Carbohydrate composition of cow's milk and plant-based milk alternatives.

The prevalence of lactose intolerance is one of the factors driving consumers toward plant-based milk alternatives (PBMA). This study aimed to analyze the carbohydrate profile of cows' milk (regular and lactose-free from both pasteurized and UHT milks; n = 80) and PBMA (n = 60) by HPLC. The study revealed that there was no significant difference in the energy content and total carbohydrate content between regular milk and lactose-free milk. Although milk and PBMA are entirely different food matrices, some PBMA types, as soya and coconut, may have energy contents and total carbohydrate contents comparable to those of milk. Furthermore, the variability observed in total carbohydrate content, as well as in carbohydrate profile, both between PBMA types and within samples of the same type, arises not only from variations in raw materials but also from the number of dilutions of the vegetable extract and the addition of different types and levels of carbohydrates, such as sucrose, fructose, or sorbitol, during PBMA manufacture. Although, milk presents a regular carbohydrate composition, differing solely between presentations (regular/lactose-free), the PBMA differs significantly between types and among the same type, not being for that reason regarded as a milk substitute.

Physiological and Technological Properties of Probiotic Lacticaseibacillus rhamnosus GG Encapsulated with Alginate-Chitosan Mixture and Its Incorporation in Whole Milk.

This study developed and evaluated chitosan-sodium alginate capsules containing the probiotic Lacticaseibacillus rhamnosus GG using extrusion and emulsification techniques. The encapsulated L. rhamnosus GG cells were also evaluated for technological and probiotic-related physiological functionalities, as well as when incorporated in UHT and powdered milk. Extrusion (86.01 ± 1.26%) and emulsification (74.43 ± 1.41%) encapsulation techniques showed high encapsulation efficiency and high survival rates of L. rhamnosus GG during 28 days of refrigeration and room temperature storage, especially emulsification capsules (> 81%). The encapsulated L. rhamnosus GG cells showed high survival rates during exposure to simulated gastrointestinal conditions (72.65 ± 1.09-114.15 ± 0.44%). L. rhamnosus GG encapsulated by extrusion and emulsification performed satisfactorily in probiotic-related physiological (pH and bile salts tolerance) and technological properties (positive proteolytic activity, diacetyl and exopolysaccharides production, high NaCl tolerance (> 91%), besides having high heat tolerance (> 76%)). L. rhamnosus GG in extrusion and emulsification capsules had high survival rates (> 89%) and did not significantly affect physicochemical parameters in Ultra-High Temperature (UHT) and powdered milk during storage. The results demonstrate that L. rhamnosus GG can be successfully encapsulated with alginate-chitosan as a protective material through extrusion and emulsification techniques. UHT and powdered milk could serve as appropriate delivery systems to increase the intake of this encapsulated probiotic by consumers.

A comparison of the quality of UHT milk and its plant-based analogs.

The aim of this study was to compare selected physical (density) and physicochemical (color, pH, titratable acidity) properties and the fatty acid profile of dairy UHT milk and its plant-based analogs, i.e., almond, soy, rice, oat, and coconut beverages. Ten products of each type were analyzed in each group. UHT milk was characterized by higher values of color lightness (L*) and titratable acidity than all plant-based milk alternatives, higher yellowness (b*) than rice drink, higher density than almond drink, and higher pH than rice and coconut drinks. In comparison with UHT milk, all plant-based beverages were characterized by higher redness (a*), soy drink was characterized by higher values of b* and chroma (C*), and almond and soy drinks had higher pH values. In the group of non-dairy beverages, the values of b* and C* were highest in soy drink, and the value of a* was highest in almond drink. Almond drink had the highest pH value, and soy and coconut drinks had the highest titratable acidity. Rice drink had higher density than oat, soy and almond drinks. The indicators characterizing the nutritional value of fat were considerably lower in coconut drink and dairy milk than in the remaining products. The values of UFA/SFA, MUFA/SFA, and DFA/OFA ratios and the indicator of nutritional value were highest in rice drink. The PUFA/SFA ratio and the total content of EFAs were highest in soy drink, and the n-6/n-3 PUFA ratio was highest in almond drink.

Characterization of the flavor profile of UHT milk during shelf-life via volatile metabolomics fingerprinting combined with chemometrics.

Ultra-high temperature (UHT) milk is popular among consumers. However, its flavor and texture change in its shelf life. Flavor is highly determinative for the success of dairy products and for consumers' willingness to buy. It is important to milk producers to ensure the optimal flavor of their products in the shelf life. In order to be able to control and predict the flavor quality of UHT milk during the shelf life, this study compared the variations in sensory quality, volatile aroma release and backbone flavor factors and developed a discriminant model to assess flavor quality based on flavouromics data of five competing milk sample during storage. Using partial least squares discriminant analysis (PLS-DA) with Electronic-nose (E-nose) data excellent classification sensitivity and specificity were achieved compared to models based on gas chromatography-mass spectrometry (GC-MS) data. The PLS-DA model using E-nose data exhibited a 100% correct classification rate for the storage period, and a 92% correct rate based on the eight variable importance in the projection (VIP) elements screened for volatile components from different groups. The discriminative model developed herein based on E-nose combined with chemometrics demonstrated advantages such as speed, efficiency, and environmental friendliness. This method shows promise as a precise tool for analyzing aroma changes in UHT milk during its shelf life, and provide support for controlling the flavor substances and milk product development.

Exploration of Novel Plasmin Inhibitor from ß-Lactoglobulin for Enhancing the Storage Stability of UHT Milk.

Plasmin-induced protein hydrolysis significantly compromises the stability of ultrahigh-temperature (UHT) milk. ß-Lactoglobulin (ß-Lg) was observed to inhibit plasmin activity, suggesting that there were active sites as plasmin inhibitors in ß-Lg. Herein, plasmin inhibitory peptides were explored from ß-Lg using experimental and computational techniques. The results revealed that increased denaturation of ß-Lg enhanced its affinity for plasmin, leading to a stronger inhibition of plasmin activity. Molecular dynamics simulations indicated that electrostatic and van der Waals forces were the primary binding forces in the ß-Lg/plasmin complex. Denatured ß-Lg increased hydrogen bonding and reduced the binding energy with plasmin. The sites of plasmin bound to ß-Lg were His624, Asp667, and Ser762. Four plasmin inhibitory peptides, QTMKGLDI, EKTKIPAV, TDYKKYLL, and CLVRTPEV, were identified from ß-Lg based on binding sites. These peptides effectively inhibited plasmin activity and enhanced the UHT milk stability. This study provided new insights into the development of novel plasmin inhibitors to improve the stability of UHT milk.

Milk serum peptidomics revealed the age gelation of direct UHT milk.

Age gelation is undesirable for direct UHT (dUHT) milk, which is closely related to protein hydrolysis. However, little information is available for the role of serum peptides during the age gelation. In this study, the composition and protein morphology of serum phase were characterized by RP-HPLC, ICP-MS and TEM. The results showed significant increases in soluble proteins, free amino acids, calcium, and phosphorus from casein micelles, indicating protein hydrolysis and peptide release into the serum phase. 23,466 peptides derived from caseins and other proteins were identified in serum phase by peptidomics. The serum peptide profiles of age gelation milk changed dramatically. Peptide fingerprinting revealed that plasmin and cathepsin contributed to the protein hydrolysis during age gelation, with a significant increase in their activity observed. 23 characteristic peptides were ultimately selected as potential indicators for age gelation. These findings provide new insights into the age gelation of UHT milk.

Effect of Thermostable Enzymes Produced by Psychrotrophic Bacteria in Raw Milk on the Quality of Ultra-High Temperature Sterilized Milk.

Ultra-high temperature sterilized milk (UHT) is a popular dairy product known for its long shelf life and convenience. However, protein gel aging and fat quality defects like creaming and flavor deterioration may arise during storage. These problems are primarily caused by thermostable enzymes produced by psychrotrophic bacteria. In this study, four representative psychrotrophic bacteria strains which can produce thermostable enzymes were selected to contaminate UHT milk artificially. After 11, 11, 13, and 17 weeks of storage, the milk samples, which were contaminated with Pseudomonas fluorescens, Chryseobacterium carnipullorum, Lactococcus raffinolactis and Acinetobacter guillouiae, respectively, demonstrated notable whey separation. The investigation included analyzing the protein and fat content in the upper and bottom layers of the milk, as well as examining the particle size, Zeta potential, and pH in four sample groups, indicating that the stability of UHT milk decreases over time. Moreover, the spoiled milk samples exhibited a bitter taste, with the dominant odor being attributed to ketones and acids. The metabolomics analysis revealed that three key metabolic pathways, namely ABC transporters, butanoate metabolism, and alanine, aspartate, and glutamate metabolism, were found to be involved in the production of thermostable enzymes by psychrotrophic bacteria. These enzymes greatly impact the taste and nutrient content of UHT milk. This finding provides a theoretical basis for further investigation into the mechanism of spoilage.

2-Heptanone, 2-nonanone, and 2-undecanone confer oxidation off-flavor in cow milk storage.

Flavor sensation is one of the most prevalent characteristics of food industries and an important consumer preference regulator of dairy products. So far, many volatile compounds have been identified, and their molecular mechanisms conferring overall flavor formation have been reported extensively. However, little is known about the critical flavor compound of a specific sensory experience in terms of oxidized off-flavor perception. Therefore, the present study aimed to compare the variation in sensory qualities and volatile flavors in full-fat UHT milk (FFM) and low-fat UHT milk (LFM) samples under different natural storage conditions (0, 4, 18, 25, 30, or 37°C for 15 and 30 d) and determine the main component causing flavor deterioration in the FFM and LFM samples using sensory evaluation, electronic nose, and headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). In addition, the Pearson correlation between the volatile flavor components and oxidative off-flavors was analyzed and validated by sensory reconstitution studies. Compared with the LFM samples, the FFM samples showed a higher degree of quality deterioration with increased storage temperature. Methyl ketones of odd carbon chains (i.e., 2-heptanone, 2-nonanone, 2-undecanone, 2-tridecanone, and 2-pentadecanone) reached a maximum content in the FFM37 samples over 30 d storage. The combined results of the Pearson correlation and sensory recombination study indicated that 2-heptanone, 2-nonanone, and 2-undecanone conferred off-flavor perception. Overall, the present study results provide potential target components for detecting and developing high-quality dairy products and lay a foundation for specific sensory flavor compound exploration in the food industry.

Phylogenetic characterization and biodiversity of spore-forming bacteria isolated from Brazilian UHT milk.

Among the milk contaminating microorganisms, those which are able to form heat-resistant spores are concerning, especially for dairy companies that use ultra-high temperature (UHT) technology. These spores, throughout storage, can germinate and produce hydrolytic enzymes that compromise the quality of the final product. This study evaluated 184 UHT milk samples from different batches collected from seven Brazilian dairy companies with a possible microbial contamination problem. The bacteria were isolated, phenotypically characterized, clustered by REP-PCR, and identified through 16S rDNA sequencing. The presence of Bacillus sporothermodurans was verified using biochemical tests (Gram staining, catalase and oxidase test, glucose fermentation, esculin hydrolysis, nitrate reduction, and urease test). According to these tests, none of the isolates presented typical characteristics of B. sporothermodurans. In sequence, the isolates, that presented rod-shapes, were submitted to molecular analyses in order to determine the microbial biodiversity existing among them. The isolates obtained were grouped into 16 clusters, four of which were composed of only one individual. A phylogenetic tree was constructed using the sequences obtained from the 16S rDNA sequencing and some reference strains of species close to those found using BLAST search in the NCBI nucleotide database. Through this tree, it was possible to verify the division of the isolates into two large groups, the Bacillus subtilis and the Bacillus cereus groups. Furthermore, most isolates are phylogenetically closely related, which makes it even more difficult to identify them at the species level. In conclusion, it was possible to assess, in general, the groups of sporulated contaminants in Brazilian UHT milk produced in the regions evaluated. In addition, it was also possible to determine the biodiversity of spore-forming bacteria found in UHT milk samples, thus opening up a range of possible research topics regarding the effects of the presence of these microorganisms on milk quality.

Influence of storage time on protein composition and simulated digestion of UHT milk and centrifugation presterilized UHT milk in vitro.

The centrifugation presterilizing UHT (C-UHT) sterilization method removes 90% of the microorganism and somatic cells from raw milk using high-speed centrifugation following UHT treatment. This study aimed to study the changes in protein composition and plasmin in the UHT and C-UHT milk. The digestive characteristics, composition, and peptide spectrum of milk protein sterilized with the 2 technologies were studied using a dynamic digestive system of a simulated human stomach. The Pierce bicinchoninic acid assay, laser scanning confocal microscope, liquid chromatography-tandem mass spectrometry, and AA analysis were used to study the digestive fluid at different time points of gastric digestion in vitro. The results demonstrated that C-UHT milk had considerably higher protein degradation than UHT milk. Different processes resulted during the cleavage of milk proteins at different sites during digestion, resulting in different derived peptides. The results showed there was no significant effect of UHT and C-UHT on the peptide spectrum of milk proteins, but C-UHT could release relatively more bioactive peptides and free AA.

Proteolytic activity and heat resistance of the protease AprX from Pseudomonas in relation to genotypic characteristics.

AprX is an alkaline metalloprotease produced by Pseudomonas spp. and encoded by its initial gene of the aprX-lipA operon. The intrinsic diversity among Pseudomonas spp. regarding their proteolytic activity is the main challenge for the development of accurate methods for spoilage prediction of ultra-high temperature (UHT) treated milk in the dairy industry. In the present study, 56 Pseudomonas strains were characterized by assessing their proteolytic activity in milk before and after lab-scale UHT treatment. From these, 24 strains were selected based on their proteolytic activity for whole genome sequencing (WGS) to identify common genotypic characteristics that correlated with the observed variations in proteolytic activity. Four groups (A1, A2, B and N) were determined based on operon aprX-lipA sequence similarities. These alignment groups were observed to significantly influence the proteolytic activity of the strains, with an average proteolytic activity of A1 > A2 > B > N. The lab-scale UHT treatment did not significantly influence their proteolytic activity, indicating a high thermal stability of proteases among strains. Amino acid sequence variation of biologically-relevant motifs in the AprX sequence, namely the Zn2+-binding motif at the catalytic domain and the C-terminal type I secretion signaling mechanism, were found to be highly conserved within alignment groups. These motifs could serve as future potential genetic biomarkers for determination of alignment groups and thereby strain spoilage potential.

Immunogold Labeling of Milk Proteins at Transmission Electron Microscopy.

Milk intended for human consumption is subjected to technological treatments to ensure its safety and storage stability. These treatments deeply modify some of its structural and nutritional characteristics. Principal modifications involve proteins that partly adsorb onto the membrane of milk fat globules upon homogenization or whey proteins that undergo denaturation and aggregation during thermal treatments. Transmission electron microscopy is a powerful approach to investigate milk ultrastructure, due to its high-resolution power. Immunogold labeling of ß-lactoglobulin and ß-casein proteins represents a sophisticated approach to examine their structure and localization following technological processes such as homogenization and UHT treatment. However, liquid milk is a very challenging matrix because of its complex multiphasic nature. To preserve both ultrastructure and antigenicity, and to obtain an efficient labelling in liquid milk samples, some precautions shall be adopted in fixation, embedding, and labeling steps as here reported.

The effects of packaging type and storage temperature on some of UHT milk quality indexes.

The objective of the present study was to illustrate the changes in physicochemical properties in ultra-high temperature (UHT) milk packed into a pouch and Tetra Brik during storage. UHT milk samples were kept at 5 and 25 °C for 3 months and regularly analyzed monthly. During storage, significant increases (p < 0.05) in titratable acidity (TA), water-soluble nitrogen (WSN), and non-protein nitrogen (NPN) when UHT milk was packed into pouch versus Tetra Brik and stored at 25 versus 5 °C. Neither type of packaging nor storage temperature affect pH values during storage. Spore-forming bacterial (SFB) count was always higher in UHT milk packed into pouch versus Tetra Brik. Refrigerated storage kept UHT milk without detectable SFB compared to UHT milk held at 25 °C. Pouch packages were responsible for the migration of phthalate derivatives [dimethyl phthalate "DMP", diethyl phthalate "DEP", dibutyl phthalate "DBP", and di-(2-Ethylhexyl) phthalate "DEHP"] into milk with significantly greater levels than milk filled into Tetra Brik. The total sensory scores were decreased significantly during storage, which was more pronounced in UHT milk filled into pouch versus Tetra Brik or stored at 25 °C versus 5 °C. It is concluded that UHT milk filled into Tetra Brik stored at 5 and 25 °C is better in terms of quality and safety indexes than such filled into a pouch.

Bisphenol determination in UHT milk and packaging by paper spray ionization mass spectrometry.

This study evaluates the use of paper spray ionization mass spectrometry (PSI-MS) for rapid determination of bisphenol A (BPA) and bisphenol S (BPS) in UHT milk and milk packaging. The packages were analyzed by cutting the cartons into triangular shapes and submitting them to PSI-MS analysis. The milk samples were subjected to a simple liquid-liquid extraction and the supernatant was deposited onto a triangular paper that was subsequently used for PSI-MS analysis. In milk, BPS and BPA levels ranged from 60.0 to 150.8 ng mL-1. The LOD and LOQ values were 1.5 and 4.8 ng mL-1 for BPA, and 4.8 and 16.0 ng mL-1 for BPS, respectively. Linearity was R2 > 0.98 for both compounds. Precision values were below 20%, and recoveries close to 100%. The PSI-MS can be used as a simple, rapid, and accurate methodology to determine bisphenols in milk and milk packaging.

Sensory Description and Consumer Hedonic Perception of Ultra-High Temperature (UHT) Milk.

Sensory characteristics of products play an essential role on the consumer' s acceptability, preference and consuming behavior choice. The sensory profiles and consumer hedonic perception for 14 UHT milk products using sensory quantitatively descriptive analysis and a 9-point hedonic scale were investigated in this study. There were significant differences in the sensory attributes intensity and liking scores among the organic whole milk, ordinary whole milk, low-fat milk, and skimmed milk (p < 0.05). Skimmed milk samples had lowest intensity scores of typical milk aroma, taste flavor and texture attributes, as well as had the lowest overall liking scores. Whole milk samples had higher sensory intensity scores than low-fat milk samples, even though no significant differences of overall liking scores were observed between whole milk and low-fat milk. Furthermore, the relationship between the sensory attribute and overall liking was demonstrated according to correlation analysis and partial least squares regression (PLSR) analysis. Overall liking increased significantly with the increasing of sweet, after milk aroma, protein-like, mellow and thick, while decreased significantly with the enhancement of cowy, cooked and whey (p < 0.05). These findings presented a potential strategy for identifying the key sensory attributes responsible for liking score differences among different kinds of UHT milk products.

Colloidal stability of milk: reinterpretation of alcohol test results by digital microscopy.

In this research communication we propose a new approach by portable digital microscopy with a 200× objective to improve the visualization of microparticles of pasteurized milk submitted to the alcohol test. Not only did the method reduce the subjectivity of the readings, but also generated high resolution images of the microparticles, which allows the creation of a specific image pattern for each type of final product. In comparison to a control pasteurized milk treatment, the results confirmed the effect and the specificity of added salts (sodium citrate, disodium phosphate or their combination) on the stability of the milk to the alcohol test. Finally, the mixture of stabilizing salts of citrate/phosphate provided the highest degree of stability to pasteurized milk among the treatments studied.

Incidence of Bacillus cereus, Bacillus sporothermodurans and Geobacillus stearothermophilus in ultra-high temperature milk and biofilm formation capacity of isolates.

The presence of mesophilic and thermophilic spore-forming bacteria in UHT milk, as well as biofilm formation in dairy plants, are concerning. The current study explored the spore-forming bacilli diversity in 100 samples of UHT milk (skimmed and whole). Through this work, a total of 239 isolates from UHT milk samples were obtained. B. cereus s.s. was isolated from 7 samples, B. sporothermodurans from 19 and, G. stearothermophilus from 25 samples. Genes encoding hemolysin (HBL), and non-hemolytic (NHE) enterotoxins were detected in B. cereus s.s. isolates. All isolates of B. cereus s.s. (12) B. sporothermodurans (38), and G. stearothermophilus (47) were selected to verify the ability of biofilm formation in microtiter plates. The results showed all isolates could form biofilms. The OD595 values of biofilm formation varied between 0.14 and 1.04 for B. cereus, 0.20 to 1.87 for B. sporothermodurans, and 0.49 to 2.77 for G. stearothermophilus. The data highlights that the dairy industry needs to reinforce control in the initial quality of the raw material and in CIP cleaning procedures; avoiding biofilm formation and consequently a persistent microbiota in processing plants, which can shelter pathogenic species such as B. cereus s.s.

Is Ultra-High Temperature Processed Milk Safe in Terms of Heterocyclic Aromatic Amines?

Herein, the presence of heterocyclic aromatic amines (HAAs) in 24 different commercial ultra-high temperature processed (UHT) milk types was investigated. The dry matter and pH values of the samples were also determined. The milk types showed significant differences (p < 0.01) regarding the dry matter, pH values, and individual HAAs and total HAAs. The milk sample dry matter and pH values were in the range of 8.56-13.92% and 6.66-6.91, respectively. The growing up milk samples had the highest dry matter and pH values. While no significant correlation between the total HAAs and dry matter was found, a negative correlation (p < 0.01) between the total HAAs and pH value was determined. Among the tested HAAs, five compounds, (IQx (up to 0.06 ng), IQ (up to 0.10 ng), MeIQx (up to 0.55 ng), MeIQ (up to 1.97 ng), and PhIP (up to 0.39 ng)) were quantified in the samples. The average total HAAs of the samples ranged from 0.13 to 0.67 ng; however, one milk sample (200 mL) contained between 10.10 and 53.35 ng total HAAs. Therefore, it was shown that protein fortification and lactose hydrolysis substantially increased the formation of HAAs in UHT milk.

Taxonomic Evaluation of the Heyndrickxia (Basonym Bacillus) sporothermodurans Group (H. sporothermodurans, H. vini, H. oleronia) Based on Whole Genome Sequences.

The genetic heterogeneity of Heyndrickxia sporothermodurans (formerly Bacillussporothermodurans) was evaluated using whole genome sequencing. The genomes of 29 previously identified Heyndrickxiasporothermodurans and two Heyndrickxia vini strains isolated from ultra-high-temperature (UHT)-treated milk were sequenced by short-read (Illumina) sequencing. After sequence analysis, the two H. vini strains could be reclassified as H. sporothermodurans. In addition, the genomes of the H.sporothermodurans type strain (DSM 10599T) and the closest phylogenetic neighbors Heyndrickxiaoleronia (DSM 9356T) and Heyndrickxia vini (JCM 19841T) were also sequenced using both long (MinION) and short-read (Illumina) sequencing. By hybrid sequence assembly, the genome of the H. sporothermodurans type strain was enlarged by 15% relative to the short-read assembly. This noticeable increase was probably due to numerous mobile elements in the genome that are presumptively related to spore heat tolerance. Phylogenetic studies based on 16S rDNA gene sequence, core genome, single-nucleotide polymorphisms and ANI/dDDH, showed that H. vini is highly related to H. sporothermodurans. When examining the genome sequences of all H.sporothermodurans strains from this study, together with 4 H. sporothermodurans genomes available in the GenBank database, the majority of the 36 strains examined occurred in a clonal lineage with less than 100 SNPs. These data substantiate previous reports on the existence and spread of a genetically highly homogenous and heat resistant spore clone, i.e., the HRS-clone.

Comprehensive Profiling of the Native and Modified Peptidomes of Raw Bovine Milk and Processed Milk Products.

Bovine milk contains a variety of endogenous peptides, partially formed by milk proteases that may exert diverse bioactive functions. Milk storage allows further protease activities altering the milk peptidome, while processing, e.g., heat treatment can trigger diverse chemical reactions, such as Maillard reactions and oxidations, leading to different posttranslational modifications (PTMs). The influence of processing on the native and modified peptidome was studied by analyzing peptides extracted from raw milk (RM), ultra-high temperature (UHT) milk, and powdered infant formula (IF) by nano reversed-phase liquid chromatography coupled online to electrospray ionization (ESI) tandem mass spectrometry. Only unmodified peptides proposed by two independent software tools were considered as identified. Thus, 801 identified peptides mainly originated from αS- and ß-caseins, but also from milk fat globular membrane proteins, such as glycosylation-dependent cell adhesion molecule 1. RM and UHT milk showed comparable unmodified peptide profiles, whereas IF differed mainly due to a higher number of ß-casein peptides. When 26 non-enzymatic posttranslational modifications (PTMs) were targeted in the milk peptidomes, 175 modified peptides were identified, i.e., mostly lactosylated and a few hexosylated or oxidized peptides. Most modified peptides originated from αS-caseins. The numbers of lactosylated peptides increased with harsher processing.