In recent years, most biofilm studies have focused on fundamental investigations using multispecies biofilm models developed preferentially in simulated naturally occurring low-nutrient medium than in artificial nutrient-rich medium. Because biofilm development under low-nutrient growth media is slow, natural media are often supplemented with an additional carbon source to increase the rate of biofilm formation. However, there are knowledge gaps in interpreting the effects of such supplementation on the resulting biofilm in terms of structure and microbial community composition. We investigated the effects of supplementation of a simulated freshwater medium with sodium citrate on the resulting structure, bacterial community composition, and microbial network interactions of an early-stage multispecies biofilm model. Qualitative and quantitative analyses of acquired confocal laser scanning microscopy data confirmed that sodium citrate supplementation distinctly increased biofilm biomass. Sequencing data revealed that the microbial community structure of biofilms grown in sodium citrate-supplemented conditions was characterized with increased relative abundance and dominance of Proteobacteria compared with that of biofilms grown in sodium citrate-free conditions. Our findings suggest that the supplementation of a low-nutrient medium with a carbon source in experiments involving multispecies biofilms may lead to structural and compositional biases of the microbial community, causing changes in biofilm phenotype. Given that the microbial systems in natural and engineered environments usually comprise consortia of highly diverse microbial communities organized in the form of biofilms, investigations focusing on multispecies biofilms has emerged as a relevant topic for elucidating the behavior of targeted microorganisms within complex systems3 where various forms of interspecies interactions are involved4. Therefore, simulating multispecies biofilm within a laboratory setting has always been considered an arduous task, which explains why most published studies have opted to investigating monospecies biofilm models or, in the best case, biofilm models involving two to three different species. Nevertheless, one of the caveat of performing in vitro experiments is associated with the choice of growth medium, regardless of which type of monospecies or multispecies biofilm model is being investigated. In situations where the composition of the inoculum is known, an appropriate medium can be selected for optimal biofilm growth. For monospecies biofilms, past studies have shown that biofilm development outcomes are significantly affected by the selected growth medium6. In oral biofilm related research, where both monoculture and multispecies biofilm models are typically employed, growth media as well as specific nutrients were also shown to affect growth rate and biofilm composition outcomes12. More specifically, ecological plaque hypothesis  proposed by Marsh assumed that the discrepancy in the microbial composition of oral biofilms impacted by both environmental factors and nutrient availability was found to contribute to the enrichment of disease-associated pathogens13. However, in experiments in which biofilm models constituting a plethora of microorganisms need to be investigated, the growth medium is generally obtained from a nonsterile natural or simulated source15.Compared to monospecies biofilm development, the establishment of multispecies biofilm model is a challenging feat, considering the existing complex biological processes linked to their high bacterial diversity17. Moreover, the speed of biofilm formation may be low in freshwater environments because unlike nutrient-rich media, nonsterile natural media usually contain lower levels of essential nutrients. For example, in nutrient-rich medium, biofilm formation generally occurs within one week, whereas in the natural river medium, at least 15 days are required for the formation of a thin patchy biofilm18.In experiments involving the use of nonsterile medium, the issue of repeatability or inconsistent biofilm outcomes may arise due to the dynamic chemical conditions of the natural source and the high diversity of initial microorganisms. For instance, the nutrient contents in freshwater environments can be affected by random factors such as weather alteration, seasonal shifts, or anthropogenic activities22 or multispecies consortia26. It is through carbon source supplementation that such studies were able to accelerate biofilm development processes needed for conducting timely quantitative and qualitative biofilm analyses, thereby providing an attractive means for experimental productivity.One strategy commonly used to accelerate biofilm formation in nutrient-poor media is the addition of an organic carbon source. In biofilm experiments using nutrient poor media, sodium citrate or sodium acetate is occasionally supplemented as the sole carbon source for accelerating biofilm development in monospecies biofilm modelsThough the addition of substrates, such as sodium citrate, is an ideal way to increase the rate of biofouling, biofilm formation, and biomass level in monospecies models, it is unclear whether such addition is similarly beneficial in multispecies models. In multispecies biofilm models, we hypothesized that the addition of sodium citrate may alter the multispecies community composition as well as its microbial succession. While sodium citrate is likely to accelerate biofilm development in multispecies biofilms, how substrate addition may affect experimental outcomes in terms of biofilm structure and microbial community composition remains unclear, thereby causing potential experimental biases.28, they are not ideal in research experiments meant to simulate freshwater environments characterized by continual flow and shear conditions, as well as constant renewal of nutrients. In the present study, we sought to investigate the effects of sodium citrate supplementation on a previously described multispecies biofilm models, using a dynamic simulated irrigation water distribution system29. The structure and microbial community composition of biofilms in the absence and presence of sodium citrate were compared at different time points of their early-stage formation.There is currently a plethora of various in vitro biofilm models used in the context of environmental or health-related research, however, the selection of either dynamic- or static-forms of systems will ultimately depend on the original conditions the selected model was meant to simulate. While, static biofilm harvesting systems are perhaps the most widely used in research due to their simplicity and other positive attributes29 and is schematized in Fig. The setup of a standardized simulated freshwater source and biofilm model has previously been described in detail29 to ensure a continuous nutrient supply for biofilm growth in each reactor. In the treatment group, sodium citrate was added to the holding tank at a final concentration of 1 mM immediately following every water replacement event. Biofilms were collected for confocal laser scanning microscopy (CLSM) observation and quantification, as well as genomic DNA extraction, at four time points: days 7, 14, 21, and 28, to investigate the shifts in biofilm structure and microbial succession during the early stages of biofilm formation. Throughout the study, biofilms of less than a month were defined as early-stage biofilms, based on a similar definition proposed by Ngan and Habimana29. The extracted DNA were sent to Novogene for 16S rRNA gene pyrosequencing and network analysis. All experiments at selected time points under both control and treatment conditions were repeated three times.For each biofilm system, the freshwater in the holding tank was replaced twice a week as previously describedBiofilm structure was qualitatively assessed from the acquired CSLM  data. All samples were first stained with 5 mM Syto 9 Green Fluorescent Nucleic Acid Stain  without affecting the original biofilm structure. Stained samples were then incubated in a dark box for 30 min. Subsequently, 3D image stacks of random areas of the biofilm samples were created. For biofilm grown in the absence of sodium citrate, an LD Plan-Neofluar 40x/0.6 Korr Ph2 M27 objective lens was used in the acquisition of biofilm stack, via confocal laser scanning microcopy, achieving × 40 magnification. For biofilms supplemented with sodium citrate, an EC Plan-Neofluar 20x/0.50 Ph2 M27 objective was used during CLSM stack acquisitions, achieving × 20 magnification. The rational for using different objectives can be explained by the level of biomass generated by the supplementation of sodium citrate, or lack thereof. We found that the × 40 objective was ideal for acquiring control biofilms stacks, however, the same objective used on biofilm supplemented with sodium citate led to loosing intricate details of the biofilm’s structural properties, which were best observed using a 20 × objective. Three to six 3D stacks were taken for each sample, depending on the sample size, to assure sufficient data for subsequent quantitative analyses.https://phlip.sourceforge.net/phlip-ml). The following quantitative parameters were evaluated: total biovolume (µm3), surface coverage (µm2), mean thickness (µm), and biofilm roughness (dimensionless)30. SPSS (IBM SPSS Statistics 24.0) was used to performed an analysis of variance for comparing biofilm groups acquired at different time points.Qualitative assessments of the biofilms were performed by processing the acquired microscopy data using Fiji software tools . The quantitative analysis of the microscopy data was performed using the image analysis tool PHLIP running in MATLAB developed by Dr. J. Xavier (29.Genomic DNA (gDNA) of biofilms grown under different conditions and sampled at selected time points were extracted using Quick-DNA Fecal/Soil Microbe Kits  as previously describedAll gDNA samples were sent to Novogene  for amplicon V3-V4 bacterial sequencing and analyses . During the transfer, all samples were kept in an isolation box with dry ice to prevent thawing and degradation. An amplicon library with fusion primers (16S V3-V4) was first constructed with dual index and adapters for PCR. Phusion High-Fidelity PCR Master Mix  was used for all PCR reactions. As a standard procedure at Novogene , PCR products underwent in-house quantification and purification procedures. A TruSeq DNA PCR-free sample preparation kit  was used following the manufacturer’s instruction for generating sequencing libraries, which were then assessed using the Qubit 2.0 Fluorometer (Thermo Scientific) and finally sequenced on an Illumina HiSeq2500 platform, generating 250-bp paired-end reads.31 following the removal of barcodes and primer sequences. Quality filtering was performed on raw reads using specific filtering conditions to obtain high-quality clean reads32 according to the QIIME quality control process33. The obtained effective tags were compared with a reference database (Gold database) using the UCHIME algorithm34 to remove chimeric sequences. Sequence analysis was performed using the UPARSE algorithm35, where sequences with a similarity index of 97% or above were assigned to the same operational taxonomic units (OTUs). Screening for species annotation to obtain a representative sequence for each OTU was performed using the GreenGene Database36 based on RDP Classifier37. The phylogenetic relationships of different OTUs, differences between dominant species in samples (groups), and multiple sequence alignments were analyzed using PyNAST v1.238 against the “Core Set” dataset in the GreenGene database.The obtained paired-end reads were merged using FLASHStatistical methods including t-test, MetaStat , LEfSe , Anosim (performed using R), and MRPP (performed using R) were used to assess the significance of differences in the structure and microbial community composition between the groups. The workflow of data analysis is provided in S1.39 in Cytoscape 3.6.0 at the following settings: Pearson correlation, Spearman correlation, Bray–Curtis dissimilarity, and Kullback–Leibler dissimilarity were calculated, and the correlation threshold was 0.3. The initial edge selection was set to include the top 100 and bottom 100 scoring edges. Edges supported by less than two metrics were removed. The significance of edges was calculated and renormalized based on 1000 bootstrap iterations, and p values were adjusted using the Benjamini–Hochberg correction for multiple tests. Only edges with p ≤ 0.05 were included in the network.Network analysis of features and taxa in each sample was performed using CoNet app 1.1.1 betaTo determine the effect of sodium citrate supplementation on fresh water biofilm structure over a four-week period, biofilms were harvested using two ABR systems run in parallel, one under the presence and another under the absence of sodium citrate (NaCi and control). The results of the qualitative and quantitative analyses of the acquired microscopy data are presented in Fig. p < 0.01) and 100 times (p < 0.01) higher on day 7 and 28 than that on the corresponding days under control conditions, which is 1.6 × 104, and 5.7 × 104 µm3, respectively. Under sodium citrate conditions, surface coverage remained in the range of 12.7 to 14.2 µm2 from day 7 to day 21 before doubling to 29.9 µm2 on day 28. Under control conditions, surface coverage doubled from day 7 to day 14 and gradually increased thereafter. Compared with surface coverage under control conditions, that under sodium citrate conditions was three times and two times higher on day 7 (p < 0.01) and day 28 (p < 0.001), respectively. Biofilm thickness quadrupled from 22 µm on day 7 to 80.3 µm on day 28 under sodium citrate conditions, whereas doubled from 7.1 µm on day 7 to 13.8 µm on day 28 under control conditions. Compared with biofilm thickness under control conditions, that in sodium citrate conditions was significantly higher between days 14 and 28 (p < 0.001). Biofilms grown under sodium citrate conditions exhibited higher average roughness value of 0.7 (p < 0.001) during day 7 to day 28. In contrast, biofilms grown under control conditions exhibited very low roughness values ranging from 0.3 on day 7 to 0.5 on day 28. The raw values of quantitative analysis of each biofilm repeat were plotted as box plots , Candidatus_Alysiosphaera (12.2%), and Amaricoccus (8.5%) in biofilms grown under sodium citrate conditions, where Mycobacterium (38.7%), Phormidium (3.1%), and Aquabacterium (2.7%) in those grown under control conditions. Changes in the microbial community composition were also observed over the course of the experiment. In biofilms grown under sodium citrate conditions, the relative abundance of the most dominant phylum Proteobacteria decreased from 76.9% on day 7 to 61% on day 14 and then gradually increased to 66.4% on day 21 and 75.0% on day 28   Fig. B.Figure Additionally, a principal coordinate analysis was performed to evaluate the separation patterns of the biofilm community structures between the control and NaCi conditions Fig. C. Based p < 0.05). Black and red edges represent positive and negative correlations, respectively. Top 100 and bottom 100 scoring edges were selected. This revealed 22 phyla in each group, exhibiting co-inclusion or co-exclusion with other phyla; of these, 19 phyla were common and three were distinct between sodium citrate conditions (A) and control conditions (B). Compared with biofilms in sodium citrate conditions (average degree = 12), those in control conditions showed a more complex network interconnection with an average of 13.8 (average degree) connections per node. In addition, the network of control conditions showed higher composition of positive correlations (38.2%) compared with the network of sodium citrate conditions (17.4%).The correlation analysis of microbial communities was performed at the phylum level, as shown in Fig. In the interaction network of biofilms grown under sodium citrate conditions, Proteobacteria represented the largest node (indicating that it was the most dominant phylum) with the highest number of co-exclusion interactions (negative degree = 15). In contrast, in the interaction network of biofilms grown under control conditions, Actinobacteria represented the largest node with the highest number of co-exclusion interactions (negative degree = 18), and Proteobacteria represented the second largest node with 11 co-exclusion interactions. Furthermore, the highest number of co-inclusion interactions was shown by the TM6 (Dependentiae) phylum (positive degree = 6) in the interaction network of biofilms grown under sodium citrate conditions and by the Chloroflexi phylum (positive degree = 10) in that of biofilms grown under control conditions.26. However, the potential biased effects caused by supplementation with such carbon sources on the structural attributes, microbial community composition, and microbial interaction networks of biofilms remain unexplored. To close this knowledge gap, we investigated the effects of a commonly used carbon source, sodium citrate, on the structure, microbial community composition, and microbial interactions of an early-stage multispecies biofilm model previously described in the context of simulated irrigation water distribution systems29. The results of our analyses revealed that the addition of sodium citrate creates a biased biofilm model in terms of an inconsistent microbial composition that may affect the outcome of the resulting biofilm.Sodium citrate and sodium acetate are the most commonly used carbon sources for supplementing natural or artificial low-nutrient media to accelerate the formation of monospecies or multispecies biofilm in laboratory-based biofilm experiments41. Bester et al.40 revealed that compared to carbon-limited conditions, carbon-replete environments led to 40% increase in planktonic cell yields of Pseudomonas sp. biofilms. Another study showed that carbon sources such as glucose, sucrose and acetate presented a significant enhancement in microalgae growth41.With respect to biofilm biomass accumulation, as expected the supplementation of a nutrient-poor artificial medium with sodium citrate successfully increased the rate of biofilm formation, as indicated by the enhanced 3D biofilm structure. Notably, the total biovolume, mean thickness, and roughness parameters increased significantly in biofilms grown in sodium citrate-supplemented medium. This finding is in accordance with findings in previous studies in which supplementary carbon source could increase microbial biomassSalmonella, Vibrio, Helicobacter, Yersinia, Legionellales, and Pseudomonas42. Such a shift in the microbial community structure may be attributable to the fact that some genera under Proteobacteria favor citrate as their sole carbon source such as Salmonella43. This shift also caused significant changes in the microbial network dynamics in terms of increased co-exclusion interactions of Proteobacteria with other phyla, suggesting the potential nutritional advantage to Proteobacteria. Previous studies have described biofilms primarily composed of Proteobacteria, could influence the overall structural attributes, due to their distinct extracellular polymeric substances (EPS) and biofilm viscoelastic properties44. In this study, the thickness and roughness of biofilms grown under sodium citrate conditions varied toward the end of the experiment on day 21 and 28, which could be explained by the potential sloughing of the biofilms in the reactor. Sloughing have been demonstrated as one of the distinct models of biofilm dispersal, for biofilm suddenly sheds a portion of its structure especially at a later stage47. However, whether the sloughing was attributable to both the significant biofilm thickness and shear conditions within the reactor or to the intrinsic viscoelastic property of the biofilms allowing for effortless sloughing still remains unclear and should be investigated in the future.In particular, comparison of the microbial community profiles of biofilms between sodium citrate-supplemented and sodium citrate-free conditions revealed that the sodium citrate supplementation resulted in experimental bias in the resulting biofilm phenotypes. Sodium citrate supplementation increased the abundance of the Proteobacteria phylum (70.0%) such that it became the most dominant phylum under sodium citrate conditions. This is a phylum of gram-negative bacteria that includes a wide variety of pathogenic genera, such as Micrococcus luteus, a species of the Actinobacteria phylum, was found to enhance the adhesion of other bacteria to the biofilm48. Two other species of the Actinobacteria phylum, Corynebacterium renale and C. pilosum, are also associated with increased adherence to various bacteria49.In sharp contrast, biofilms grown under sodium citrate-free conditions showed less susceptibility to sloughing under experimental shear conditions, indicating that the high abundance of Actinobacteria may have increased the cohesiveness of the biofilm. Previous investigations on Actinobacteria biofilms have revealed that these biofilms are highly adherent and may promote the recruitment of other cells to its structure. In one recent study, 50, the viscoelasticity property of biofilms serves a protective role from external stresses in the form of chemical or mechanical challenges51. More specifically, most biofilms in their natural habitats are exposed to external forces applied in a compressive, tensile or shear modes51. Given that biofilm EPS are characterized as a multi-component biological material54, biofilm developments is influenced in a time-dependent manner with series of shifts in elastic and viscous properties. Since biofilm viscoelasticity properties are closely related to biofilm structure and composition, the present study provides a caveat in which researchers should also consider the effects of nutrient supplementation when studying biofilm viscoelasticity and their related response to induced stresses; and by doing so, avoiding potential biased outcomes.Future studies are clearly warranted to further elucidate the impact of microbial community structure on the viscoelastic property of multispecies biofilms. The observed differences in the microbial community profiles caused by sodium citrate supplementation in low-nutrient media may lead to experimental biases in studies focusing on the structural properties of biofilms or specific microbial communities. In the former case, supplementation with sodium citrate or another carbon source to accelerate the turnover of multispecies biofilm models for testing biofilm mitigation strategies could result in the design or engineering of antifouling features that may be inefficient in natural conditions. As an important feature in many types of biofilms including oral, environmental and medical biofilmsIn a microbial ecological context, the beneficial faster turnover of the biofilm model resulting from carbon source supplementation may cause a serious misrepresentation of the microbial community intended for fundamental investigations. In this study, sodium citrate supplementation significantly decreased the relative abundance of Actinobacteria among other phyla. Such biases may hamper accurate fundamental studies on biofilms, especially multispecies biofilm models, in specific simulated environmental conditions.Although it is understandable that the use of carbon source to accelerate the time-consuming biofilm formation in experimental models is essential, the results of this study suggest the importance of carefully considering the potential biases caused by carbon source supplementation, as they may significantly affect the experimental results. The potential biases in biofilms grown in supplemented media include shifts in the biofilm structure and microbial composition compared with those in biofilms grown in non-supplemented media.Our study revealed that adding sodium citrate, a carbon source, to an early-stage multispecies biofilm model significantly affects the biofilm 3D structure, characteristics, bacterial community composition, and network interconnection, thus generating a biased biofilm model for the investigation. The findings provide caveats for investigators intending to accelerate biofilm growth by adding a carbon source in their low-nutrient media for generating multispecies biofilm models.Supplementary Figures.


Clamshells), preserving 84.8% of the initial antioxidants content. Fruit packed in LEO films exhibited the greatest weight loss and rot incidence, and poor surface color. CS:CH and GSE films controlled the fruit respiration rate and weight loss, therefore they are materials with adequate barrier properties for blueberries conservation. Bags formulated with GSE showed adequate barrier properties to maintain fruit quality attributes without the incidence of rottenness, being an interesting option for blueberries exportation.Blueberries are highly appreciated for their high antioxidant content but are also particularly susceptible to fungal deterioration. In this work, corn starch and chitosan, byproducts of the fishing industry, as well as active compounds obtained from citrus processing waste were used to obtain active biodegradable film packaging. Blueberries were packed in corn starch–chitosan (CS:CH) films and in active films containing lemon essential oil (LEO) or grapefruit seed extract (GSE). The effects of film packaging on the quality parameters of berries and the fungal incidence of disease during storage were studied and compared to benchmark materials. A conservation assay simulating transport and commercialization conditions was conducted. Blueberries packed in CS:CH films showed antioxidant capacity values closer to those packed in commercial PET containers ( According to the United Nations (UN) on average 13.8% of the food produced worldwide is lost after harvest and during transportation, storage, and processing . BecauseMany bioplastics from biopolymers such as starch, cellulose, and proteins, synthetized plastics from biomass (PLA) or produced by microorganisms (PHAs and PHBs) have been extensively studied as alternative materials for food packaging ,6,7,8. AVaccinium corymbosum L.) are recognized as an excellent source of natural antioxidants, which have proven health benefits for prevention and treatment of neurodegenerative and cardiovascular diseases, diabetes, and cancer, among others [Blueberries  bags, was conducted. The evolution of the fruit main quality attributes as well as the rot incidence of mold in the packed berries during cold storage and thermal abuse were analyzed, considering typical transport and commercialization conditions of the fresh product. Finally, the correlation among the packaging materials properties and fruit quality parameters was also studied.The aim of this work is to assess the performance of biodegradable sustainable active packaging with active compounds obtained from fishing and citrus industry processing waste on fresh blueberries preservation. A comparative study between the biobased materials and benchmark synthetic plastic packaging systems, PET w/w in all formulations.Chitosan of 444 kDa molecular weight and 85% deacetylation degree was provided by Parafarm . Corn starch with 25% amylose content was purchased from Glutal (Argentina), grapefruit seed extract (GSE) was provided by Euma SAICIYF , and lemon essential oil (LEO) was supplied by Litoral Citrus S.R.L. . Acetic acid  was used to solubilize chitosan powder and glycerol  was used as a plasticizer at 25% w/w solution. On the other hand, a 4% w/w corn starch (CS) suspension was gelatinized at 90 °C for 20 min in a thermostatic bath. Both biopolymers were mixed in a 75:25 CS:CH proportion. Glycerol was added as a plasticizer at 25% w/w. Two essential oils were chosen because of their antimicrobial activity: lemon essential oil (LEO) and grapefruit seed extract (GSE). The essential oils were added at 3% w/w in formulations according to a previous work [2 and dried in a convection oven  at 50 °C until constant weight. The films were removed from the plates and thermo-sealed with an impulse sealer  to obtain the active biodegradable packaging.Active biodegradable films were obtained by casting method from filmogenic suspensions formulated with chitosan and corn starch, which were optimized in a previous work . Brieflyous work . The filClamshells,  and commercial bags suitable for modified atmosphere  were used as benchmark packaging systems. Barrier and mechanical properties of flexible film materials are included in Synthetic PET containers, commonly called Clamshells (CL).In order to determine the effects of the active biodegradable packaging on the main quality attributes of commercial fresh blueberries under refrigeration condition, fresh Emerald blueberries cultivated in Salto Grande  were used. Fruit was harvested with an optimum maturity degree, selected by size, shape, and color, discarding the damaged or altered fruit. The selected blueberries were packed in five different containers: 75:25 corn starch-chitosan biodegradable film (CS:CH); biodegradable active film with 3% GSE (GSE3); biodegradable active film with 3% LEO (LEO3); commercial bags for modified atmosphere (MA) and PET All containers were filled with 25 berries each (approximate weight: 55 g), and both biodegradable films and MA commercial bags were sealed. In this case, 5 replications were made for each treatment. A batch of 1.5 kg of blueberries was used in this experiment.Ship transport conditions simulation assays for blueberries exportation were conducted in a cooling chamber with controlled temperature and relative humidity  during 30 days. Subsequently, samples were kept for 7 days at 20 °C to simulate shelf storage and marketing conditions. The fruit quality attributes were analyzed throughout the whole storage period.Fruit weight loss was determined at the end of cold storage and after 7 days of storage at 20 °C. An OHAUS SP 602 scale  was used and the result was expressed as percentage of weight loss with respect to the initial sample weight. The informed values correspond to the average of ten replicates.Blueberries firmness tests are based on skin toughness measurements considering puncture force and penetration or deformation of the fruit. Several authors have commented on the reproducibility of puncture tests thus a similar probe than that used by other authors was selected for the puncture tests ,42,43. FSurface color was evaluated using a Minolta CR-300 colorimeter  with D65 illuminant on the CIELab color scale. The value of L represents the luminosity and varies from 0 (black) to 100 (white); the parameter a* indicates green (−) to red (+), while b* varies from blue (−) to yellow (+). Three measurements per fruit were performed and the informed values correspond to the mean of 25 fruit per sample, at the beginning and at the end of the test period. Color differences (ΔE) were calculated as described in a previous work  with resThe titratable acidity (TA) was measured by potentiometric titration at pH 8.3 with a pH-meter  applying the method 942.15 of the Association of Official Analytical Chemists . Considering that the main organic acids present in blueberries are citric and succinic acid, exhibiting higher content of the first one, the titratable acidity was expressed as citric acid content per 100 g of fruit (g citric acid per 100 g fruit).Total soluble solids , were determined by the refractometric method , using a digital Abbe type refractometer, with temperature compensator .For each determination, three samples of 5 g from a lot of 25 blueberries randomly selected at the beginning and the end of storage time were analyzed.2 measurement and a non-dispersive infrared CO2 sensor. The gases concentration (%) inside the glass jars was measured every hour for 8 h at 25 °C. CO2 and O2 percentages were plotted as a function of time and data were linearly regressed. According to Fonseca et al. [−1 h−1 and calculated in terms of CO2 produced and O2 consumed as follows:2 and sCO2 are the slopes corresponding to the regression lines of O2 and CO2 percentage concentration as a function of time (h), w (kg) is the fruit weight, and V (mL) is the available headspace volume in the container.The respiration rate of the fruit was determined by a quasi-stationary state method placing 120 g of fruit in a 1.5 L capacity glass container. This was hermetically sealed and connected by a septum to a CheckMate 3 Dansensor gas meter . The equipment has a zirconium sensor for Oa et al. , the res2 produced to O2 consumed by the product. Tests were carried out, at least in duplicate, at the beginning and at the end of the storage time.In addition, the respiratory quotient (RQ) was determined as the proportion of COBlueberries are valued nutritionally for their high content of antioxidants . Thus, tg for 10 min at 4 °C in a Rolco 2036 centrifuge .First, ethanolic extracts were prepared. Pulp tissue frozen with liquid nitrogen was ground to a powder in a grinder . Approximately 0.1 g of sample were weighed and diluted with 5 mL of ethanol . The suspension was vortexed and centrifuged at 12,000× −1) and 0.0033 g of K2S2O8 (MW = 270 g mol−1) to a final concentration of 7 mM ABTS and 2.45 mM of K2S2O8. A total of 5 mL of this solution was stored in dark at room temperature for 12 to 16 h without agitation to favor the formation of the ABTS *+ radical. Then, an aliquot of the solution was taken and diluted with ethanol until an absorbance of 0.7 ± 0.05 at 734 nm was reached. Subsequently, 50 μL of the ethanolic extracts of the samples and 1 mL of the ABTS *+ radical solution prepared above were mixed under constant agitation and after 6 min the absorbance at 734 nm was measured in a spectrophotometer . As blank control sample, 50 μL of ethanol were placed with 1 mL of the ABTS *+ solution. For the measurements to be reliable, the percentage of reduction of the absorbance must be between 20 and 80% with respect to the target. The calibration curve was performed with Trolox reagent , a water-soluble analog of α-tocopherol, as standard. The determinations were made at least in triplicate using independent extracts. The results were expressed as Trolox equivalent antioxidant capacity (TEAC) by fresh weight in mg kg−1.To quantify the antioxidant capacity (AC), a solution was prepared with 0.0192 g of the ABTS reagent . The samples were homogenized by vortexing for 3 min at 20 °C before 100 μL of 20% Na2CO3 (w/v) in 0.1 eq L−1 of NaOH was added. The tubes were vortexed and incubated at 20 °C for 1 h in a dark place. Absorbance at 760 nm was measured in a spectrophotometer . For quantification, a calibration curve was made with chlorogenic acid , the predominant phenolic compound in blueberries, using a standard solution of 224 μg/mL. Samples were measured at least in triplicate using independent extracts. The results were expressed as equivalent milligrams of chlorogenic acid per kilogram of fresh weight (mg kg−1).Likewise, the content of total phenolic compounds (TPC) was quantified using the Folin-Ciocalteu technique. For each sample, 150 μL of fresh tissue ethanolic extract were placed in tubes with distilled water  and 50 μL of 1 eq LThe post-harvest infection of the fruit at the end of the refrigerated storage was evaluated by macroscopic observation. The first visible infection point was counted as indicative of rot and the result was expressed as a percentage of deteriorated fruit with respect to the total analyzed ,48. LotsThe obtained results were statistically analyzed using the Statgraphics Plus software for Windows 5.1  performing the analysis of variance (ANOVA). Likewise, the means comparison was carried out using the Fisher’s minimum difference test (LSD) with a confidence level of 95%. Results were subjected to principal components analysis (PCA) and cluster analysis (CA). Data were analyzed using the Infostat software v2011 .The evolution of weight loss of blueberries after the two storage stages is shown in Clamshell, CL) (p < 0.05). In contrast, the samples packed in active films LEO3 had weight losses similar to CL, since they were inefficient barriers to water vapor during the first storage stage. Moreover, when fruit was transferred to room temperature after 30 days of refrigerated storage samples packed in active films LEO3 exhibited the highest weight loss, probably due to structural defects induced in the composite films by the thermal shock that could affect WVP of films.Blueberries packed with CS:CH and GSE3 films exhibited lower weight losses than the control  in firmness among all samples (p > 0.05) changes in firmness during thermal abuse  were observed in the L and b* parameters, except for the fruit packed with LEO active films and MA. This was also evidenced through the color differences (ΔE) calculated with respect to the initial values. The ΔE values obtained were 2.39 ± 0.11, 3.21 ± 0.46, and 2.35 ± 0.44 for the blueberries packed in clamshells, flexible biodegradable bags of CS:CH and the active ones containing 3% GSE (GSE3), without significant differences among them (p > 0.05). On the other hand, fruit packed in the active flexible bags with LEO at 3% and those packed in the bags for modified atmosphere (MA) presented statistically higher (p < 0.05) ΔE values, 4.73 ± 0.84 and 5.86 ± 1.40 respectively, without significant differences between them.Regarding surface color parameters, it has been reported that the luminosity (L) and chromaticity parameter (b*) values can be affected by the natural waxy layer covering the berries surface known as bloom ,46. The bloom contains various lipidic components, mainly triterpenoids and diketones [Blueberries iketones . Its maiiketones . TherefoFurthermore, Perdonés et al.  indicateWith respect to the fungal decay, the packed Emerald blueberries did not show mold growth at the end of the refrigerated storage period (30 d). However, when fruit was exposed at room temperature for 7 days the microorganisms present in the fruit manifested . The besp > 0.05) variations were found in the ratio between the TSS and TA of the fruit. Contrastingly, fruit packed in commercial materials , showed a significant (p < 0.05) decrease in TA  and active films (GSE3 and LEO3) maintained the maturity index during the storage under refrigeration condition, since no significant , flavones, and their glycosides. Other phenolic compounds present in berries are hydrolysable tannins, phenolic acids, lignins, among others ,78,79.Cantín et al.  and Giug2 > 0.99). As it was expected, both the TPC and AC decreased, indicating as loss percentage for each parameter in 2 permeabilities and that the active films did not show any antioxidant activity per se [The antioxidant capacity correlated satisfactorily with the total phenolic compounds content . Accordingly, Remberg et al.  stated tBoth CS and CH are polysaccharides that, being hydrophilic in nature, present poor barrier properties with relatively high values of solubility and permeability to water vapor . This bep < 0.05) different because of the variations in the total solid content of the formulations , among others. In addition, thickness may alter optimal heat-sealing conditions (time and temperature) used in manufacturing containers. However, no modification of the sealing conditions were needed for LEO3 film bags.Clamshells were omitted in this comparison since they are perforated containers and no barrier properties values could be assigned to them.The conglomerate analysis indicated that the mechanical and barrier properties were similar between the biodegradable films: CS: CH and GSE3 active film a. The diConsidering the poor performance of LEO3 films on blueberries preservation assay, these samples were not included in the principal component analysis (PCA). This analysis shows that the first two principal components (PC1 and PC2) explained the 86.7% of the total variance with a cophenetic coefficient of 0.982 b. PC1 waRegarding the nature of the packaging material, positive values of PC2 related fruit attributes that conditioned its shelf-life such as RI, RQ, firmness, and chromaticity parameters b. Furtheclamshells, allowing to maintain the quality attributes of blueberries and to reduce the rot incidence with the additional advantage of the biodegradable nature of the proposed system.In summary, blueberries packed in flexible bags with synthetic films (MA) presented a distinctive behavior in the physiological quality parameters that negatively impact the attributes that limit the fruit shelf-life. Flexible bags obtained by heat-sealing active biodegradable films are a viable alternative to the use of clamshells may be recycled and incinerated for energy recovery, while composability should be preferred for biodegradable active films depending on the waste management systems applied.The development of more sustainable materials for the packaging industry comprises a more holistic approach to consumer good production systems, tending to a circular economy. Driven by a global environmental awareness, biobased and biodegradable materials aiming to waste generation reduction, climate change prevention, and nonrenewable energy sources use minimization are key players. Chitosan and corn starch-based bio-packaging active films with GSE are fully biobased materials mostly derived from agricultural waste that successfully preserved blueberries under transport and market conditions for 37 days. The renewable, biodegradable, and non-toxic character of the raw materials are positive features that could contribute to a low environmental impact by reducing food spoilage and plastic waste in landfills, reducing greenhouse gases emissions, providing added value to agricultural by-products, and lessening petroleum resources depletion. Nonetheless, a life cycle analysis (LCA) of the herein studied materials is recommended for future research since some biopolymers and biobased packaging systems have shown poorer environmental impact results than their plastic counterparts depending on the assumptions made and the selected application of the materials ,88. CareThe economic constraints in the production of biodegradable films based on CS:CH and active agents is an important challenge in the large-scale development of such active packaging. On the one hand, regarding the process scale up of the active films obtained on a laboratory scale by casting the film forming dispersions, the simplest technique is the use of tape-casting. However, to meet market demands the use of industrial scale technologies adapting available equipment for synthetic materials, such as extrusion or compression molding is required . In thes2 of film. In view of the filmogenic dispersions composition and molding ratio, the minimum cost in raw materials corresponding to CS:CH films would be 4.59 $/m2 while for GSE3 films it rises to 10.68 $/m2, due to the active compound inclusion.On the other hand, the cost of essential oils and production along with the complicated processing steps make it less attractive for food packaging. As regards the production cost, raw materials market prices were considered to estimate CS:CH and GSE3 films cost: $0.8/kg for CS , $76.95/Lastly, regarding the legal considerations related to the approval of containers in contact with food, it is important to note that the active ingredients used have GRAS status.The present study demonstrated that the biodegradable films based on corn starch and chitosan and the active film containing GSE reduced the post-harvest weight loss of packed blueberries during their refrigerated storage in comparison to CL PET containers. In addition, in contrast to MA plastic bags, all the biodegradable packaging materials were effective in controlling the fruit respiratory rate during storage without changes in respiratory patterns. Although the antioxidant capacity decreased during storage, blueberries packed in CS:CH biodegradable films showed values closer to those packed in CL, the losses being, with respect to the initial content, 15.2 and 5.6% respectively.Biodegradable bags, except those with LEO3, showed reduced blueberries rot incidence compared to those packed in the commercial containers (CL).The conservation assay aimed to simulate the real conditions under which blueberries are transported until displayed on shelves for consumers. Such conditions are crucial for proper fruit preservation contemplating that blueberries are high nutritional and valued food that are seasonally exported from one hemisphere to the other having to undergo large periods of time under refrigerated conditions before reaching the market. Considering a limit weight loss value acceptability of 8%, the shelf-life of packed fruits would be acceptable after 30 days of storage in all cases. However, simulating the thermal abuse that would occur at the commercialization points (30 days at 1 °C and 7 days at 20 °C) the containers containing LEO would not meet this quality criterion. Besides, if the criteria for estimating shelf-life is restricted to the absence of rot, only active formulations containing GSE comply with this requirement throughout the analyzed period. Although MA packaging also presented low rot incidence after storage, it has proven to alter the respiratory conditions of the fruit leading to anaerobiosis with significant detriment of the fruit antioxidant capacity.Overall, despite the limitations presented by the biodegradable materials herein studied, these are valuable in light of developing more sustainable packaging materials for organic products, especially considering that these are biobased and biodegradable materials derived mostly from food industry waste compounds. As we have previously argued, CS:CH films present similar and improved characteristics in preserving blueberries quality attributes in comparison with CL benchmark containers, while GSE3 would be a promising alternative to MA films. Future research should consider the potential effects of using a combination of materials to design bio-packaging systems with improved outcomes.The results obtained in this work serve as a starting point for the development of biodegradable packaging for highly perishable products. This is an integral research that contemplates not only the use of active biodegradable bags but also evaluates their performance under real conditions of transport and commercialization, also considering the costs and possible scaling of the process.


The aim of the present study was to investigate the beneficial effects of dietary supplementation with oil palm frond (leaf) (OPF) with and without oil palm meal (OPM) on nutrient intake and digestibility, ruminal fermentation and growth performance in goats.Six female crossbred goats were fed for 28 days of 3 diet treatments; 100% para-grass (T1); 50% para-grass + 50% OPF (T2), and 30% para-grass + 50% OPF + 20% OPM (T3). Body weight, rectal temperature, respiratory rate, and urine volume, food intake, dry matter intake and water intake were measured daily. Nutrient digestibility was determined from five consecutive days of last week in each diet. Ruminal fluid, urine and blood were collected at the end for determination of rumen protozoa and volatile fatty acid contents, urinary allantoin excretion, blood cell count and chemistry profiles.Goats fed T2 and T3 showed higher dry matter and nutrients intakes while protein digestibility was suppressed compared with those for T1. Crude fat digestibility declined in T2 but maintained after adding the OPM (T3). High fat intake by giving OPF and OPM corresponded to a higher ruminal acetate/propionate ratio (C2/C3) and serum cholesterol level. An increased urinary allantoin/creatinine ratio was found in T2 and T3 compared with T1, implying an increased number of ruminal microbes.Increased dry matter intake in T2 and T3 suggested that oil palm by-products are partly useful as a replacement for para-grass in goats. Replacement with the by-products increased plasma cholesterol level, which suggested that these products are a useful energy source. Changes in rumen parameters suggested an increased microbial number and activity suitable for acetate production. However, the limited digestibility of protein implies that addition of high protein feeds may be recommended to increase body weight gain of goats. Brachiaria mutica) is easily grown in Thailand and has been used by the farmer as a basic feedstuff for goats. However, other local plants such as white popinac  have also been added to avoid para-grass shortage or improve palatability. In South East Asia, where oil palm tree planting is sometimes the major industry, oil palm frond (OPF) and its by-products after oil extraction may be an interesting resource as feed for animals.Goat production plays an important role in the rural economy in many developing countries because goats produce essential food items such as milk and meat. Although there are many factors such as feed and environment that affect the growth and reproductive performance of animals, it is preferable to use agricultural and natural resources specific in those regions of the world. Naturally, para-grass (ad libitum increased crude protein (CP) intake when compared with Napier grass [The OPF as well as palm tree by-products  have been extensively studied as a feed for ruminants. A study in goats showed that OPF or Napier grass + OPF er grass . The PO er grass . Feedinger grass . Howeverer grass . Goats fer grass . These dIn the southern region of Thailand, palm trees have been extensively grown to make PO for biodiesel fuel under the Chaipattana Foundation. Thus, products including palm leaf and by-products of palm fruits after oil extraction known as OPM that contained kernel, mesocarp, endocarp and palm shell have been used as feed for goats. The present study was carried out to investigate the beneficial effects of dietary supplementation with OPF with and without OPM on nutrient intake and digestibility, ruminal fermentation and growth performance in goats. The findings of this study could provide more information on the benefits of these products to improve nutritional management in goat farming.The protocol of this experiment was carried out in according to the institutional guidelines and was approved by Animals Care and Use Committee, Faculty of Veterinary Science, Chulalongkorn University . Six healthy Black Bengal crossbred mature female goats with average body weight of 15.08±0.98 kg and age between 8 to 9 month old that belonged to Chaipattana Foundation raised at Phetchaburi province were used. All animals were kept in individual metabolic cages (2×1 m. shaped pens with an opened top and plastic floor) under a shaded barn for the entire studying period. The goats were also able to see and communicate with other goats for socialization. The animals were randomly allocated into 3 groups  and were assigned as two replications of 3×3 Latin-square designs. After 1 week of cage acclamation, each group of animals was randomly and rotationally assigned to three different diets. The diets were as follow: 100% of para-grass , 50% of para-grass + 50% OPF  and 30% of para-grass + 50% OPF + 20% OPM . The compositions of the para-grass, OPF and OPM are shown in Brachiaria Mutica) was grown naturally without fertilizer and was cut daily in the morning (09:00 to 10:00). The cutting length was around 30 cm long. The OPF was obtained from palm trees grown inside the foundation mainly for biodiesel production. The whole leaf was cut and only the leaflet was chopped into pieces of approximate 2.5 cm long before feeding to the goats. The OPM was obtained from the PO fruit after oil extraction. In brief, the fruit moisture was removed by boiling with PO and then the fruit was compressed in order to extract the PO. The residue after oil extraction was sieved to remove the big pieces of shell and the remaining was used as OPM to feed the goats.The para-grass  and respiratory rate (RR), which were measured every day before morning meal, were averaged for last two weeks of each diet. The RT and RR were recorded using digital thermometer and stethoscope, respectively during 08:00 to 09:00 along with the collection of ambient temperature and humidity to obtain the temperature humidity index.Daily food intake (FI), dry matter intake (DMI) and water intake (WI) were daily recorded and averaged for the entire 28 days.In order to evaluate the nutrient digestibility, samples of both feed offered and refusals and all feces were collected daily for 5 consecutive days at the end of each treatment and the data were averaged. The feces were dried in microwave oven at 400 watts of heat intensity for 30 min and weighted for the five consecutive days during the last week of each diet experiment. Samples collected in each day were mixed thoroughly and kept frozen at −20°C for later chemical analysis.Collection of ruminal fluid, urine and blood was performed at the end of last week of each diet experiment for determination of rumen protozoa and VFA contents, urinary allantoin excretion and blood cell count including chemistry profiles. Samples collected were kept frozen at −20°C for later chemical analysis.Rumen fluid samples were collected using an orogastric tube connected to a syringe while the goats were under a few minutes of physical restrain by experienced personnel. Approximately 20 mL of fluid was taken 2 h after morning meal. For determination of VFA concentrations, the ruminal fluid was filtered through two layers of cheesecloth, then 2 to 3 drops of toluene were added to inhibit fermentation. Filtered samples were then frozen at −20°C for later analysis2SO4 per urine 100 mL) added to prevent nitrogen loss. Final urine pH was kept below 3. Total urine volume was measured separately by day and night times and then combined. All urine samples were diluted with distilled water to minimize the crystallization of urinary compounds and then kept in a refrigerator.Total urine was collected using plastic containers beneath the cage floor with 10% sulfuric acid solution (13 mL of 10% HApproximately 6 mL of blood was collected from the left jugular vein into a syringe and put into the ethylenediaminetetraacetic acid-added and heparinized tubes (3 mL each) at 09:00 on the same day as the ruminal fluid collection. The samples were kept on ice for later analysis of complete blood count and chemistry profiles.All diets were dried overnight in oven at 60°C until weights were constant to determine dry matter (DM). Samples after drying and grinding were used to determine diet composition. The DM, crude fat, CP, and crude ash were determined by the method of AOAC .The chemical compositions of the OPF  and OPM  were estimated as percentage of DM.In order to obtain the nutrient digestibility, the composition in feces was analyzed along with diets. The total digestible nutrient (TDN) was calculated according to Lofgreen . The totThe protozoa counting and determination of the types were analyzed following the reported method by Dehority . One ml On the day of measurement, a 5 mL of rumen fluid was thawed, and was mixed with 1 mL of 25% of meta-phosphoric acid and formic acid (3:1) in a centrifuge tube. The mixed samples, after being allowed to precipitate for 30 min, were centrifuged at 4,000 rpm for 20 min, and the clear supernatant was collected into another tube, a part (1 μL) of which was injected into gas chromatography , equipped with a 30 m×0.25 mm×0.50 μm film fused silica capillary columns . Injector and detector temperatures were 225°C and 250°C, respectively. Carrier gas-Helium total and column flow were 35.8 and 1.56 mL/min, respectively.The allantoin was analyzed by colorimetric method according to Young and Conway . The uriThe complete blood cell count was determined using an automated analyzer . The blood chemistries including blood urea nitrogen, glucose, alanine transferase (ALT), alkaline phosphatase (ALP), total protein, albumin, cholesterol, and triglyceride were analyzed by automated analyzers .All data are represented as the mean and standard error of mean (n = 6). The differences among diet treatments were compared using analysis of variance based on Latin square model of SAS program. The pairwise comparisons were performed using Duncan new multiple rank test. Difference was considered significant when p-value was less than 0.05.The compositions of experimental diets  are shown in The total heat index during the experiment was not different among treatments , but WI was higher in T3. The body weight and % BW change of goats were not different among three groups .The FI recorded for 28 days of treatment period was significantly smaller in T2 and T3 than in T1, where T3 was significantly smaller than T2 (p<0.05). The DMI was higher in T2 and T3 than in T1 (p<0.05).The levels of RT and RR were not different among groups.The average FI, DMI, and nutrient intakes during the last week (week 4) are shown in The nutrient digestibility measured on the last 5 days of each treatment periods are also shown in Dasytricha spp. for T3 was higher than that for T1 and T2 (p<0.05).The species and number of protozoa and VFA contents in the rumen fluid are shown in On ruminal VFA profiles, although there was no difference for each acid concentration among three groups, the C2/C3 ratio was significantly increased by replacement with oil palm-related compounds (p<0.01), and that was the largest in T3.The urinary allantoin/creatinine ratio was significantly increased by replacement with oil palm-related compounds (p<0.001).The complete blood cell counts were not different among three groups . Blood cSince goats are unable to digest a large amount of plant cell walls because of a relatively short transit time in the rumen , goats pThe palm leaves, with typical stiff tree leaves, and their by-products have been widely used as feed for ruminants, especially in areas rich in palm trees planting . The proThe PO resources involve many kinds of by-products; i) palm kernel expeller, in which the palm kernels were ground and screwed by pressing with and without flaking and cooking, ii) PKC, which is made from palm kernel oil via solvent extraction, iii) palm oil sludge, iv) palm pressed fiber, v) OPF, vi) oil palm trunks, and vii) whole fruit after oil extraction known as OPM. The OPM has a high content of DM and crude fat.When para-grass was replaced with palm-related by-products to make experimental diets (T2 and T3), CP contents were adjusted at a similar level ca. 11%), which was reasonable because the daily requirement of feed protein is 11% for meat producing goats with average body weight of 80 to 120 pounds [%, which Our results showed reduction in feed intake when replacing with OPF in the diets with or without OPM. However, DMI was higher in T2 and T3 than T1, suggesting better palatability of the by-products than para-grass alone. Higher DMI was also reported in a previous study, in which goats were fed a combination of palm frond and Napier grass . AlternaAlthough OPF contains high cellulose, hemicellulose and lignin , lignoceProtein digestibility was reduced in the diets replaced with OPF. However, there is discrepancy with previous studies which reported a higher protein digestibility obtained by adding PO , and no Isotricha spp., when OPM was fed. The Isotricha spp. and Dasytricha spp. which are Holotrich ciliates can synthesize and degrade carbohydrates and sugars. In term of nitrogen metabolism, Holotrichs can both synthesize amino acids and utilize preformed amino acids released by proteolysis from ingested bacteria or plant materials [The urine data suggest the possibility of an increased conversion of feed proteins into microbial proteins by replacement with palm-related compounds because the ratio of urinary allantoin/creatinine was significantly higher in T2 and T3. The increase in allantoin urinary excretion may be related to an increased DMI in T2 and T3. A study with calves showed that purine derivative excretion was positively related to feed intakes . Our resaterials . Thus, caterials . Neverthaterials . Howeveraterials . It is sIn addition to reduced protein digestibility, the crude fat digestibility was also reduced when OPF was fed in T2. This limited reduction digestibility in T2 may not be significant because it was not confirmed in T3, where the fat content was higher than T2. However, the lower fat digestibility may also be due to changes in rate of gastrointestinal passage or rumen environment, by a similar mechanism discussed for protein digestibility. However, fat digestibility in T3 was preserved compared with that in T2. Adding PO to diets in goats was reported to increase EE digestibility . This fiThe data on ruminal short-chain fatty acid measurement showed C3 tended to be lower after giving palm-related compounds, resulting in higher C2/C3 ratio. Higher C2/C3 ratio supports the data that the intake of ADF and NDF was increased as shown in Palm-related compounds when given to goats did not affect the general health as monitored by complete blood cell counts and chemistry profiles, which were in normal reference ranges. Increased ALT and ALP may be related to fat degradation and utilization. High fat content in palm-related compounds increased plasma triglyceride and cholesterol levels, which support palm by-product fat as a good energy source. The PO contained 83% of triglyceride among 84% of neutral lipids when extracted with organic solvent . SimilarThe PO frond will be suitable as a roughage source in the areas of palm plantation since it is a low-cost by-product available throughout the year as an eco-feed. Partial replacement of para-grass with the by-products increased plasma cholesterol level, which suggested that these products are a useful energy source. Changes in rumen parameters suggested increased microbial number and activity suitable for acetate production. However, the limited digestibility of protein implies that the addition of high protein feeds may be needed to increase body weight gain of goats.


WHO’s haemoglobin cutoffs to define anemia were based on five studies of predominantly White adult populations, done over 50 years ago. Therefore, a general re-examination of the existing haemoglobin cutoffs is warranted for global application, in representative healthy populations of children and adults. Such data are scarce in low-income and middle-income countries; however, a 2019, large-scale, nationally representative survey of children and adolescents aged 0–19 years in India  offered an opportunity for this re-examination. Using this survey, we aimed to assess the age-specific and sex-specific percentiles of haemoglobin and cutoffs to define anaemia in the CNNS population.2 and haemoglobin S); and history of smoking. We considered age-specific and sex-specific 5th percentiles of haemoglobin derived for this healthy population as the study cutoff to define anaemia. We compared these with existing WHO cutoffs to assess significant differences between them at each year of age and sex for quantifying the prevalence of anaemia in the entire CNNS sample.For this population-based study, we constructed age-specific and sex-specific haemoglobin percentiles from values reported for a defined healthy population in the CNNS, which used rigorous quality control measures during sample collection and in the laboratory analyses. To obtain a healthy population, we excluded participants with iron, folate, vitamin B12, and retinol deficiencies; inflammation; variant haemoglobins (haemoglobin Avs 30·0% with WHO cutoffs).Between Feb 24, 2016, and Oct 26, 2018, the CNNS survey collected blood samples from 49 486 individuals. 41 210 participants had a haemoglobin value, 8087 of whom were included in our study and comprised the primary analytical sample. Compared with existing WHO cutoffs, the study cutoffs for haemoglobin were lower at all ages, usually by 1–2 g/dL, but more so in children of both sexes aged 1–2 years and in girls aged 10 years or older. Aanemia prevalence with the study cutoffs was 19·2 percentage points lower than with WHO cutoffs in the entire CNNS sample with valid haemoglobin values across all ages and sexes (10·8% with study cutoffs These findings support the re-examination of WHO haemoglobin cutoffs to define anaemia. Our haemoglobin reference percentiles, derived from healthy participants in a large representative Indian survey, are suitable for national use in India. Substantial variations in the 5th percentile of haemoglobin values across the 1–19 years age range and between sexes argue against constructing common cutoffs in stratified age groups for convenience.None. In 2000, in addition to adjustments for altitude and smoking, the age group 5–14 years was split to define a lower haemoglobin cutoff for children aged 5—11 years, on the basis of the US National Health and Nutrition Examination Survey 2 data.7 No separate cutoffs were defined for infants younger than 6 months; the reference value for age 2—6 months was added in a later WHO document.8The existing WHO cutoffs to define anaemia were first proposed in 1968, from five studies of predominantly white adult populations in North America and Europe;3 Haemoglobin cutoffs for anaemia similar to WHO recommendations were reported in several studies but tended to be lower for infants, young children, premenopausal women, and older people and higher for men. The haemoglobin cutoffs in children and adolescents from African and Asian regions were substantially lower (1–2 g/dL) in some datasets. However, limited conclusions can be made regarding the normal reference ranges because only a few studies had specifically excluded individuals with iron deficiency or inflammation.3 There were also uncertainties regarding the optimal testing methods to measure haemoglobin concentration.4 Although anaemia is defined functionally, its diagnosis is presently based on a haemoglobin cutoff that is statistically derived rather than on functional or health outcomes, which would be ideal. The present haemoglobin cutoffs for anaemia, derived 50 years ago, need reassessment to potentially enhance the evaluation of determinants, burden quantification, and the management of anaemia at the individual and population level.A 2019 review of 60 global studies, reported between 1975 and 2018, evaluated haemoglobin variation across the lifecycle.4 One suggested approach is to define anaemia by haemoglobin concentrations lower than the reference range  in a representative and healthy population, using stringent exclusion criteria.4 Such data are scarce, especially for children residing in low-income and middle-income countries (LMICs). A 2019 conference abstract reported summary haemoglobin data from 25 countries for preschool children  and women of reproductive age from the Biomarkers Reflecting Inflammation and Nutritional Determinants of Anaemia (BRINDA) project.9 Their definition of healthy was based on the exclusion of iron and vitamin A deficiency, inflammation detected through biomarkers, and malaria. In this study, we aimed to assess the age-specific and sex-specific percentiles of haemoglobin and cutoffs to diagnose anaemia in children and adolescents aged 1–19 years, including additional rigorous criteria to define a healthy population, using the 2019 quality controlled, nationally representative Comprehensive National Nutrition Survey (CNNS) in India.10WHO is indeed re-examining the appropriateness of the existing haemoglobin cutoffs to define anaemia, on the basis of relevant global evidence.The CNNS was done under the aegis of the Indian Ministry of Health and Family Welfare in collaboration with UNICEF and the Population Council . The CNNS was designed to provide nationally representative and comprehensive nutritional profiling of preschoolers (aged 0–4 years), school-age children (5–9 years), and adolescents (10–19 years), based on biological sample assessment and multiple anthropometric measures. Because blood samples were not collected for children younger than 12 months, haemoglobin percentiles were analysed in children and adolescents aged 1–19 years.10 Briefly, the CNNS used a multistage, stratified, probability proportion to size cluster sampling design to select a nationally representative sample of households and individuals aged 0—19 years across all 29 states of India and the capital Delhi. Households with one or more individuals aged 0—19 years were randomly selected from rural and urban primary sampling units. Children or adolescent members were classified into three age strata , and only one child or adolescent was selected from each stratum per household. Children or adolescents who had a chronic illness, physical disability, mental illness, cognitive disability, or any ongoing acute illness  were excluded from the survey.The detailed survey design and methods are published elsewhere.Ethical approvals were obtained from the Ethics Committee of the Postgraduate Institute for Medical Education and Research  and the Institutional Review Board of the Population Council. Written, informed consent was obtained from caregivers of children aged 0—10 years. For adolescents aged 11—17 years, written informed consent was obtained from their caregivers and written informed assent obtained from the participants. Adolescents aged 18—19 years provided their own consent.2 and haemoglobin S) were considered, and those with abnormal values for these biomarkers  were done only for children older than 5 years. All participants aged 1—19 years with available haemoglobin values and with no history of smoking were included in the initial filter . Among t19 Trained phlebotomists collected venous blood from children aged 1–4 years (8 mL) and 5–19 years (10 mL) for estimating micronutrient concentrations and biomarkers for non-communicable diseases (5–19 years only). Haemoglobin was estimated by the conventional best practice of drawing venous blood and using the cyanmethaemoglobin method of automated photometric estimation.20 Biochemical analyses were done in a commercial laboratory with stringent quality control measures . Rigorous control and monitoring systems were included in the standard operating procedures for quality assurance of biomarker data. First, an internal quality control sample was used for each batch of 20 survey samples. Second, for external quality assurance, a subset of samples was sent to other participating laboratories monthly for comparison testing. The SRL laboratories also participated in the Bio-Rad and CDC external quality assurance scheme. Third, on a weekly basis, a percentage of samples were split and reanalysed.10 Apart from haemoglobin, the following blood biomarkers, with a defined role in haematopoiesis, were used for selecting healthy participants for the purpose of defining haemoglobin cutoffs for the diagnosis of anaemia: erythrocyte folate, serum concentrations of ferritin, transferrin receptor, cyanocobalamin, retinol, zinc, C-reactive protein (CRP), creatinine, total cholesterol, albumin and glycosylated haemoglobin, and haemoglobin variants (haemoglobin S and haemoglobin A2). Stool samples were examined for the presence of parasites. The laboratory estimation methods for the biomarkers and the cutoffs used for specific diagnoses are summarised in 19Sociodemographic details and history of previous illness  in the preceding 2 weeks were recorded in a pretested pro forma. The blood sample collection procedure and protocols followed are detailed elsewhere.2 tests. We applied a generalised additive model for location scale and shape to estimate LMS  values and penalised smooth percentiles for altitude adjusted haemoglobin over age separately for boys and girls in analytical sample 1. We adopted the Box-Cox-Cole-Green transformation to estimate location, scale, and shape parameters.21 The 5th percentile of haemoglobin distribution was considered as the haemoglobin cutoff for the diagnosis of anaemia in this study. These cutoffs were compared with the existing WHO haemoglobin cutoff values to assess significant differences by evaluating if the 95% CI of the smoothed 5th percentile (haemoglobin cutoff of this study), at each age and sex, included the WHO cutoff value. Furthermore, we estimated age-specific and sex-specific anaemia prevalence using appropriate survey weights (with 95% CIs) from the entire CNNS data with valid haemoglobin samples, using cutoff values from both WHO and this study. The anaemia prevalence estimates using WHO and this study’s haemoglobin cutoffs were considered to be significantly different if their 95% CIs did not overlap. We used the statistical software R, version 3.6.1, with gamlss library for the entire analysis.Differences in sociodemographic parameters between the entire sample of participants with a valid haemoglobin and the healthy participants chosen in analytical sample 1 were assessed with ANOVA and χThe funder had no role in study design, data collection, data analysis, data interpretation, writing of the report, or the decision to submit for publication.Between Feb 24, 2016, and Oct 26, 2018, the CNNS survey collected blood samples from 49 486 individuals, 41 210 of whom had a haemoglobin value. After general and sample-specific exclusions , 8087 pa22 with means ranging from −0·8 Z score to −1·2 Z score for various anthropometric indices. Two thirds of participants were from the two richest wealth quintiles, and the primary sample had more boys than girls (3–10% higher) and more rural than urban residents (9–10% higher). Compared with excluded participants, the analysed children were slightly older (by 0·2 years) in the 1–4 years age group and slightly younger (by 0·4 years) in the 10–19-years age group; their haemoglobin (0·2–0·5 g/dL), anthropometry (0·1–0·2 Z score), and wealth index were also higher. There were marginally more Muslim participants in the included participants than in those excluded, and minor regional variations.The sociodemographic characteristics of the primary analytical sample and their comparison with those of the non-analytic sample, in three broad age groups  are summarised in The mean haemoglobin concentrations at various ages were almost identical in girls for the five analytical samples  p 4. LikWe assessed the sex-specific smoothed haemoglobin (g/dL) percentiles from ages 1–19 years in the analytical sample 1  p 3. TheThe age-specific and sex-specific haemoglobin cutoffs to define anaemia, from this secondary analysis of the CNNS, were lower throughout ages 1–19 years than the WHO recommended cutoffs. For both sexes and across the ages, the WHO cutoff was outside the 95% CI of this study’s cutoffs . The difvs 22·2 in girls), almost similar in both sexes for 5—9 years (14·5 in boys vs 16·1 in girls), and substantially higher for girls older than 10 years (22·6 in girls vs 8·9 in boys for ages 10—14 years and 32·6 in girls vs 11·5 in boys for ages 15—19 years).We compared the anaemia prevalence using the haemoglobin cutoffs from this study and WHO applied to all children and adolescents aged 1–19 years in the CNNS with valid haemoglobin estimates, excluding those with history of smoking  to substantially bias the national representativeness of our primary analytical sample. The findings remained stable in various sensitivity analyses. We excluded the most common pathological conditions that contribute to lower haemoglobin concentrations in public health settings. These included inflammation, common haemo-globinopathies, haematopoietic nutrient deficiencies , intestinal parasitosis, high risk of renal dysfunction, and cardiometabolic risk biomarkers (hypercholesterolaemia and impaired glucose homoeostasis as surrogates for risk of adiposity and the attendant inflammatory response). Subclinical malaria, though not formally examined by a peripheral smear examination, was effectively excluded through negative inflammatory biomarkers, and negligible (0·26%) history of malaria in the 2 weeks preceding the survey. We chose the 5th percentile cutoffs to define anaemia for comparability with published literature;9 these cutoffs are marginally higher than the suggested 2·5th percentile.4 The 5th percentile is also a more conservative cutoff; if the 2·5th percentile was chosen as the cutoff, the anaemia prevalence would also be lower than our estimates and further from prevalence estimates based on the WHO cutoff.The finding of substantially lower percentiles and anaemia diagnostic cutoffs in our study than those recommended by WHO when using a defined healthy sample population from the CNNS is intriguing and offers an accurate and current estimate of specific haemoglobin norms for India. Haemoglobin wasestimated from venous blood by the most accurate cyan-methaemoglobin estimation method. Although mixed conclusions have emerged in various studies, datasets—including those from India in field settings—indicate a higher haemoglobin estimate (up to 0·6-0·9 g/dL) in venous blood than in capillary samples.3 Higher haemoglobin concentrations in boys during adolescence have been ascribed to greater muscle mass and testosterone.26 This aspect, as well as the onset of menstruation in girls, could partly explain the greater difference in anaemia prevalence between adolescent boys and girls. In secondary analyses from the US National Health and Nutrition Examination Surveys, the mean adult haemoglobin concentrations of Black, Hispanic, and Asian individuals were lower than those of White individuals.28 Evidence from high-income countries also suggests that haemoglobin cutoffs to define anaemia in children aged 6 months to 2 years should be lowered.3 Anaemia cutoffs in children and adolescents from African and Asian regions were substantially lower (1-2 g/dL) in several datasets compared with WHO recommended cutoffs; however, limited conclusions are possible on norms because few studies had specifically excluded individuals with iron deficiency or inflammation.3 Therefore, scarce data exist to directly compare with our findings, especially from LMICs. A conference abstract has reported summary data on preschool children  among selected healthy participants who had no iron or vitamin A deficiency, inflammation (CRP or α1-acid glycoprotein biomarkers), or malaria (if measured).9 This resulted in 17–88% data loss, and the age-adjusted and country-adjusted mean of haemoglobin concentration was 11·7 g/dL (SE 0·14), with substantial country heterogeneity (p<0·001). The haemoglobin concentration of the pooled countries at the 5th percentile was 9·4 g/dL (range 7·9 g/dL for Pakistan to 11·3 g/dL for USA). The abstract concluded that a single haemoglobin cutoff to define anaemia might not work for every country. By comparison, our study’s mean and 5th percentile of haemoglobin concentrations for children aged 1–5 years were largely comparable. The differences in exclusion criteria and age group (absence of children aged 6–12 months in CNNS) might have slightly increased our estimates.The observed age and sex patterns of haemoglobin concentrations are in consonance with global literature.29 Children and adults living in India have been characterised by a muscle-thin but high-adipose body composition compared with those in other settings.32 Haemoglobin concentrations are positively associated with skeletal muscle density, mass, and strength, and inversely with fat mass.34 Estimates indicate that only 17% of children and 38% of adolescents in India achieve the recommended levels of physical activity36 and, in one subnational study, fewer than 10% of adults (aged ≥20 years) engaged in recreational physical activity.37 This is relevant, because the aerobic requirements for increased muscular energy expenditure provide relevant haematopoietic feedback to the bone marrow, even among athletes with good muscle mass,38 and the promotion of physical fitness and exercise per se elevates haemoglobin concentration.39 In public health settings, these unconventional factors are ignored for establishing causes and instituting interventions for anaemia. This might partly explain the observed proportion (30–48%) of “anaemia of other causes”19 and the relatively static burden of mild anaemia in India, despite decades of anaemia control programmes.Other plausible explanations within the physiological realm could partly account for our study’s haemoglobin cutoffs being lower than the WHO cutoffs based on White participants. Geographical differences could be partly related to the heritability of haemoglobin, which has been shown to explain 34–43% of variation in one Italian study.9 Second, we could not evaluate a few other haematopoietic nutrient deficiencies , but these are rare and unlikely to affect the estimates in public health settings. We also could not evaluate chronic inflammation through serum α1-acid glycoprotein measurements, nor could iron stores be evaluated in detail because CNNS did not collect data on these parameters. Third, the survey study design precluded the inclusion of infants (aged 0–12 months) and also the correlation with robust functional markers for validating anaemia cutoffs. Fourth, even though exposure to infections in the preceding 2 weeks was an exclusion criteria, lingering post-infectious altered erythropoiesis could not be ruled out.The following limitations of our study merit consideration. First, our stringent exclusion criteria resulted in 80% loss of sample; however, this loss is within the range observed in the BRINDA analysis we previously described.7 and its responsiveness to appropriate public health interventions. A substantially lower burden than that currently estimated might also economically permit an in-depth evaluation for a precise aetiological diagnosis. Substantial variations in anaemia cutoffs with age and sex argue for use of haemoglobin centile curves, analogous to growth curves, to prevent misclassification errors. Mounting evidence exists of high heterogeneity in haemoglobin cutoffs among healthy children from different countries.9 Therefore, WHO should urgently initiate a relook at the one-size-fits-all definition of anaemia and issue updated evidencebased guidelines.India should adopt these lower haemoglobin cutoffs instead of the present WHO cutoffs for diagnosing anaemia, because of the contemporary context specificity, national representativeness, accounting for an overwhelming majority of pathological conditions, and rigorous quality control for haemoglobin estimation. These cutoffs might provide a closer estimate of the true burden of anaemia in the country than that calculated by using WHO cutoffs, its grading as a public health problem (mild instead of severe),In conclusion, on the basis of high-quality national data from a recent survey, this study provides contemporary age-specific and sex-specific haemoglobin reference centiles for children and adolescents aged 1–19 years, which are suitable for national use in India. Compared with WHO recommendations, these cutoffs resulted in a substantially lower prevalence of anaemia, which is probably a more accurate reflection of the burden responsive to relevant public health interventions. These findings will contribute to WHO’s efforts to reexamine the applicability of a one-size-fits-all definition of anaemia for different regions and ethnic populations.Appendix 1Appendix 2Appendix 3Appendix 4Appendix 5