCC 7090 and ATCC 9763) used as a model cell exposed to a toxic ZEN dose. The preparation at a dose of 5 mg/mL showed the adsorption of ZEN present in model systems at concentrations between 1 µg/mL to 100 µg/mL. The highest degree of adsorption was established for ZEN concentrations of 1 µg/mL and 5 µg/mL, becoming limited at higher doses of the toxin. Based on the Langmuir model of adsorption isotherms, the predicted maximum ZEN adsorption was approx. 190 µg/mL, regardless of pH. The growth of three strains of S. cerevisiae yeast cells in the medium with ZEN at concentrations within the range of 1.56 µg/mL-100 µg/mL was analyzed to determine the minimum inhibitory concentration. The growth of all tested strains was especially limited by high doses of ZEN, i.e., 50 and 100 µg/mL. The protective effect of the tested preparation was noted in relation to yeast cells exposed to toxic 100 µg/mL ZEN doses. The highest yeast cell growth (app. 36% percentage) was noted for a S. cerevisiae ATCC 9763 strain compared to the medium with ZEN but without preparation. More detailed tests determining the antitoxic mechanisms of the A. pullulans preparation are planned in the future, including cell culture bioassays and animal digestive tract models.

Co-occurrence of T-2 and HT-2 Mycotoxins and α and ß Anomers of Their Glucosides in Wheat and Oat Grains.

The aim of this study was to simultaneously determine T-2 and HT-2 toxins and the α and ß anomers of their glucosides to assess their content in wheat and oat grains harvested in Poland (2020-2022). Of 298 wheat samples, only 14 (5%) contained the sum of the T-2 and HT-2 toxins (average 34.2 µg/kg; 10.6-67.7 µg/kg). In oat (n = 129), these compounds were detected much more frequently (70% of samples) at an average level of 107.5 µg/kg (6.9-949.1 µg/kg). The sum of T-2 and HT-2 glucosides was detectable in 3% of the wheat (average 16.3 µg/kg; 7.1-39.4 µg/kg) and 65% of the oat samples (average 35.1 µg/kg; 4.0-624.1 µg/kg). Following the study, T-2-3-α-glucoside was identified as the only naturally occurring anomer, while both anomers of HT-2-3-glucosides were detected with higher contents and occurrence rates of HT-2-3-ß-glucoside than the α anomer of this compound.

The Development, Validation, and Application of a UHPLC-HESI-MS Method for the Determination of 17 Cannabinoids in Cannabis sativa L. var. sativa Plant Material.

Cannabinoids are an important group of secondary metabolites found in the plant Cannabis sativa L. The growing interest in the use of hemp in food production (e.g., hemp teas, hemp cookies) makes it necessary to develop a method for determining these compounds in the plant, both fresh and dried. The selection of a suitable extraction liquid for the extraction of cannabinoids and the development of a method for the determination of 17 cannabinoids is a prelude to the development of an effective method for the extraction of these compounds. In the present study, a novel, simple, and efficient method was developed and validated for the determination of up to 17 cannabinoids in fresh plant parts (inflorescences and leaves) of Cannabis sativa L. and in dried material, including hemp teas. Analyses were performed using ultra-high-performance liquid chromatography-Q-Exactive Orbitrap mass spectrometry setup operating with a heated electrospray interface (UHPLC-HESI-MS). Based on the comparison, methanol was selected as the best for the extraction of cannabinoids from fresh and dried material. The efficiency and validity of the method were assessed using certified reference material (dried Cannabis) and confirmed by z-score from participation in an international proficiency test conducted by ASTM International for dried hemp.

Low Molecular Weight and High Deacetylation Degree Chitosan Batch Alleviates Pathogenesis, Toxin Accumulation, and Fusarium Gene Regulation in Barley Leaf Pathosystem.

Fusarium graminearum is a cosmopolitan fungal pathogen that destroys cereal production, in terms of loss of yield and grain contamination with mycotoxins, worldwide. Chitosan is a natural biopolymer abundant in the environment with proven antifungal properties that also acts as a plant immunity elicitor. Despite a number of articles, there is a lack of systematic comparison of antifungal activity of diverse batches of chitosan. The current study aimed to test the inhibitory effects of a collection of diverse chitosan samples on the growth and production of F. graminearum toxins, validated by changes in the Fusarium transcriptome. Experiments included testing antifungal activity of different chitosan samples, the application of the best performing one in vitro to investigate the impact on F. graminearum growth, followed by analyzing its effect on Fusarium toxins accumulation, and Fusarium transcriptomics in the barley leaf pathosystem. Confirmatory antifungal assays revealed that CS_10, a specific batch of chitosan, retarded Fusarium growth with an application concentration of 200 ppm, significantly reducing toxin synthesis and disease symptoms in Fusarium-inoculated barley leaves. RNA-Seq analysis of F. graminearum in barley leaf pathosystem exposed to CS_10 showed a list of differentially expressed genes involved in redox balance, cell respiration, nutrient transport, cell wall degradation enzymes, ergosterol biosynthesis, and trichothecenes production. The genes functioning in these essential pathways are discussed and assigned as critical checkpoints to control Fusarium infections. The results suggest some important molecular targets in F. graminearum that may be suitable in gene-specific targeting or transgene-free methods, such as spray-induced gene silencing during host-pathogen interactions.

Recent advances and opportunities related to the use of bee products in food processing.

Nowadays, natural foods that can provide positive health effects are gaining more and more popularity. Bees and the products they produce are our common natural heritage that should be developed. In the article, we presented the characteristics of bee products and their use in industry. We described the development and importance of beekeeping in the modern world. Due to their high nutritional value and therapeutic properties, bee products are of great interest and their consumption is constantly growing. The basis for the use of bee products in human nutrition is their properties and unique chemical composition. The conducted research and opinions confirm the beneficial effect of bee products on health. The current consumer awareness of the positive impact of food having a pro-health effect on health and well-being affects the increase in interest and demand for this type of food among various social groups. Enriching the daily diet with bee products may support the functioning of the organism. New technologies have appeared on the market to improve the process of obtaining bee products. The use of bee products plays a large role in many industries; moreover, the consumption of bee products and promotion of their medicinal properties are very important in shaping proper eating habits.

Toxico-pathological effects of ochratoxin A and its diastereoisomer under in ovo conditions and in vitro evaluation of the toxicity of these toxins against the embryo Gallus gallus fibroblast cell line.

Herein, we conducted a comparative study on the embryotoxicity of ochratoxin A (OTA) and its diastereomer 2'R-ochratoxin A (2'R-OTA) under in ovo conditions, as well as assess the in vitro embryotoxicity of these substances together with ochratoxin B and α-ochratoxin, using chicken (Gallus gallus domesticus) embryo cell lines. In ovo tests involved egg incubation of 8 different groups (i.e., control "0"-no puncture or injection (standard incubation); "00"-punctured eggs without injection; "OTA 0.25," "OTA 0.50," "OTA 0.75," "2'R-OTA 0.25," "2'R-OTA 0.50," "2'R-OTA 0.75"-eggs containing OTA or 2'R-OTA at 0.25, 0.50, and 0.75 µg/egg concentration, respectively). The results confirmed OTA's impact on early and late embryo mortality, where chick hatchability decreased with increasing toxin dosage. Both OTA and 2'R-OTA demonstrated embryotoxicity, however, in the case of the highest OTA diastereomer dose, nearly 11% higher chick hatchability was observed compared with the group that received OTA. 2'R-OTA dosage did not reduce parameters chick quality compared to chicks hatched from control group eggs. OTA concentrations were higher than 2'R-OTA detected in chicken organs such as liver and kidney, whereas 2'R-OTA concentrations were higher in blood serum and heart. The presented studies highlighted the differences in the ability to accumulate toxins in certain organs, which, to a certain extent, may affect the potential toxicity on individual organs. Additionally, during in vitro tests, when assessing the cytotoxic effects of OTA and its analogues toward the chicken embryonic cell line in an MTT assay, the cell metabolic activity was inhibited to a comparable extent at 27-times higher concentration of 2'R-OTA than OTA (0.24 µM). Also, comparably lower toxicity was attributed to the remaining OTA derivatives.

Trichoderma versus Fusarium-Inhibition of Pathogen Growth and Mycotoxin Biosynthesis.

This study evaluated the ability of selected strains of Trichoderma viride, T. viridescens, and T. atroviride to inhibit mycelium growth and the biosynthesis of mycotoxins deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEN), α-(α-ZOL) and ß-zearalenol (ß-ZOL) by selected strains of Fusarium culmorum and F. cerealis. For this purpose, an in vitro experiment was carried out on solid substrates (PDA and rice). After 5 days of co-culture, it was found that all Trichoderma strains used in the experiment significantly inhibited the growth of Fusarium mycelium. Qualitative assessment of pathogen-antagonist interactions showed that Trichoderma colonized 75% to 100% of the medium surface (depending on the species and strain of the antagonist and the pathogen) and was also able to grow over the mycelium of the pathogen and sporulate. The rate of inhibition of Fusarium mycelium growth by Trichoderma ranged from approximately 24% to 66%. When Fusarium and Trichoderma were co-cultured on rice, Trichoderma strains were found to inhibit DON biosynthesis by about 73% to 98%, NIV by about 87% to 100%, and ZEN by about 12% to 100%, depending on the pathogen and antagonist strain. A glycosylated form of DON was detected in the co-culture of F. culmorum and Trichoderma, whereas it was absent in cultures of the pathogen alone, thus suggesting that Trichoderma is able to glycosylate DON. The results also suggest that a strain of T. viride is able to convert ZEN into its hydroxylated derivative, ß-ZOL.

Recent Research on Fusarium Mycotoxins in Maize-A Review.

Maize (Zea mays L.) is one of the most susceptible crops to pathogenic fungal infections, and in particular to the Fusarium species. Secondary metabolites of Fusarium spp.-mycotoxins are not only phytotoxic, but also harmful to humans and animals. They can cause acute or chronic diseases with various toxic effects. The European Union member states apply standards and legal regulations on the permissible levels of mycotoxins in food and feed. This review summarises the most recent knowledge on the occurrence of toxic secondary metabolites of Fusarium in maize, taking into account modified forms of mycotoxins, the progress in research related to the health effects of consuming food or feed contaminated with mycotoxins, and also the development of biological methods for limiting and/or eliminating the presence of the same in the food chain and in compound feed.

Variation in Tocochromanols Level and Mycotoxins Content in Sweet Maize Cultivars after Inoculation with Fusarium verticillioides and F. proliferatum.

A major problem in maize production is the contamination of the grain with Fusarium spp., mainly F. proliferatum and F. verticillioides and their secondary metabolites-mycotoxins. Under biotic stress conditions, caused by a fungal pathogen, plants initiate a series of defense mechanisms that may cause quantitative and qualitative changes in the composition of phenolic compounds. We analyzed the resistance of four sweet maize cultivars (Syngenta Group: Overland, Sweetstar, GSS 8529, Shinerock) to the infection with Fusarium verticillioides and F. proliferatum isolates, along with fumonisins B1, B2, and B3 grain contamination and the levels of tocopherols and tocotrienols accumulated. Differences in ear rot levels were found between the cultivars and isolates used. The phenotypic evaluation positively correlated with the concentrations of fumonisins. The results obtained also indicate a significant dependence on tocochromanols content in sweet maize cultivars tested on the infection of plants with Fusarium isolates and fumonisin biosynthesis. Further studies are needed to investigate the mechanisms of the plant reaction and the effect of different levels of tocopherols and tocotrienols on Fusarium resistance and grain contamination with mycotoxins.

Delving the Role of Caralluma fimbriata: An Edible Wild Plant to Mitigate the Biomarkers of Metabolic Syndrome.

Metabolic syndrome (MS), commonly known as syndrome X or insulin resistance syndrome, is a collection of risk factors for cardiovascular diseases and type II diabetes. MS is believed to impact over a billion individuals worldwide. It is a medical condition defined by visceral obesity, insulin resistance, high blood pressure, and abnormal cholesterol levels, according to the World Health Organization. The current dietary trends are more focused on the use of functional foods and nutraceuticals that are well known for their preventive and curative role against such pathological disorders. Caralluma fimbriata is one such medicinal plant that is gaining popularity. It is a wild, edible, succulent roadside shrub with cactus-like leaves. Besides its main nutrient contents, various bioactive constituents have been identified and linked with positive health outcomes of appetite-suppressing, hypolipidemic, antioxidant, hepatoprotective, and anticancer potentials. Hence, such properties make C. fimbriata an invaluable plant against MS. The current review compiles recent available literature on C. fimbriata's nutritional composition, safety parameters, and therapeutic potential for MS. Summarized data in this review reveals that C. fimbriata remains a neglected plant with limited food and therapeutic applications. Yet various studies explored here do prove its positive health-ameliorating outcomes.

Role of Lactic Acid Bacteria in Food Preservation and Safety.

Fermentation of various food stuffs by lactic acid bacteria is one of the oldest forms of food biopreservation. Bacterial antagonism has been recognized for over a century, but in recent years, this phenomenon has received more scientific attention, particularly in the use of various strains of lactic acid bacteria (LAB). Certain strains of LAB demonstrated antimicrobial activity against foodborne pathogens, including bacteria, yeast and filamentous fungi. Furthermore, in recent years, many authors proved that lactic acid bacteria have the ability to neutralize mycotoxin produced by the last group. Antimicrobial activity of lactic acid bacteria is mainly based on the production of metabolites such as lactic acid, organic acids, hydroperoxide and bacteriocins. In addition, some research suggests other mechanisms of antimicrobial activity of LAB against pathogens as well as their toxic metabolites. These properties are very important because of the future possibility to exchange chemical and physical methods of preservation with a biological method based on the lactic acid bacteria and their metabolites. Biopreservation is defined as the extension of shelf life and the increase in food safety by use of controlled microorganisms or their metabolites. This biological method may determine the alternative for the usage of chemical preservatives. In this study, the possibilities of the use of lactic acid bacteria against foodborne pathogens is provided. Our aim is to yield knowledge about lactic acid fermentation and the activity of lactic acid bacteria against pathogenic microorganisms. In addition, we would like to introduce actual information about health aspects associated with the consumption of fermented products, including probiotics.

In Vitro Effects of Lemon Balm Extracts in Reducing the Growth and Mycotoxins Biosynthesis of Fusarium culmorum and F. proliferatum.

The objectives of this research were to obtain the extracts of lemon balm (Melissa officinalis) using supercritical CO2 (SC-CO2) and methanol as co-solvent and evaluate the antifungal activity of those extracts against two selected strains of Fusarium species (Fusarium culmorum and Fusarium proliferatum). The extraction conditions were set at 40 and 60 °C and 250 bar. The obtained extracts were characterized in terms of antifungal activity on potato dextrose agar media (PDA). The results showed that the extraction parameters had different effects on mycelium growth and mycotoxins biosynthesis reduction. All studied lemon balm extracts (1, 2.5, 5, 7.5, and 10%) inhibited the growth of F. proliferatum and F. culmorum mycelia compared to the control. The lemon balm extracts significantly reduced ergosterol content and synthesized mycotoxins in both tested strains. These findings support the antifungal activity of lemon balm extracts against F. proliferatum and F. culmorum. However, more research on other Fusarium species is needed, as well as in vivo applications, before considering lemon balm extracts as a natural alternative to synthetic fungicides.

Uncovering the Industrial Potentials of Lemongrass Essential Oil as a Food Preservative: A Review.

The food industry is growing vastly, with an increasing number of food products and the demand of consumers to have safe and pathogen-free food with an extended shelf life for consumption. It is critical to have food safe from pathogenic bacteria, fungi, and unpleasant odors or tastes so that the food may not cause any health risks to consumers. Currently, the direction of food industry has been shifting from synthetically produced preservatives to natural preservatives to lower the unnecessary chemical burden on health. Many new technologies are working on natural prevention tools against food degradation. Lemongrass is one such natural preservative that possesses significant antimicrobial and antioxidant activity. The essential oil of lemongrass contains a series of terpenes that are responsible for these activities. These properties make lemongrass acceptable in the food industry and may fulfill consumer demands. This article provides detailed information about the role of lemongrass and its essential oil in food preservation. The outcomes of the research on lemongrass offer room for its new technological applications in food preservation.

Dynamics of Deoxynivalenol and Nivalenol Glucosylation in Wheat Cultivars Infected with Fusarium culmorum in Field Conditions─A 3 Year Study (2018-2020).

Fusarium head blight (FHB) caused by pathogenic species of Fusarium fungi is one of the most important diseases of cereal plants and a factor contributing to losses in plant production. The growth of FHB-associated species is often accompanied by biosynthesis of secondary metabolites─mycotoxins, which serve as a virulence factor. The aim of the study was to evaluate the ratios between deoxynivalenol (DON) and nivalenol (NIV) and their derivatives in the ears of six cultivars of winter wheat with varying resistance to FHB, taking into account a range of factors (weather conditions, location, cultivar, and year) after inoculation with Fusarium culmorum, during a 3 year field experiment, 2018-2020. The presence of toxins in the ears was measured within 21 days of inoculation. The toxins were found in the ears as soon as on the third day from the start of the experiment, whereas relative humidity higher than 80% was a decisive factor for FHB incidence. All wheat cultivars showed the ability to biotransform DON and NIV present in the ears to glucosides, that is, deoxynivalenol-3-glucoside (DON-3G) and nivalenol-3-glucoside (NIV-3G). The levels of these metabolites showed significant correlation with the levels of their basic analogues. In most cases, higher levels of DON and NIV in wheat ears and higher levels of their metabolites were observed, but the relative levels of DON-3G/DON and NIV-3G/NIV at relatively high levels of toxins were lower compared to the ear samples with relatively low toxin levels. The presented results are the first studies, which systematically correlate a variety of wheat cultivars with their extent to glucosylate trichothecenes.

Changes in the microbiological quality and content of biogenic amines in chicken fillets packed using various techniques and stored under different conditions.

The aim of this study was to determine the biogenic amines (BAs) formed in chicken breast meat packaged using different techniques (AP, Hi-O2-MAP or VP) during the storage under different conditions (cold room or display case), to correlate the microbiological quality (TPC, LAB, Pseudomonas spp. and Enterobacteriaceae) of chicken meat with BAs formation and to assess the suitability of selected biogenic amines as indicators of chicken meat spoilage. The initial TPC of chicken fillets was 2.57-3.04 log cfu/g. Over time a systematic significant (p ≤ 0.05) increase in TPC was observed to >7.5 log cfu/g (AP and VP; display case) determined on day 9. It was found that cadaverine and tyramine dominated in quantitative terms in chicken fillets, regardless of packaging technique and storage conditions (166.00 mg/kg in AP meat in cold room on day 9 and 175.03 mg/kg on day 9 in MAP meat in display case, respectively). Taking into account the BAI, high and significant (p ≤ 0.05) correlation coefficients (from 0.51 to 0.95) were obtained with all analyzed indicators of microbiological quality. The concentration of cadaverine, putrescine contents or BAI can potentially serve as chemical quality indicator for freshness of chicken meat.

Transformation of Selected Fusarium Toxins and Their Masked Forms during Malting of Various Cultivars of Wheat.

This study investigated the impact of malting of six wheat cultivars inoculated with Fusarium culmorum on the dynamics of content changes of selected Fusarium toxins. The grains of all the tested cultivars showed a high content of deoxynivalenol (DON), zearalenone (ZEN), and their derivatives, whereas nivalenol (NIV) and its glucoside were found only in the Legenda cultivar. Our experiments confirmed that the malting process of wheat grain enables the secondary growth of Fusarium, and mycotoxin biosynthesis. The levels of toxins in malt were few-fold higher than those in grain; an especially high increase was noted in the case of ZEN and its sulfate as the optimal temperature and pH conditions for the biosynthesis of these toxins by the pathogen are similar to those used in the grain malting process. This is the first paper reporting that during the malting process, biosynthesis of ZEN sulfate occurs, instead of glycosylation, which is a typical modification of mycotoxins by plant detoxication enzymes.

Updated Review of the Toxicity of Selected Fusarium Toxins and Their Modified Forms.

Mycotoxins are one of the most dangerous food and feed contaminants, hence they have significant influence on human and animal health. This study reviews the information reported over the last few years on the toxic effects of the most relevant and studied Fusarium toxins and their modified forms. Deoxynivalenol (DON) and its metabolites can induce intracellular oxidative stress, resulting in DNA damage. Recent studies have also revealed the capability of DON and its metabolites to disturb the cell cycle and alter amino acid expression. Several studies have attempted to explore the mechanism of action of T-2 and HT-2 toxins in anorexia induction. Among other findings, two neurotransmitters associated with this process have been identified, namely substance P and serotonin (5-hydroxytryptamine). For zearalenone (ZEN) and its metabolites, the literature points out that, in addition to their generally acknowledged estrogenic and oxidative potentials, they can also modify DNA by altering methylation patterns and histone acetylation. The ability of the compounds to induce alterations in the expression of major metabolic genes suggests that these compounds can contribute to the development of numerous metabolic diseases, including type 2 diabetes.

Cannabinoids-Characteristics and Potential for Use in Food Production.

Scientific demonstrations of the beneficial effects of non-psychoactive cannabinoids on the human body have increased the interest in foods containing hemp components. This review systematizes the latest discoveries relating to the characteristics of cannabinoids from Cannabis sativa L. var. sativa, it also presents a characterization of the mentioned plant. In this review, we present data on the opportunities and limitations of cannabinoids in food production. This article systematizes the data on the legal aspects, mainly the limits of Δ9-THC in food, the most popular analytical techniques (LC-MS and GC-MS) applied to assay cannabinoids in finished products, and the available data on the stability of cannabinoids during heating, storage, and access to light and oxygen. This may constitute a major challenge to their common use in food processing, as well as the potential formation of undesirable degradation products. Hemp-containing foods have great potential to become commercially popular among functional foods, provided that our understanding of cannabinoid stability in different food matrices and cannabinoid interactions with particular food ingredients are expanded. There remains a need for more data on the effects of technological processes and storage on cannabinoid degradation.

Transformation of Selected Trichothecenes during the Wheat Malting Production.

The transformation of deoxynivalenol (DON), nivalenol (NIV), and their glucosides (DON3G and NIV3G) during the malting of grains of two wheat varieties was studied. The concentration of DON3G and NIV3G started to increase significantly before the concentration of DON and NIV increased. This may reflect the transformation of the parent mycotoxin forms into their glucosides due to xenobiotic detoxification reactions. After a sharp rise during the last 2 days of the process (day 6 and 7), the DON concentration reached 3010 ± 338 µg/kg in the Legenda wheat-based malt and 4678 ± 963 µg/kg in the Pokusa wheat-based malt. The NIV concentration, at 691 ± 65 µg/kg, remained the same as that in the dry grain. The concentration of DON3G in the Legenda and Pokusa wheat-based malt was five and three times higher, respectively, than that in the steeped grain. The concentration of NIV3G in the Legenda wheat-based malt was more than twice as high as that in the steeped grain. The sharp increase in the concentration of DON at the end of the malting process reflected the high pathogen activity. We set aside some samples to study a batch that was left undisturbed without turning and aeration, for the entire period of malting. The concentration of DON in the malt produced from the latter batch was 135% and 337% higher, for Legenda and Pokusa, respectively, than that in the malt produced from the batch that was turned and aerated. The NIV concentration was 22% higher in the latter batch.