Knowing country-specific predictors of smoking behaviour for adolescents is crucial for successful smoking prevention programs. This study aims to assess demographic and socio-cognitive variables related to smoking initiation among Saudi male adolescents.Longitudinal data were collected at T1 (baseline) and at T2 (followup at 6 months) using a self-administered questionnaire. We assessed smoking behaviour and related demographic variables and socio-cognitive variables. Chi-squared tests and independent-samples t-tests were used to identify differences in baseline characteristics between smokers and non-smokers at T1. Furthermore, non-smokers at T1 were included in logistic regression analyses to examine the predictors of smoking initiation between T1 and T2.At T1, the non-smokers who were included in further analysis were 523 (84.9%) of whom 48 (9.2%) had initiated smoking at T2. They differed significantly from non-initiators, including having a more positive attitude towards smoking, reporting more social norms, modelling and pressure to smoke, having a lower self-efficacy to refrain from smoking and higher intention to smoke in the future . The regression analysis revealed that: adolescents with disrupted-families, being of low academic achievement, with relatively high monthly-income families, having more smoking-peers, high-perceived pressure to smoke from parents (p=0.002) and teachers (p=0.001), have smoking supportive-norms of parents and having high intention to smoke in the future (p<0.001) were at higher risk of being smokers.Findings suggest that health-promoting programs should address strengthening of self-efficacy and enhancing refusal skills against modelling of peers, pressure and norms of parents. Smoking is the number one preventable cause of death3.Smoking remains one of the major public health problems. It is associated with different types of morbidities, including cancers, heart and coronary diseases, and lung disease; almost all systems of the body are negatively affected by smoking4; approximately 40% of smokers start by this age5 and it is estimated that 88% of adults who smoke daily started smoking by the age of 18 years6. Early smoking initiation is associated with difficulty in quitting, being regular smokers as adults7 and with susceptibility to addiction8.Smoking experimentation and initiation mostly occurs during adolescence10 integrates several cognitive models to understand and change health behavior. It originated from the Attitude-Social influence-Self efficacy (ASE) model, which is based on the Theory of Reasoned Action11. The model incorporates insights from the Transtheoretical Model12, the Theory of Planned Behavior13, Social Cognitive Theory14, the Precaution Adoption Model15, and goal setting theories16. The model assumes that intention is the most proximal predictor of behavior, which in turn is influenced by a person’s attitude comprising cognitive and emotional advantages and disadvantages, social influence beliefs , and self-efficacy. Additionally, the most recent version acknowledges pre-motivational and post-motivational determinants10.The Integrated Change Model (I-Change Model)17. In 1994 the report of the Surgeon General of the United States Department of Health and Human Services clearly showed that smoking initiation was associated with psychological and social factors18. Using longitudinal data, De Vries et al.19 found that the social influence constructs , in additon to self-efficacy and intention were significant predictors of adolescent smoking behavior. In a review with 53 longitudinal studies, published between January 1984 and August 2015, ninety-eight potential predictors were identified, including increased age/grade, poor academic performance, lower socioeconomic status, intention to smoke in the future, smoking family members, smoking friends, and exposure to smoking promoting films and tobacco promotion efforts, against which high self-efficacy was found to be protective20.Investigation of smoking behavior predictors among adolescents is an essential step to develop and design a successful smoking prevention program. The social influence approach to study smoking behavior was developed for the first time by Evans in 1976 when with colleagues addressed the impact of social pressure to smoke from parents, media, and peers21 in their cross-sectional study found that having smoking friends, parental smoking, exposure to cigarette advertisements in mass media, and higher pocket money were risk factors for smoking initiation. Al-Makadma et al.22 in their survey documented that paternal smoking was the most significant factor to explain smoking onset; their findings were supported by Alsubaie23 who also found age, studying in private schools, having friends who smoke, perceived poor health and dissatisfaction with life as predictors of smoking onset.In Saudi Arabia, several studies investigated smoking predictors among adolescents. Results were similar to those found in international studies. Al-Zalabani et al.The aim of this paper is to investigate the predictors of smoking intiation among Saudi male adolescents to guide the development of smoking prevention programs for this target group.24.As part of the development of a smoking prevention program targeting school going adolescents in Taif, Saudi Arabia, secondary schools were approached to participate in a two-armed cluster randomized controlled trial. Nine schools were randomly selected to represent the control group. To select participants, students were given the chance to pick one of two papers in which either ‘Included’ or ‘Excluded’ were written. Out of the 707 included, twenty-four students did not fill in the questionnaire, resulting in 683 (96.6%) participants. In the current study, we analyze the data of the control group only for whom baseline (T1) and at 6 months (T2) longitudinal data were obtained. Only boys were included in the study, since the educational system in Saudi Arabia is gender specific and smoking is officially not considered a problem for girlsThe data were collected with the approval of the General Directorate of Education, school health program and school masters. Participants were given the right not to participate or stop at any time, as was explained prior to filling in the questionnaire.25. Translation to Arabic and back-translation was done by a public-health expert, with some modifications to fit with Saudi norms and culture. The questionnaire was pretested in a focus group discussion for participants from the same selected schools and accordingly some adaptations were made. The questionnaire assessed demographics, attitudes, social influences, self-efficacy, intention not to smoke in the future and smoking behavior.The questionnaire used was a modified version of the European Smoking Prevention Framework Approach (ESFA) based on the I-Change ModelDemographic factors included were: age (months), school area , school type , family monthly income (US$) , daily pocket money (US$) , academic performance for the last year final exam , and family structure .Attitude was assessed using a 7-point Likert scale with 9 items: 1) very pleasant/very unpleasant, 2) very desirable/very undesirable, 3) makes me feel very relaxed/makes me feel very stressed, 4) very much more confident to be part of the crowd/ very much less, 5) very much friendly/very much unfriendly, 6) very much sociable/very much unsociable, 7) tastes really very nice/tastes really very horrible, 8) friends pay much more attention if I smoke/pay much less attention, and 9) much more easier to start talking with others/much more difficult; coded as  .19. Social modeling was assessed by asking respondents to indicate the smoking behavior of important people in their environment   . Social norms were measured by asking whether respondents believed that these people felt that they should smoke or not  . To measure perceived social pressure to smoke the respondents were asked whether they countered pressure to smoke from the same eight people  .Social influence beliefs were measured using three constructs: social modeling, social norms, and perceived social pressureSelf-efficacy was assessed using a 7-point Likert scale by asking the adolescents to indicate how difficult they found it not to smoke in a variety of situations . The situations measured were: with another who smokes, with friends who smoke, when offered a cigarette by someone, or by a friend, on the way home from school, while watching TV, doing homework, going out with friends, stressed, upset, depressed, nervous, worried, and when shopping .Intention not to smoke was asked by four items on a 7-point scale to assess whether adolescents had the intention to smoke in the next 6 months, the next year, the next five years and in the future  .25; any inconsistency in response was resolved by recoding to the most unfavorable response25.Smoking status was defined when the adolescent indicated that he had smoked at least once a week or daily, and when he reported that he smoked 100 cigarettes or more, unless he reported that he had quit. An adolescent was coded as a non-smoker when he had quit smoking, smoked monthly/occasionally, or experimented with smoking. An algorithm with four questions was used to validate the self-reported smoking status  in Model 2, and demographics, motivational constructs, and intention in Model 3. The variables, which were significantly related to the outcome in Model 1, were also considered to be included in Model 3, but did not appear in the final Model 3 as they were no longer significant. In all analyses, the dependent variable was smoking status at T2, where only non-smokers at T1 were included to address smoking initiation between wave 1 (T1) and 2 (T2). As sensitivity analysis, the three final logistic regression models were repeated with the general estimated equation (GEE) analysis, accounting for the nesting of adolescents within schools (exchangeable structure).Assumptions were checked using variance inflation factors (VIF>10 indicates a (multi) collinearity problem), Cook’s distances , and tests on quadratic terms .All data analyses were performed using IBM SPSS Statistics for Windows . Two-sided p-values ≤0.05 were considered statistically significant.Of the 707 adolescents, 683 (96.6%) filled in the questionnaire at both time intervals; 67 of these were excluded from the analysis either for not fully completing the questionnaire or for missing important demographic and cognitive variables, resulting in 616 out of 707 (87.1%) participants included in the analysis.The mean age of the included adolescents (n=616) at T1 was 13.5 years (SD=0.49). The vast majority 565 (91.7%) lived in urban areas and most studied at public schools 540 (87.7%). The distribution of the respondents based on family structure was: 520 (84.4%) lived in a stable family with father and mother, while 96 (15.6%) lived in disrupted families, including 46 (7.5%) with father and mother but the father had another wife, 32 (5.2%) had divorced parents, and of the remaining 18 (2.9%) one or both parents had passed away. As for daily pocket money, 48.7% got less than US$2. The families of 217 (35.2%) participants earned more than US$2400 per month, 146 (23.7%) between US$1600 and US$2400, 104 (16.9%) between US$800 and <US$1600, and 149 (24.2%) earned <US$800. At T1, there were 523 (84.9%) non-smokers and 93 (15.1%) smokers. Smokers at T1 were more often from a disrupted family, had more daily pocket money, and belonged less often to the higher third of the class than non-smokers  .To identify the predictors of smoking onset, only the non-smokers at baseline 523 (84.9%) were included in further analyses. Of these non-smokers at T1, 475 (90.8%) had not initiated smoking at T2, whereas 48 (9.2%) initiated smoking between T1 and T2. Adolescents who did not initiate smoking scored on average significantly higher on self-efficacy, and lower on attitude towards smoking and intention to smoke than smoking initiators . The analysis of social influence items showed that smoking initiators scored significantly higher than non-initiators on all items assessed , except for the social pressure from the mother (p=0.32) and sister (p=0.68), and modeling of mother (p=0.35) and sister (p=0.09). Analysis of the perceived social influence of parents and peers led to significant differences between the two groups for the three constructs assessed .In order to identify the predictors of smoking onset, we ran three logistic regression models, where all assumptions were met. If we only consider demographics (Model 1), adolescents with low academic performance, who were members of a disrupted family and had a relatively high family monthly income were more vulnerable to initiate smoking than others. From Model 2, including demographics and motivational constructs, adolescents with more smoking peers  were at higher risk of being smokers too. Also, those with high perceived social norms of parents and with pressure to smoke from parents (mostly the father) or teachers were more likely to smoke. Adding intention to demographics and motivational constructs (Model 3) suppressed the effect of peers modeling, and teachers’ pressure, while perceived pressure and norms of parents remained significant in the final model .The sensitivity analyses with GEE, accounting for nesting of adolescents within schools, showed similar results for all three models (intra-class correlation ICC<0.001).26. In line with other studies27, smoking prevalence was higher among respondents from disrupted families. Smokers and non-smokers varied significantly in received daily pocket money, while family monthly income revealed the trend that smoking prevalence was higher among respondents of higher socioeconomic status, in contrast to findings of similar studies in other countries that documented that uptake is higher among those with low socioeconomic status29. Yet, our result is consistent with the findings from local studies30. This may indicate that the smoking epidemic is at an early stage in Saudi Arabia and, as according to Lopez et al.31, smoking is initially highest among people in higher socioeconomic strata, an alarming fact that the tobacco control in Saudi Arabia should consider.Our findings about smoking behavior and academic performance support the findings of other studies and reviews in which smoking was more prevalent in adolescents with lower academic performance32 showing the importance of the social influence approach in understanding adolescent smoking behavior.Smokers and non-smokers differed significantly for all assessed social influence constructs, except for perceived modeling and pressure to smoke from mothers and sisters, which is likely to be due to the nature of the Saudi community where males and females are raised separately with boys being more attached to fathers and girls to mothers. Smokers perceived higher pressure to smoke, more often had smoking parents (mostly fathers), peers and teachers, experienced smoking supportive norms, had positive attitudes towards smoking, less self-efficacy to refrain from smoking and a high intention to smoke in the future. Our findings are in line with several international studies33, revealed a significant impact of perceived parental norms and pressure to smoke and modeling of peers; findings also reported by other local23 and international studies34. Model 3 added intention to the previous factors and revealed that intention to smoke in the future was a highly significant predictor of smoking initiation; findings also supported by other studies35. Parental norms and pressure remained significant in this model; this finding was also found in another study that reported that social influence can act directly on behavior32.To examine the predictors for smoking initiation we ran logistic regression analyses. Model 1 with only sociodemographic factors revealed that academic performance and family structure were significant predictors. Model 2, which added the motivational factors36, an approach that we also followed.Our study had some limitations. First, only boys were included, because smoking among females was not considered a public health problem in Saudi Arabia; hence, we could not get approval from girls’ schools. Secondly, six months is considered a relatively short period for behavioral change. Thirdly, we could not biologically validate self-reports of smoking behavior. However, it is clearly documented that there is high correlation between biochemical assessment of adolescent smoking behavior and self-reports, if confidentiality is preserved and anonymity is assuredFinding of this study can help the school health program and tobacco control program in Saudi Arabia in shaping smoking prevention programs for adolescents. Coping with pressure to smoke, refusal skills development and enhancing self-efficacy are essential elements to be considered for better outcomes from anti-smoking interventions.


Celiac disease (CD) is an autoimmune disorder characterized by intolerance to dietary gluten in genetically predisposed subjects. Iron deficiency anemia (IDA) is a common sign in CD, being the only abnormality in approximately 40% of celiac patients. A multifactorial etiology leads to IDA in CD. The two main causes are the villous atrophy of the mucosa at the site of iron absorption (the duodenum) and the resulting inflammation, which triggers the mechanism that leads to the anemia of chronic disease. Until now, it has been unclear why some patients with CD continue to have IDA despite a careful gluten-free diet (GFD) and the normalization of villous atrophy. Furthermore, some celiac patients are refractory to oral iron supplementation despite the healing of the mucosa, and they thus require periodic intravenous iron administration. The Marsh classification evaluates the degree of inflammation and villous atrophy, but it does not assess the possible persistence of ultrastructural and molecular alterations in enterocytes. The latter was found in CD in remission after adopting a GFD and could be responsible for the persistently reduced absorption of iron and IDA. Even in non-celiac gluten sensitivity, anemia is present in 18.5–22% of patients and appears to be related to ultrastructural and molecular alterations in intestinal microvilli. It is possible that a genetic component may also play a role in IDA. In this review, we evaluate and discuss the main mechanisms of IDA in CD and the possible causes of its persistence after adopting a GFD, as well as their therapeutic implications. Celiac disease (CD) is an autoimmune disease involving the small bowel mucosa that is triggered by the ingestion of gluten in genetically predisposed subjects ,2. The dCD is a global health-care problem ,4,5. TheThe clinical presentation of CD has a wide spectrum of manifestations, both intestinal and extra-intestinal, involving one or more organs . SeveralThe clinical manifestations of CD can be directly related to autoimmunity or can indirectly derive from intestinal inflammation and/or malabsorption .12. Chronic inflammation may also play a role in the development of anemia [Anemia is one of the most common clinical manifestations of CD and may be present in over half of patients at the time of diagnosis ,13. AnemIron deficiency anemia (IDA) is the most commonly recognized type of anemia in patients with CD ,14. IDA 12 malabsorption can also occur, but it mostly does in celiac patients with more extensive lesions [Folate and vitamin B lesions ,17. The Folate is primarily absorbed in the jejunum, which is commonly affected by CD. In patients with CD, the risk of folate deficiency is high, reaching up to 20–30% at CD diagnosis ,18,19. T12 requires the formation of a complex with the intrinsic factor produced by the stomach. The terminal ileum is the primary site of absorption of vitamin B12 [The absorption of vitamin Bamin B12 .12 deficiency of between 8 and 41% has been reported [12 deficiency are not yet fully clear. In addition to specific intestinal mucosal damage due to CD, they include other gastrointestinal disorders such as chronic autoimmune gastritis (with the reduced production of the intrinsic factor) frequently associated with CD, intestinal bacterial overgrowth , and disorders of the distal tract of the small intestine (with the reduced absorption of vitamin B12).At CD diagnosis, a prevalence of vitamin Breported . In CD, 12 has been found to be 15.7–18.3% and 4.3–8%, respectively [In pediatric patients with CD, the range of the prevalence of low levels of folate and vitamin Bectively ,20,21.Returning to the topic of IDA, blood loss from intestinal lesions can be considered an adjunctive cause that should be excluded.It is worth noting that anemia in CD is caused not only by malabsorption or blood loss but also by other factors. Anemia of chronic disease (ACD), a form of anemia associated with chronic inflammation, may occur in up to a quarter of patients with CD ,23,24. SIt has been reported that pro-inflammatory cytokines—in particular interferon-gamma (IFN-y), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α)—trigger the synthesis of the iron regulatory hormone, hepcidin ,27. HepcIt is likely that IDA and ACD can coexist in CD before diagnosis and before adopting a gluten-free diet (GFD), whereas after a prolonged period on a GFD, the role of ACD is likely to be minimal or absent. In clinical practice, it is possible to evaluate whether IDA or ACD prevails by using several laboratory parameters  13,23,2,223,24.It is noteworthy that IDA, like other clinical manifestations, can persist in a proportion of patients with CD that experience mucosal healing and the resolution of intestinal malabsorption after excluding gluten from the diet ,14,28,29This review primarily focuses on published studies performed on IDA in patients with CD selected from a computer search of the literature  from January 1990 to January 2020, performed by three authors , and using the following keywords: “celiac disease,” “anemia,” “microcytic anemia,” “macrocytic anemia,” “megaloblastic anemia,” “iron deficiency,” “micronutrient deficiencies,” “iron-deficient anemia,” ”IDA,” “anemia of chronic disease,” “ACD,” “persistent anemia in celiac disease,” “refractory anemia,” “gluten-free diet,” and “GFD.” Additional studies were identified through a manual review of the reference lists of the selected studies and review articles. Clinically significant full-text articles were selected for this review.IDA is a relatively frequent condition caused by iron loss or reduced iron absorption. It is defined as reduced values of hemoglobin and mean corpuscular volume, as well as low serum iron and ferritin ,31. As fp = 0.0002) [p < 0.001). They also showed that the frequency of potential CD was higher in patients unresponsive to iron supplementation than controls.Since patients with IDA are more likely to have CD than the general population, serum screening tests for CD should always be performed in these patients. A cross-sectional study on 290 patients (age more than 12 years) diagnosed with IDA showed that tTGs were found in 32 (11%) of these patients ( 0.0002) . Shahria 0.0002) . They foData on the prevalence of IDA at the time of CD diagnosis and after starting a GFD, both in adult and pediatric patients with CD, are summarized in Kolho et al. reported IDA in 25% of Finnish adult patients at the time of CD diagnosis, all with partial or total villous atrophy . Akbari 12 deficiency in 25.6% of cases, and ACD in 10.2% of cases) [A recent Turkish retrospective analysis of 195 adult patients at the time of CD diagnosis showed that anemia was found in 60.5% of patients  . In an If cases) . In anotf cases) . An imprf cases) . In a cof cases) .p = 0.007). Furthermore, at the time of CD diagnosis, the body mass index (BMI) was lower in the first than in the second group . After one year on a GFD, the villous height–crypt ratio was lower in patients with anemia than those without anemia  and IEL was higher in patients with anemia than without  [p = 0.032) [p < 0.01), respectively. They reported that the lag time between the onset of symptoms and the diagnosis of CD was higher in patients with CD alone  [12 deficiency in 13.6%, folate deficiency in 10.7%, mixed nutritional deficiency in 16.5%, and ACD in 3.9% [p = 0.004; median ferritin values were 5 versus 20 ng/mL, p = 0.002, respectively). The hypothesis that the degree of villous atrophy correlates with anemia severity was partially confirmed by Harper et al. After analyzing a cohort of 405 adult celiac patients at the time of CD diagnosis, they showed that degree of villous atrophy did not influence the proportion of anemic patients. Conversely, there was a significant difference in the proportion of patients with iron deficiency  [p = 0.0003) between an increase in hemoglobin concentration and a decrease of histological scores of duodenal lesions after adopting a GFD. They concluded that the hemoglobin value normalizes 6–12 months after starting a GFD alone as a consequence of the normalization of alterations in the intestinal mucosa. Conversely, after 12 months on a GFD, only 50% of patients had recovered from iron deficiency [Duodenal villous atrophy impairs iron absorption, and IDA may be the only clinical sign in adults with CD . Chronic= 0.054) . In a co= 0.054) . Similar= 0.032) . Conside < 0.05) . In a ca in 3.9% . Hemoglo> 0.001) . They deficiency . In a stp = 0.046) in the Synergy 1 group, whereas no significant difference was observed in the placebo group. The reduction of hepcidin was probably due to the potential anti-inflammatory effect of prebiotics. However, no differences in morphological or biochemical blood parameters  were observed after intervention in either group [Studies aimed at understanding why there is a partial response to a GFD are lacking. Efthymakis et al. investigated the diagnostic role of small bowel capsule endoscopy in 26 adult celiac patients with persistent IDA, despite them following a GFD . In 23% er group .Helicobacter pylori) and increased need for iron . A non-negligible role in causing IDA in CD patients could be played by an inadequate low-iron diet, given that a GFD does not generally include enriched/fortified iron from wheat-based foods.The leading causes of IDA in celiac patients include iron malabsorption, chronic intestinal inflammation that is typical of CD, and the concomitant presence of other inflammatory bowel diseases  and giardiasis) or extraintestinal diseases , as well as gastrointestinal occult blood losses . The iron to be absorbed must be converted from the Fe3+ form to the ferrous form (Fe2+) by the combined action of duodenal cytochrome B (DCYTB), a ferrireductase that is present on the apical membrane of duodenal enterocytes, or dietary reducing agents such as ascorbic acid [2+ form is then transported across the apical duodenal membrane by the iron symporter, divalent metal transporter 1 (DMT1) [2+ is subsequently transferred to the blood via the iron exporter, ferroportin, which is also called metal transporter 1 (MTP 1); MTP 1 is localized in the basolateral membrane (3+) ions to distant target tissues. Prior to transferrin binding, ferrous (Fe2+) ions must be reconverted to ferric (Fe3+) ions. Excess iron can be deposited in the liver, and then it can be released and used later [Non-heme iron is mainly present in the diet as the ferric form  ,53,55. D1 (DMT1) ,14,55. Fmembrane . For traed later ,57. By iIron absorption is reduced in patients with CD due to duodenal villous atrophy (the duodenum is the specific site of iron absorption) . VillousInflammatory states are often accompanied by microcytic anemia, and they can lead to the alteration of iron homeostasis . At leasIncreased serum levels of acute-phase proteins, such as the C-reactive protein (CRP), are rare in CD, although the gliadin-dependent activation of mononuclear cells of the lamina propria mucosa causes the hyperproduction of pro-inflammatory cytokines, such as IFN-γ and IL-6 ,63,64, wH. pylori infection, esophagitis, erosive duodenitis, and microscopic enteropathy. The association between IDA in celiac patients and H. pylori infection remains controversial [Causes of iron loss must be excluded, even in celiac patients. Causes of bleeding from the lower digestive tract should be excluded by an occult blood stool test and subsequent colonoscopy . Esophagoversial ,73,74,75H. pylori and IDA in 362 adult patients with CD, and they found H. pylori infection in 21% of cases [H. pylori infection in 32.1% of cases [H. pylori, among which 47% had IDA [H. pylori infection did not significantly differ between celiac adult patients with or without IDA [H. pylori, taking gastric biopsies to diagnose H. pylori infection should be a routine part of the diagnostic workup to identify the causes of IDA in patients with CD [Cuoco et al. assessed the relationship between of cases . In a coof cases . Demir e had IDA . On the hout IDA . Given t with CD ,76.H. pylori infection is found in over 50% of patients with unsolved IDA [H. pylori infection [H. pylori eradication could resolve IDA [H. pylori is not fully known [H. pylori and could be involved in heme binding and/or uptake [H. pylori leads to an inflammatory state that damages tissue, resulting in nutrient release, including iron. In this model of a parasitic relationship, the bacterium competes with the host for iron, progressively decreasing the iron stores, with IDA ensuing over time [An active lved IDA ,77,78. Anfection . In theiolve IDA . The patly known , and varly known ,82,83,84r uptake . Kirschever time .H. pylori infection, Giardia infection, IgA deficiency, hypogammaglobulinemia, autoimmune enteropathy, common variable immune deficiency, eosinophilic gastroenteritis, collagenous sprue, and Crohn’s disease [Microscopic enteropathy with an increased number of intestinal IELs can be associated with IDA. The increase in IELs, in addition to being a characteristic histological feature of CD, has been reported in various other disorders such as  disease ,88.Giardia lamblia must be considered as a possible cause of IDA; although giardiasis is rare, its histopathological and serological picture may resemble that of CD. In some cases, G. lamblia can survive for long periods and can lead to partial or complete villous atrophy and crypt hyperplasia, which closely resemble CD [G. lamblia [Potential infection with emble CD ,90. It h lamblia . Giardia lamblia .Other rare causes of anemia that can be associated with CD include hemolysis, myelodysplastic syndrome, chronic renal insufficiency, aplasia (mainly drug-induced), congenital hemoglobinopathy, and erythropoiesis disorders.The factors that could explain the persistence of anemia, and in particular of IDA, in patients with CD despite following a GFD have not yet been identified or clarified. Excluding non-adherence to a GFD as the main cause, the presence of ultrastructural and/or molecular alterations of enterocytes or specific genetic factors would seem to be involved in the persistence of IDA after adopting a GFD and oral iron supplementation.Concerning anemia that is refractory to oral iron supplementation in celiac patients despite the recovery of duodenal mucosa after adopting a GFD, it is conceivable that the ultrastructural and molecular alteration of the enterocytes could play a role. In many patients with CD on a GFD, the histologic evaluation of duodenal biopsies finds no evidence of significant mucosal lesions. However, in some adult patients mucosal healing is not always complete. In a study on 27 pediatric celiac patients (and five controls), Stenling et al. investigated duodenal biopsies by low, medium, and high power scanning electron microscopy, both before and after adopting a GFD. They demonstrated the persistence of ultrastructural lesions in enterocytes, such as disrupted and decreased glycocalyx, and irregular or absent microvilli after adopting a GFD when biopsies were investigated by medium or high power scanning electron microscopy, whereas low power scanning electron microscopy did not detect these lesions. Of the 27 patients (13 girls and 14 boys), seven were between 6 and 30 months, six were between three and seven years, and four were over 10 years at the time of the first biopsy. Dietary instructions were given to the patients and their families by a trained dietician . SimilarUltrastructural and molecular alterations of enterocytes would also appear to be responsible for IDA in patients with non-coeliac gluten sensitivity (NCGS). IDA was found in approximately 20% of patients with NCGS ,97. NCGSSeveral studies have evaluated the possible genetic predisposition to IDA in CD. Barisani et al. evaluated iron regulatory proteins in 25 celiac patients compared to 10 controls and six iron-deficient patients. They collected duodenal biopsies from all these patients and investigated DMT1, DCYTB, ferroportin 1 (FP1), hephaestin, and transferrin receptor 1 (TfR1) expression, as well as iron regulatory protein (IRP) activity. They showed that in all patients, including celiac patients, body iron stores mainly influenced the expression of these proteins. They demonstrated that DMT1, FP1, hephaestin, and TfR1 expression are similarly regulated in CD and non-CD patients. Thus, CD alone did not influence the expression of these proteins involved in iron absorption . SimilarA multifactorial etiology may lead to anemia in celiac patients ,49. At tIn patients with CD and on a gluten-free diet, the persistence of IDA can be due to various conditions, such as a lack of adherence to the GFD, the involuntary intake by the patient of food erroneously contaminated with gluten, to the intake of a low-iron GFD , or to the persistence of atrophy of the intestinal mucosa, which leads to an impairment of iron absorption. In patients with CD and persistent IDA despite a careful GFD, the persistence of villous atrophy should first be excluded by the histology of duodenal biopsies. If mucosal healing has occurred, possible occult blood loss from other parts of the intestinal tract should be investigated by colonoscopy and then bowel capsule endoscopy to exclude other intestinal diseases. In addition, the physiological causes of IDA, such as iron loss through copious menses in fertile women, gastrointestinal occult blood loss, and urinary iron losses due to intense exercise-induced hemolysis in athletes, should be considered . Further


An inter-laboratory proficiency test was organized to assess the ability of participants to perform shotgun metagenomic sequencing of cold smoked salmon, experimentally spiked with a mock community composed of six bacteria, one parasite, one yeast, one DNA, and two RNA viruses. Each participant applied its in-house wet-lab workflow(s) to obtain the metagenomic dataset(s), which were then collected and analyzed using MG-RAST. A total of 27 datasets were analyzed. Sample pre-processing, DNA extraction protocol, library preparation kit, and sequencing platform, influenced the abundance of specific microorganisms of the mock community. Our results highlight that despite differences in wet-lab protocols, the reads corresponding to the mock community members spiked in the cold smoked salmon, were both detected and quantified in terms of relative abundance, in the metagenomic datasets, proving the suitability of shotgun metagenomic sequencing as a genomic tool to detect microorganisms belonging to different domains in the same food matrix. The implementation of standardized wet-lab protocols would highly facilitate the comparability of shotgun metagenomic sequencing dataset across laboratories and sectors. Moreover, there is a need for clearly defining a sequencing reads threshold, to consider pathogens as detected or undetected in a food sample. Foodborne illnesses associated with pathogenic microorganisms are a global public health and economic challenge. In 2018, 26 European Member States reported 5146 food-borne and waterborne outbreaks with 48,365 human cases and 21.2% increase in the overall number of outbreak-related deaths, in comparison to 2017 [Shotgun metagenomics provides the potential for detection, identification, and characterization of pathogens in food ,5, in thShotgun metagenomic sequencing includes a wet-lab part, followed by sequence data analysis. Concerning the wet-lab protocols, comparative studies were performed to assess the implementation of different strategies for sample handling ,9, nucleVarious pipelines for the pre-processing, assembly , clusterwww.compare-europe.eu), involving 11 participants from the EU and Singapore. All participants received the same food sample spiked with a mock community composed of six bacteria, three viruses, one parasite, and one yeast. Each participant processed the sample using in-house wet-lab protocol(s), up to the production of the metagenomic dataset(s). Metagenomic datasets were both submitted to a COMPARE data-hub [To access the suitability of shotgun metagenomics to detect and possibly quantify microorganisms belonging to different domain in a food sample, a proficiency test (PT) was carried out as part of the COMPARE project , using a sterile scalpel and sterile petri dish. An amount of 0.2 g was transferred to individual sterile Nunc screw cap tubes. Subsequently, each tube was kept on ice and spiked with 50 µL of a mock microbial community, consisting of six bacteria, one parasite, one fungus, and 10 µL of heat-inactivated viruses (one DNA and two RNA viruses) .The tubes were kept on ice during preparation. Cell counts of the bacteria and the fungus were determined using a Petroff counting chamber, under a light microscope, counting cells in two diagonal corners on two separately prepared slides. The parasite oocysts were obtained from Waterborne Inc. in PBS, containing 1.2 × 108 cells . After spiking, each tube was vortex-mixed and placed at −80 °C, before shipping to each PT participant with a freezer pack kept at −80 °C, until packaging. The frozen samples were supplemented by another 50 µL of the virus mix, without additional mixing.The wet-lab protocols used by the participants differed from each other. These are summarized in g at 4 °C. The pellet was resuspended in 2 mL of glycine buffer and filtrated using a cascade of 5, 1.2, and 0.45 mm filter pores . The recovered filtrates were incubated for 1 h at 37 °C, with 2000 Units of OmniCleave EndonucleaseTM (Lucigen corporation) and 100 mL of MgCl2 (100 mM). For M30, M31, and M32, the samples were centrifuged at 5000 rpm for 20 min at 4 °C, and the pellet was used for nucleic acid extraction. For M08, the sample was homogenized in liquid nitrogen using a ceramic mortar and pestle, followed by the gradual addition of 2.5 mL TE buffer. The homogenate was then centrifuged at 4000× g for 10 min and the supernatant was filtered on a 0.45-µm disc HPF Millex syringe filter , followed by the nuclease treatment. Subsequently, 200 µL of the sample were used for DNA extraction. For M11 and M13, the samples were homogenized in liquid nitrogen, using a ceramic mortar and pestle, followed by the gradual addition of 2.5 mL TE buffer. The homogenate was then centrifuged at 4000× g for 10 min, and the supernatant was subjected to three cycles of the freeze–thaw method  and submitted to DNA extraction. For M12, the sample was homogenized in liquid nitrogen, using a ceramic mortar and pestle, followed by the gradual addition of 2.5 mL TE buffer. The homogenate was then centrifuged at 4000× g for 10 min, and the supernatant was filtered on a 0.45-µm disc HPF Millex syringe filter , followed by nuclease treatment. Subsequently, 200 µL of the sample was transferred to a new tube and mixed with 600 µL of TRIzol Reagent , 160 µL of chloroform, followed by RNA extraction. Finally, for M27 and M28, the samples were submitted to a disintegration step, using the Covaris cryoPREP CP02 [A total of 27 metagenomics datasets obtained by shotgun metagenomic sequencing of spiked salmon were submitted as part of the PT. Among the 27 samples tested, 7 were not pre-processed and directly submitted to nucleic acid extraction, while 9 were pre-processed by using a bead beating protocol with TissueLyser, with modifications by each participant. Moreover, for M33 and M34, two milliliter of glycine buffer 0.05 M (pH 9) were added to the spiked salmon and homogenized in a potter grinding tube. Then, the pH was decreased to 3 by adding HCL. An equal volume of chloroform-butanol (v/v) was added and mixed by vortexing. After centrifugation, the supernatant was treated with Cat-Floc T  and precipitated with polyethylene glycol 6000 (PEG 6000) , for 1 h at 4 °C, and centrifuged for 20 min at 11,000× For 18 samples, the target nucleic acid was DNA . In 2 sa® miniMAG® . Moreover, in 2 samples, the RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen) and in 4, the RNeasy Mini Kit (Qiagen). For M12, the use of the kit was associated to Tag labeling and Sequence-Independent, Single-Primer-Amplification (SISPA). In 5 samples, the extracted RNA was reverse transcibedusing the cDNA Synthesis System Kit , while in 4, the SuperScript IV Reverse Transcriptase  was used.In 9 samples, the target nucleic acid was RNA . In one ® DNA Library Prep Kit Illumina (n = 3), the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®  (n = 2), and the GeneRead DNA Library kit Qiagen (n = 2)  and in one, the Nextera™ DNA Flex Library Preparation kit  was used. The libraries for the remaining 7 samples were prepared using the TruSeq (n = 2) .All samples were sequenced in paired-end, except two , and thehttps://www.mg-rast.org/mgmain.html?mgpage=project&project=mgp86519 in the MG-RAST server, under the project labelled as Food metagenomic ring trial 2018. The metadata associated with the metagenomic datasets are those detailed in All metagenomic datasets included in this PT are publicly available on The workflows including DNA as the target nucleic acid were exploited in the modelling and bio-statistical analysis described below, while RNA-based metagenomic datasets were only used to evaluate the detection performances, since the lack of information on RNA copy numbers prevented the possibility of computing quantitative comparisons. For the bio-statistical analysis, similar wet-lab steps were categorized together .https://www.mg-rast.org) [Filtering, trimming, and taxonomic classification of raw reads were performed using MG-RAST (ast.org)  and the ast.org) . The staast.org)  and DESeast.org) .Before proceeding with the statistical analysis, the read counts were normalized using DESeq2 , taking The expected proportions of the microorganisms in the mock community and those detected in the metagenomics datasets submitted as part of the PT, were compared using the following approach. The expected relative abundances of the mock community were calculated, based on the cell counts of each spiked microorganism . Such coTo visually compare the expected and the experimentally obtained compositions of the mock community in the DNA metagenomic datasets, we plotted the relative abundances in the form of bar plots. Differences in the mock community composition among samples and between each sample and the expected composition, were also evaluated by computing the Bray-Curtis beta-diversity displayed using the heat map. Samples were then ranked based on their distance  from the expected composition of the mock community.The statistical comparison of the species abundances obtained with different experimental parameters was performed with DESeq2. Specifically, starting from the normalized read counts, DESeq2 was used to compute the negative binomial generalized linear models for each taxon and, hence, to test the impact of the variables of interest.p-value was <0.0001 was read length-sequencing platform . In all other cases, the p-value was ≥0.0001 and both variables were hence included in the model. All in all, the tested variables were pre-processing protocol, DNA extraction kit, library preparation strategy, sequencing depth, and sequencing platform. The advantage of using a multiple regression model that considers all predictors  at the same time is that, when evaluating the effect of one predictor, it adjusts for the effect of all others. The resulting estimates of this model, sometimes called ‘partial effects’, hence, indicate the effect that each experimental variable has on the abundances of a certain species, when all other variables are fixed. Statistical significance was assessed using the Likelihood Ratio Test for multiple comparisons and the Wald’s test for pairwise comparisons. Moreover, the impact of each workflow on the detected mock community microorganisms’ abundances was evaluated by performing a pairwise two-samples t-test between the DESeq2 normalized read counts, obtained with each pair of workflows. For bacteria, the parasite and the yeast, such a t-test was computed by considering the mean value and the variance of the abundances detected in the workflows’ samples. Since only one sample was tested by applying WF12, in this case, the sample mean of each species was set to the value of its abundance in that sample and the sample variance was estimated as the average of the variances observed for that species, in all other workflows. Finally, the sample size was set to the average number of samples present in the other workflows. In this approximated test, we assume that the variance of each microorganism’s abundance among the samples within a workflow is the same for all workflows with only small deviations , as shown in We evaluated a multiple regression model in which we considered all experimental parameters in order to detect the effect of each variable, after adjustment for all others. To select the model variables, we first estimated the relationship between the variables using Fisher’s exact test for categorical data and the slope of a linear regression model for numerical variables. The only pair of variables for which the The reads belonging to different taxonomic domains were quantified in the 27 metagenomics datasets, submitted as part of the PT .C. parvum and S. cerevisiae) ranged between 2.957% (M34) and 80.567% (M11). The reads assigned to the domain bacteria ranged between 18.823% (M11) and 95.202% (M36), and those assigned to viruses ranged between 0.031% (M19) to 38.344% (M33)  . These rThe quantification of the mock community microorganisms, in terms of relative abundance, differed among the DNA metagenomic datasets . As detaC. parvum was detected decreased from 100 to 67% in the metagenomic dataset from RNA, increasing the detection threshold from one to 10 reads. The same result applied to F. nucleatum and P. freudenreichii detected in 100% of the metagenomic datasets from RNA, by considering one read as the detection threshold, while in 78% 10 reads were considered as the detection threshold  followed by M38 , and thiAmong the variables investigated in the workflows, those impacting on the abundance of one or more microorganisms spiked in the salmon were the pre-processing protocol, the DNA extraction protocol, the library preparation strategy, and the sequencing platform.C. parvum, E. coli, F. nucleatum, P. freudenreichii, S. enterica, and S. aureus  .The results for each workflow (WF), starting with DNA as a target nucleic acid were compared by performing a pairwise approximated t-test, in which we tested if the mock community microorganism’s abundances detected by each pair of workflows differed.F. nucleatum. The average abundance of the yeast was significantly higher for the samples processed using WF4, including a bead beating protocol with TissueLyser than for WF6, while for B. fragilis it was the opposite  is a freely available and user-friendly web resource, while more powerful bioinformatic tools could have been used. The outputs of this PT should contribute to speed up the application of shotgun metagenomic in food microbiome studies and in food safety risk assessment, but for both, a quality assurance approach consistent with regulatory standards is mandatory. At present, individual research institutes set the internal reference standards, but the lack of universal reference organisms and genomic material makes it difficult to compare assay performances between different laboratories . Hence, The main methodological constraints to be overcome are the lack of harmonized and validated methods, the low sensitivity in detecting certain taxa in the sample, or the fact that the results obtained strongly depend on the choice of wet-lab workflow and the bioinformatics pipelines, as shown by our results. The scientific community must agree on the basic requirements to consider a dataset meaningful for both bioinformatic and biostatistics analysis. If such requirements would be clearly defined, then different workflows could be applied for the wet-lab part, as far as those requirements are achieved, exploring further protocols to reduce shotgun metagenomic sequencing costs and improving efficiency .In this PT, the RNA viruses were hardly detected or not detected at all in the metagenomic datasets. There are different possible reasons for the observed failure to detect viruses. This is likely caused by the low relative spiking input of the viruses, in comparison to the final overall nucleic acid content, which was intended to resemble a situation of exogenous viral contamination. However, a pilot sequencing of a salmon aliquot after sample preparation with one-sixth (10 µL) of the virus mix, resulted in a very low recovery for the viruses. Therefore, before the samples were sent out to the participants, another 50 µL  was added to the frozen sample. Moreover, it should be mentioned that the necessary heat-inactivation of the virus mix could cause the virus particles to be destroyed, leading to a release of unprotected viral nucleic acids. Ultimately, this could have caused a loss of viral nucleic acids, before sample processing by the participants, due to exposure to the nucleases present in the salmon matrix. To solve the issue of low virus concentration, which is common to food and human samples, it was suggested that an RNA extraction be carried out and then to combine targeted with untargeted sequencing, spiking the variably sized panels  of short primers into the reaction mixtures, at the reverse transcription step . FollowiS. aureus  on the abundances of a certain species, when all other variables were fixed, which did not reflect what happened in real life, where each step of the wet-lab protocol impacts on the following. However, since each participant applied in house wet-lab protocols without restriction, the resulting variables in the metagenomic datasets were many, and the multi regressing modeling approach allowed us to collect meaningful results, species by species. This limitation might explain why sequencing coverage did not turn out to be a key variable affecting the relative abundance of the spiked organisms, although the six metagenomic datasets closest to the expected composition of the mock community were all sequenced at ≥8 Gbp. Clear indications on the sequencing coverage threshold to be achieved in metagenomic investigations are lacking. However, Ni et al., 2013  stated tThe investments in research on shotgun metagenomics are justified by the fact that the results achieved in both human and food sectors translate in the identification of potential pathogens in the ecosystems where those pathogens are in real life, and as a matter of fact, the interaction between pathogens and their ecosystems affects both pathogen survival and multiplication ability ,44. If sIn conclusion, our results showed that there are huge differences in the workflows applied in the wet-lab part of shotgun metagenomic sequencing at the international level, while there is a need for harmonized and validated protocols. Despite the differences between wet-lab protocols, all microorganisms of the mock community, belonging to the different domains  were successfully detected in the metagenomic datasets obtained from spiked samples, suggesting that different wet-lab protocols could be successfully applied to reach the same result. Nonetheless, since the percentages of metagenomic datasets in which the mock community members were detected changed according to the number of reads selected as the detection cut-off level, this parameter should be clearly defined to use shotgun metagenomic sequencing in food microbiome studies, and in food safety risk assessment. There are many valuable papers on the application of shotgun metagenomics, but the lack of transparent information on the technical details of both the wet-lab and bioinformatic procedures are delaying the full implementation of this powerful sequencing approach.


Functional nutrition, which includes the consumption of fruit juices, has become the field of interest for those seeking a healthy lifestyle. Functional nutrition is also of great interest to the food industry, with the aims of improving human health and providing economic prosperity in a sustainable manner. The functional food sector is the most profitable part of the food industry, with a fast-growing market resulting from new sociodemographic trends , which often includes sustainable concepts of food production. Therefore, the demand for hurdle technology in the food industry is growing, along with the consumption of minimally processed foods, not only because this approach inactivates microorganisms in food, but because it can also prolong the shelf life of food products. To preserve food products such as fruit juices, the hurdle technology approach often uses non-thermal methods as alternatives to pasteurization, which can cause a decrease in the nutritional value and quality of the food. Non-thermal technologies are often combined with different hurdles, such as antimicrobial additives, thermal treatment, and ultraviolet or pulsed light, to achieve synergistic effects and overall quality improvements in  juices. Hence, hurdle technology could be a promising approach for the preservation of fruit juices due to its efficiency and low impact on juice quality and characteristics, although all processing parameters still require optimization. Functional foods are products with added active substances, which if consumed in adequate quantities not only provide basic nutrition, but also have a positive impact on general health or contribute to reduction of the risks for developing certain diseases . ConsumeHurdle technology is a set of methods used for inactivation of microorganisms in food preservation. Hurdles are food preservation factors, which are combined to achieve certain food quality and stability, in terms of the temperature, pH, redox potential, water activity, preservatives, and competitive microorganisms. Today, more than sixty hurdles are known to have an effect on food stability and quality, as well as microbial stability . Not allTo preserve food using hurdle technology, it is necessary to create a hostile environment for microorganisms. This environment can either cause the death of microorganisms or slow down their growth, largely depending on how microorganisms react to the treatment. These reactions are being researched as methods of food protection, together with the effects of hurdle technology on homeostasis, stress reactions, metabolic exhaustion of microorganisms, and multitarget preservation, all of which are essential for food safety.The use of only a single technology may cause damage to microorganisms that they may recover from during food storage. However, if this recovery is effectively prevented by using a combination of additional hurdles, the microbial cells will not be able to grow and higher inactivation levels could be reached. In this respect, most of the current research studies are focused on high-power ultrasound (HPU), pulsed electric field (PEF), and high-pressure processing (HPP) . These tIn today’s world, there is a growing trend toward health and fitness. Accordingly, the food industry has directed their efforts towards producing healthier and more nutritious products that will satisfy consumer demands. One way to go about this is to design completely new products, while another way is to add functional ingredients to already existing products. Functional ingredients are substances that are beneficial for our health, e.g., carotenoids, phenolic acids, flavonoids, polyunsaturated fatty acids, probiotics, minerals, and vitamins. Because of the fast-paced Western lifestyle, people have less time to eat fresh fruit and prefer to consume fruit juice instead. Hence, juice represents an excellent vehicle for the delivery of bioactive ingredients.Some fruit juices are already highly valued because of their unique nutritive composition. However, new products in the fruit sector are constantly being designed for several reasons. People are willing to purchase healthier products, such as those that are organically made, those they find appealing, and new and different fruit juices. The demand for functional fruit juices is increasing rapidly. Superfruits with high levels of bioactive compounds, such as goji berries, areola, noni, and acai, are already added to some fruit juices, as well as prebiotics, probiotics, omega-3 fatty acids, botanicals, and isoflavones from soy, in order to make products functional ,10. In 2The economic value of juice production was confirmed over the last 5 years with statistical data from the European Union (EU), where production of juices not made from concentrate increased by 5.4%, while production of freshly squeezed juices grew by 4.8%. Simultaneously, the production of nectars and other types of fruit beverages declined by 3.8% and 1.6%, respectively . In thisFruits and their juices contain significant amounts of different BACs, which individually or mutually have a significant impact on improving human health. The most common types of BACs in the majority of fruit juices are polyphenols, which are antioxidants with anti-inflammatory activities. Polyphenols bind free radicals, participate in the inhibition of pro-oxidative processes, and significantly influence chronic inflammation or lessen the burden of various medical pathologies, such as cardiovascular diseases, forms of cancers ,15,16,17Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus reuteri, Bifidobacterium breve, Lactobacillus gasseri, Lactobacillus crispatus, and others [In addition to the usual nutritional value, in order to provide functionality and added value to fruit juices there is the possibility of adding prebiotics and probiotics, which have been examined recently. The term “probiotic” refers to mixtures made of one or more bacterial species that act positively on the health of the host to which they are introduced by improving the properties of the hosts intestinal microflora ,21,22. Td others . Selectid others ,29. DespMalpighia emarginata D.C.), cashew apple , yellow mombin (Spondias mombin), pineapple (Ananas comosus L.), acai (Euterpe oleracea), and camu-camu (Myrciaria dubia) fruit. The results revealed that the pasteurization did not affect the total polyphenolic content or antioxidant activity of either formulation but caused a 7% reduction of ascorbic acid. A higher carotenoid content in the pasteurized samples as compared to the control sample was explained by the combination of homogenization and heat treatment, which caused disruptions of cell membranes and the protein–carotenoid complex, making carotenoids more accessible for extraction.Currently, consumers demand that fruit juices are minimally processed, contain no additives, and have an extended shelf life. Recent studies have confirmed that consumers are often ready to pay a higher price for premium products of high sensory quality; therefore, there is currently high motivation to develop and produce premium quality juices. Such fruit juices are produced directly by squeezing or cold-pressing and are not subjected to any further processing—they are simply stored at a temperature of 2–5 °C, meaning they last for only a few days (1–3 days). On the other hand, the results of a major study involving over 160 US and EU fruit juice producers stated that microbiological safety is a key factor in product protection within a market. As many as 92% of manufacturers have encountered subsequent contamination (yeast or mold) in their products . FunctioNon-thermal processing technologies are able to extend the shelf life while retaining the nutritional and sensory value. These technologies operate at lower temperatures and with shorter processing times, while assuring microbiological safety, inactivation of the enzymes, and higher stability of the BACs in the juice. Recently, for the purposes of extending the shelf life and retaining the quality of juices, combinations of thermal and non-thermal technologies were tested as “hurdle” technologies. Here, these technologies are applied in a certain sequence, so that each of the non-invasive (“milder”) processing conditions gives the best results in terms of extending the shelf life and sensory quality of fruit juices ,36. In tIn recent decades, there has been an extensive increase in the number of commercially available foods processed using non-thermal technologies. For instance, food products processed using non-thermal technologies such as HPP have been marketed in Japan since 1990 and in the United States and Europe since 1996 . At the −1) in a very short time (μs to ms) to food placed between two electrodes. Here, the main parameters are temperature and time, electric field strength, and energy input [The application of pulsed electric field processing technology as an alternative to pasteurization of fruit juices has shown good results, without significant deterioration of nutritional or sensory juice quality ,42. PEF gy input .Microbial inactivation by PEF is based on the rupture of microbial cells due to differences in the potential outside vs. inside the cells . When thThe efficiency of PEF depends on the experimental conditions, food composition, and electrical conductivity of the food. If the conductivity is higher than 6 μs, the food is harder to process with this technology . AdditioLiquid foods without bubbles are the most suitable for PEF treatment due to the presence of charged molecules that can transfer electric charges formed during treatment . This meLycopersicum esculentum cv. Raf) increased concentrations of individual carotenoids  [Many studies have confirmed the superior polyphenolic, mineral, and free amino acid contents, as well as better preservation of native color in PEF-treated juices as compared to thermally treated juices. High-intensity PEF (HIPEF) was able to improve the bioaccessibility of bioactive antioxidants in several functional beverages ,48. Recearotene) . In partarotene) . On the arotene) . In concAs mentioned earlier, PEF can inactivate microorganisms in juices while retaining quality , which makes it an advantageous method for preserving foods over an extended shelf life ,58. The Another study aimed to evaluate and compare the effects of processing via high-intensity pulsed electric fields (HIPEF) vs. thermal pasteurization 90 °C/60 s) on the nutritive and physicochemical parameters of date juice (variety Bou-Hattem) before and after storage for 5 weeks at 4 °C. In comparison to pasteurization, HIPEF better preserved the physicochemical characteristics and even caused an increase in the concentrations of some bioactive compounds in fruit juices. Moreover, HIPEF-treated date juices, in comparison to untreated and thermally processed samples, retained quality better during storage. A possible reason could be the improved extraction of intracellular contents due to permeabilization of plant cells, which is enhanced by PEF  s on the.Non-enzymatic browning reactions were slowed down by PEF, which resulted in a low concentration of hydroxymethylfurfural (HMF) in treated date juice. HMF, as an indicator of non-enzymic browning, is probably a result of Maillard reaction in the presence of a reducing sugars  and acids under high temperature . The slo−1; “PEF Low” pulse numbers of 243, and “PEF High” of 1033) and subsequently macerated. The grape juice treated with PEF had better yield during the first four days of maceration than untreated juice. Enhancement of juice yield and improvement of mass transport of juice from the grape pulp is credited to the PEF permeabilization of cell membranes. Juices that were processed with “PEF low” had increased vitamin C content during the first two days of maceration. However, when the maceration was longer than 8 days, PEF-treated juices exhibited higher degradation of vitamin C than non-treated juices. It seems that the higher release of vitamin C in early maceration increased the available total vitamin C for oxidation due to the higher susceptibility to oxidation and degradation with prolonged maceration. “PEF high” samples showed an increase of total phenols after two days of maceration. “PEF low” and “PEF high” juices had higher contents of malvidin-3-O-glucoside (anthocyanin) after eight days of maceration as compared to control samples, suggesting that PEF has the potential to maximize the release of phenolics much more effectively than conventional maceration alone. In addition, the maceration process was successfully shortened from 14 to 8 days, thus confirming the potential of PEF in grape juice processing [Another study on Pinot noir grape juice was done to evaluate exposure to PEF and the effects this has on the quality and nutritive value . Grapes have a number of anthocyanins, phenolic acids, flavonols, flavan-3-ols, and stilbenes. Prolonged maceration is commonly used to increase the concentrations of these compounds, which are released from grape skins, while PEF can be used to accelerate this process. To that end, after preparation the grape juice was treated with PEF  at low frequencies (20–100 kHz). Due to the high power, ultrasonic waves in juice cause physiochemical changes, accelerating biochemical reactions and causing enzymatic and microbial inactivation [The use of ultrasound (US) as an alternative to pasteurization in fruit juices has great potential, as the acoustic energy is immediately transmitted through the entire volume of juice, which significantly shortens the treatment time. In addition, its lower energy consumption and potentially lower costs makes this method appropriate for industrial usage . Fruit jtivation . Expansitivation . The coltivation . Studiestivation ,67. The tivation . This tetivation ,69. TablHPU can provoke microstructure changes of treated juice, particularly the rupturing of cellular walls. More precisely, the physical force of ultrasound causes the dispersion of the intracellular components into the juice, causing higher injury to the cellular structures, which occurred through elongating the HPU processing time . These sThe addition of various prebiotic carbohydrates in orange juice, such as xylooligosaccharides (XOS), has been introduced through high-intensity ultrasound (HIUS) for the engineering of functional beverages with similar sensory characteristics to those of fresh products . The quaHPU treatment may prevent the activity of browning enzymes such as PPO and POD, maintain the taste and nutritional value, and the improve stability of juice with respect to conventional heat treatment . The effThe positive aspects of ultrasound treatment were documented in a study on the effects of HPU on passionfruit juice. The juice was treated with a frequency of 20 kHz for 10 min, after which it was stored with untreated samples at 4 and 10 °C for 10 days. Samples were taken every other day and their color, pH, ascorbic acid content, and microbial count were measured. Results of this experiment showed that HPU did not cause sufficient cell damage to start immediate microbial inactivation. However, sonication treatment successfully reduced the aerobic mesophilic bacteria and yeast counts. Due to HPU treatment, the color parameters lightness (L*), a* (redness), and yellowness (b*) of passionfruit juice changed and the color was lighter and greener. The degradation of ascorbic acid was significant, but was lower than the decrease caused by pasteurization, hence making the HPU treatment a better method for preservation of fruit juices. Further studies showed that the juice was stable for 10 days after sonication because there were no significant changes in microbial quality of the product, as opposed to non-sonicated samples . Aside fNon-clarified strawberry juice was exposed to HPU and thermal treatment (TT) in order to compare their influences of the quality and nutritional value during 10 days of storage. The evaluated properties were color, total soluble solids, total acidity, microbial growth, total phenolic content, and antioxidant activity (DPPH assay). The juice was sonicated at 40 kHz for 10 and 30 min at 90 °C for 60 s. All HPU samples showed a constant color right after the treatments, which started to degrade during storage (the largest drop was observed on the third day). TT samples had higher color changes after the treatment compared with untreated samples, but after the initial change the color remained stable over the storage period. All samples showed no “off odors”, but HPU samples were sweeter than TT juice, probably due to the breakage of cellular structures that contained sugars. HPU and TT samples had similar total soluble solid contents, total acidity, and nutritive value, while TT inhibited microbial growth better than HPU .Conversely, the negative side effects of ultrasound on juice quality can include changes of color, viscosity, formation of free radicals and off flavors, as well as degradation of lipids. In some juices, sonication reduced the amount of ascorbic acid, anthocyanins, and carotenoids. These factors, as well as the cost of ultrasound treatment, affect (and limit) the application of ultrasound in the food industry .Due to the low lethality of ultrasound, a number of microorganisms, especially bacteria spores, can survive HPU treatment. To insure a higher lethality, HPU is often combined with pressure, heat, or both at the same time. These combinations are more energy-efficient than ultrasound alone, but cannot be used for preservation of thermosensitive products. For example, thermosonication can accelerate enzymatic inactivation and sterilization by shortening the treatment time and decreasing the intensity of sterilization and the corresponding negative side effects . MoreoveHigh-pressure processing is considered the best non-thermal method used for food preservation. Food products that can be processed with HPP are low- to medium-moisture semisolids and solids (vacuum packed), high-moisture solids (vacuum packed), and high-moisture liquid food (bottled). Products are packed before the process, which decreases the chance of postcontamination. After this, products are placed in baskets and lowered into a vessel. The vessel is then filled with water and the products are treated with high hydrostatic pressure, which can be as high as 800 MPa. The pressure is evenly spread throughout the vessel, which assures that the product will be properly processed . By applGovernmental authorities in the USA, Europe, and Canada have approved the use of HPP for reducing pathogens in juices and extending their shelf life. With an annual growth of at least 1%, fruit beverages show the fastest growth in the HPP food market, so such products may hold a higher market share than meat products that utilize HPP technology. However, the main technological limitations of HHP are the high investment costs for the equipment and the batch operation mode. The application of HPP for the preservation of functional fruit juices and beverages over the last 5 years is outlined in Lactobacillus rhamnoses GG probiotic bacteria were preserved. Some studies even show that after 30 days of storage, the bacteria had a content of over 108 CFU/mL, with a well-preserved nutritive profile. Hence, it is not surprising that consumers showed tendencies toward purchasing HPP-treated probiotic products rather than thermally treated products [When HPP  was compared with pasteurization , reduced numbers of bacteria and yeast were shown in mango juice, and the shelf life of the juice was prolonged for up to 60 days while stored at 4 °C. Furthermore, losses of ascorbic acid, carotenoids, and total phenols were reduced as compared to control samples . HPP hasThe inactivation of quality degradation by endogenous enzymes in fruit juices is needed to obtain a high-quality product. The effects of HPP on the inactivation of various enzymes in fruit juices, e.g., PPO, POD, pectinmethylesterase (PME), β-glucosidase, polygalacturonase, lipoxygenase, amylase, and hydroperoxide lyase, have been extensively studied . One recBioaccessibility is another important nutritive parameter for evaluation of functional food products as fruit juices. A recent study confirmed that the bioaccessibility of phenolics in fruit juice may be enhanced by modulation of the process parameters and the composition of the food matrix. A comparison of the high-pressure homogenization  and T80 (80 °C/30 min) and T90 (90 °C/30 s) thermal treatments with the addition of milk was associated with phenolic bioaccessibility and activity in kiwi and pomelo juices. Both HPP and TT increased the contents of phenols in both juices, although the T80 sample was an exception because of the phenolic degradation in pomelo juice. Unfortunately, the bioaccessibility of free and total phenols did not increase as a result of the applied treatments. Moreover, in some samples the total phenolic content even decreased, while the addition of milk improved the bioaccessibility of free and total phenols. Here, the exception was soy milk, where the number of total phenols decreased. In kiwi juice samples, the best bioaccessibility was with the addition of semiskimmed milk, while in pomelo juice this resulted from the addition of the whole milk. The authors explained that the improvements were due to the fact that some phenols can create bonds with milk proteins, while others can create bonds with lipids. To conclude, under the tested circumstances the kiwi juice had better phenolic activity and bioaccessibility than pomelo juice .Green asparagus juice was tested in terms of its microbial contamination, physiochemical properties, antioxidant activity, and changes in BAC content in HPP vs. TT treatments  and control (no treatment). The researchers used HPP at 200, 400, and 600 MPa for 10 and 20 min. HPP at 400 and 600 MPa reduced the number of microbes  to the same values as the TT. Moreover, the physiochemical properties and pH did not change during the HPP treatment, which had more BACs  in comparison to the TT .Apple juice samples (elephant apple) were treated with HPP (600 MPa/5 min) and TT (80 °C/60 s) for extended storage . SamplesCloudy apple juice was treated with HPP (600 MPa for 3 min) and TT (85 °C for 5 min) to examine changes in the quality and nutritive value. TT caused significant color changes, with an increase in L* and decreases in b* and a* CIELab values. Sugars and acids in the samples remained similar after both treatments. TT-treated juices showed higher amounts of aldehydes, ketones, alkanes, alcohols, and organosulfur compounds than HPP and untreated samples did. These likely resulted from Maillard and oxidative reactions, which are propagated with heating. The results showed that better retention of natural juice color was possible in HPP samples, while TT samples showed better inactivation of PPO and POD .The advantages of using HPP in the food industry, aside from food conservation, include improved antioxidant properties in food components. For example, in strawberry juice, when both treatments HPP and TT are combined, this will decrease the total monomeric anthocyanin (TMA) and vitamin C contents. However, results showed that the TMA and vitamin C contents did not decrease with HPP but did during TT . AnotherEscherischia coli in strawberry juice. Samples were treated with HPP at 200–400 MPa for 2 min; HPU with 120 μm, 24 kHz at T = 25–55 °C; PEF with electric field intensity at 25–35 kV cm−1 and treatment time = 5–27 μs; and TT at T = 72 °C. The focus was on the processing conditions needed for each technology to give a 5-log microbial reduction from a starting inoculum level of roughly 106 CFUmL−1. HPP gave 5.75-log reduction with 300 MPa|t = 1 min; HPU (thermosonication) gave a 5.69-log reduction with 55 °C, t = 3 min; PEF gave a 5.53-log reduction with 35 kV cm−1, t = 27 μs, 350 mL/min flow rate, 2 μs pulse width in monopolar mode; and TT contained no detectable E. coli. The authors concluded that the above non-thermal parameters had crucial roles in the microbial reduction ability, which was equivalent to thermal pasteurization.The HPP, HPU, and PEF samples were tested for their ability to reduce the power of the microbial load of trans lycopene decreased, while cis-lycopene isomers increased. The best HPP treatment was achieved at 200 MP, with a total lycopene content of 776.94 μg/100 g. The best HPU treatment was at 400 W, with a total lycopene content of 920.52 μg/100 g. The β-carotene and ζ-carotene contents were higher in HPP and HPU than in untreated samples, while HPP and HPU showed slight increases in the bioaccessibility of carotenoids. The highest lycopene bioaccessibility in HPP samples was obtained for all-trans (6.86%) and isomers (13.35%) at 500 MPa. In HPU samples, bioaccessibility peaks for all-trans (9.71%) and of isomers (15.82%) were achieved at 800 W. Afterwards, the HPP lycopene was fully liberated from the protein bonds and well dispersed in the tomato juice, making it fully available. On the other hand, ultrasound treatment caused less damage to the cell membranes, which resulted in lower pigment release. Therefore, it was concluded that HPP increases the antioxidant activity, while HPU slightly decreases it [The two technologies (HPP and HPU) were compared and tomato juice was treated with both to see which process had a better effect on the stability and in vitro bioaccessibility of carotenoids. Tomato juice was subjected to different HPP treatments at 200, 300, 400, and 500 MPa. HPU samples were treated with 200, 400, 600, and 800 W, with a constant frequency of 25 Hz. With the increase of pressure, the contents of all-eases it , which wLactobacillus species). These are valid methods for extending the shelf life and retaining the quality of a juice. The specificity of the hurdle concept is reflected in the synergistic effect of various mechanisms for inhibition or inactivation of target microorganisms [In fruit juice manufacturing, important hurdle methods can be combined, which include: (i) application of low or high temperature; (ii) manipulation with water activity; (iii) acidity; (iv) redox potential; (v) application of chemical or natural preservatives; and (vi) application of competitive microorganisms . CDs are allowed as additives in the food industry as natural antibrowning agents in fruit and vegetables juices . The α- The concentrations of α- and β-CDs that were added to the juice were 10, 30, and 40 mM; and 5, 10, and 15 mM, respectively. The juice samples were exposed to HPP and browning was measured with the CIELab method. The control sample was untreated apple juice, while the rest of the samples were treated with HPP at 300, 400, and 500 MPa for 5 min at T = 22 °C. The browning index was calculated from L*, a*, and b* parameters, and phenolic compounds were measured using UHPLC. The results showed that HPP treatment did not cause color changes. Phenolic levels were higher in HPP samples, as increased pressure fostered extraction process. The majority of samples that contained 30 mM of α-CD had the least browning, while samples with 10 mM of α-CD showed the same effect but at 400–500 MPa. It was concluded that browning enzymes degraded with exposure to HPP and with the addition of α-CD, with the best results reached at 500 MPa exposure and 30 mM α-CD. For β-CD, the best results were observed with 15 mM, as higher concentrations were not tested due to limited water solubility. It was recommended that the use of β-CD can be accompanied by processing at 500 MPa [Bacillus subtilis spores were studied in Ringer solution at different pH values (4 and 7), sugar levels (5% and 10%), and conductivity levels (4 and 15 mScm−1), as well as under various PEF operating conditions regarding the specific energy (up to 350 kJ/kg), electric field strength (6–11 kV/cm), and inlet temperature . The results revealed that the higher inlet temperature (80 °C) at 9 kV/cm and 10% of sugar content, which increased the electrical conductivity of the medium, was sufficient for successful microbial inactivation (3-log reduction) [Bacillus subtilis spores are more heat-sensitive in acidic environments [Some studies have shown limited inactivation of bacterial endospores under PEF treatment; however, when combined with thermal energy, successful inactivation could be achieved . Inactivduction) . Additioronments .E. coli O157:H7. Blueberry juice was treated with different temperatures  and power settings  for 10 min. Afterwards, samples were treated with HT (80 °C), TS (40 °C/560 W), MT (350 MPa/40 °C), MS (560 W/5 min/350 MPa), or MTS  for 5, 10, 15, and 20 min. Firstly, HT inactivated PPO (−2% after 5 min) and E. coli well, with 80% retention of anthocyanins. TS exposure slightly decreased the E. coli load by 0.17 log, with 23% PPO activity and 99% preservation of anthocyanins after the same length of time as for HT. MTS preserved 98% of anthocyanins and showed a 5.85-log reduction, with 11% PPO activity. Hence, combined treatment with sonication, high pressure, and mild thermal treatments has potential for the production of safe and nutritious blueberry juice [The combined application of various technologies was further researched in blueberry juice . The aut−1, 600 μs, T < 45 °C), and subsequently the juice sample was sonicated using an ultrasound bath cleaner . The obtained results revealed that the acidity, soluble solids content, pH, electrical conductivity, and color parameters after treatment were not significantly changed in comparison to the control (untreated sample). As this hurdle concept resulted in decreased viscosity and increased cloud values as compared to control samples, the authors concluded that combined treatment with PEF and ultrasound might be used for the processing of grapefruit juice.The combined effects of ultrasound and PEF on quality of grapefruit juice were studied . Grape jAliclyclobacillus acidoterrestris spores in apple juice. The juice was pretreated at 600 MPa for 15 min to weaken the spores. After the pressure treatment, the partially damaged spores were exposed to power ultrasound at 24 kHz and 20.2 WmL−1 for 60 min to reach the final spore reduction of 4.2 log. Nevertheless, the HPP-75 °C treatment was found to be the most effective for inactivating spores, resulting in 3.3-log reductions after 10 min vs. no inactivation for thermosonication (TS) and thermal processing [A recent study combined the use of HHP and ultrasound at 75 °C to achieve inactivation of ocessing .M. domestica cv. Fuji) were evaluated [The effects of combined treatment with US and HPP on enzymes , physicochemical properties, bioactive compounds, antioxidant activity, and microorganisms in apple juice , and the addition of essential oil components -limonene) were employed to reduce spoilage in a clear apple juice [Leuconostoc spp. and five Saccharomyces spp. were tested, with different inactivation levels achieved for each treatment and strain. The authors concluded that combined processes could be useful alternatives for prolonging the shelf life of food products after moderate-intensity heat treatment, which would diminish the undesirable effects of high temperatures on food quality.A combination of different hurdles, such as mild heat (54 °C for 10 min) or PEF treatments  sole UV-C; (ii) sole HPU; (iii) exposure of 25 min of HPU and 5 min of UV-C radiation; (iv) exposure of 5 min of HPU and 25 min of UV-C; (v) exposure of 5 min of UV-C and 25 min of HPU; (vi) exposure to TT at 95 °C (control); and (vii) exposure of TS in a thermostatic water bath at 95 °C. Spore inactivation was higher when ultrasound and thermal treatment were applied in combination than when each procedure was used separately. The use of UV-C alone severely reduced the number of spores by 5 logs in a short period of time. For the above parameters, the combined treatment (i) showed around a 5-log reduction; (ii) showed a low 1-log reduction; (iii) showed a 2-log reduction; (iv) showed a 5-log reduction; (v) showed a 4-log reduction; (vi) showed a 4-log reduction; and (vii) showed a 5-log reduction. HPU treatment alone had the lowest ability to reduce the microbial count, while thermal treatment (control) was comparable to sole use of UV-C, sole use of thermosonication, and combined treatment with 5 min of HPU and 25 min of UV-C. It was concluded that the results of this study provided new options for assuring food safety and quality in food manufacturing.to 70 °C . Acidic Saccharomyces cerevisiae and E. coli in prickly pear juices. They found that S. cerevisiae was more resilient to pH reduction and addition of preservatives than E. coli, so the PEF was mainly used to inactivate S. cerevisiae. Interestingly, here pH was not just used to inactivate microorganisms, but also to increase their sensitivity to PEF treatment. The highest reduction of activity was accomplished with treatment parameters of 15 ls, 50 Hz, 36 kV cm−1 [In 2015, a group of Mexican and Spanish scientists used combined alterations in pH, preservatives, and PEF treatment to inactivate  kV cm−1 .−1 with 4 µs bipolar pulses at 200 Hz for 1800 µs, HPP conditions were 400 MPa at 40 °C for 5 min, while TT was performed at 90 °C t = 1 min. After the treatments, the bioaccessibility of all carotenoids was reduced in the samples, except in the case of cis-violaxanthin and neoxanthin, which increased by 79% in PEF and HPP samples. Losses of carotenoids were the highest during the TT, while samples with milk had the best results for bioaccessibility.To test how technology modifies bioaccessibility, researchers mixed 75% of blended fruit juice  with 17.5% of water, milk, or soy milk and 7.5% of sugar, then the mixture was treated with PEF, HPP, or TT to evaluate the influence of carotenoids and antioxidant activity on bioaccessibility. PEF conditions were 35 kV cmJuices are very important food commodities in the global market. Their preservation needs to account for microbial safety and thermally sensitive biologically active compounds. Pasteurization is a thermal treatment that has assured microbial safety in the past, but not without losses of the nutritive value of the juices. The hurdle technology approach, involving the combination of various preservation factors, has the potential to satisfy both aspects and to safely preserve the natural value of foods over extended periods of time.Functional juices can be engineered from a number of biologically active ingredients, such as polyphenols, which have known antioxidative activity; or probiotic lactic acid bacteria, which need to be processed so that their positive aspects are retained in the final product.Current markets demand juices with native nutritive value, sensory appeal, and an extended shelf life, but without additives. It is difficult to preserve products in such a way with the usual thermal treatments , as quite often higher temperatures have detrimental influences on the nutritive value of foods. Hence, there is an ongoing need to combine alternative non-thermal technologies for juice preservation. The pulsed electric field approach is an electrotechnology and a good option for the preservation of liquid foods that is able to ensure microbial safety and preserve the nutritive value of foods. However, it might need to be combined with other methods to achieve full microbial inactivation. Ultrasound-based technologies are valid options for microbial inactivation in juices, however as with PEF, sole application of such technology is not sufficient to achieve inactivation of microbes.To reach the full potential of this technology, it is often combined with short thermal treatments to achieve complete product safety. Although it is generally good for preserving the nutritive and sensory attributes of foods, ultrasound still can generate “off flavors” in juices, undesired changes in viscosity, formation of free radicals, and lipid degradation ,128. HigAlthough there have been numerous attempts to identify the ideal combination of technologies and their parameters in the processing of juices, there is still a gap in the research in terms of combining two of the most promising hurdle technology techniques, such as high-pressure processing and pulsed electric field techniques. This is somewhat unexpected, as both of these technologies are sustainable, selective, and thermally sensitive—processing factors that are currently cherished in food manufacturing. Some shortcomings that need to be overcome with these technologies include improvements in microbial inactivation, larger numbers of tests at the industrial scale, lowering implementation costs, and providing more funding to the scientific community for experimentation with innovative industry solutions.