Mediterranean Milk Ladder: Integrating a Healthy Eating Plan While Reintroducing Cow’s Milk

The process of gradually reintroducing food allergens into an individual’s diet is referred to as a food allergen “ladder”, and the most recent edition of the original Milk Allergy in Primary (MAP) Care Guidelines, as well as the International Milk Allergy in Primary Care (IMAP), includes a shortened, improved, and international version with specific recipes, indicating the exact milk protein content, as well as the duration of heating and the temperature for each step of the ladder. Food allergen ladders are being used increasingly in clinical practice. The aim of this study was to develop a Mediterranean milk ladder based on the principles of the Mediterranean eating pattern. The protein content delivered in a portion of the final food product in each step of the ladder in the Mediterranean version corresponds to that provided in the IMAP ladder. Different recipes for the various steps were provided to increase acceptability and variety. Quantification of the total milk protein, casein content, and beta-lactoglobulin by Enzyme-linked immunosorbent assay (ELISA) could detect the gradual increase in concentrations, but the accuracy of the method was affected by the presence of the other ingredients in the mixtures. When developing the Mediterranean milk ladder, a key consideration was to reduce the amount of sugar by using limited amounts of brown sugar and substituting sugar with fresh fruit juice or honey for children aged older than one year. The proposed Mediterranean milk ladder includes principles of (a) healthy eating based on the Mediterranean diet and (b) the acceptability of foods across different age groups.


Introduction
The EuroPrevall birth cohort study spanning nine European countries indicated that 0.54% of children present a challenge-proven cow's milk allergy (CMA) [1]. National incidences are reported to range from 1% (in the Netherlands and United Kingdom) to <0.3% (in Lithuania, Germany and Greece) [1]. Children diagnosed with IgE and non-IgE mediated CMA generally have a good prognosis, with successful reintroduction of particularly baked forms of milk in the first five years of life [2,3]. Delaying the reintroduction of cow's milk can lead to a decrease in the quality of life of both the children and their families, as well as impairment of the children's physical health [4][5][6].
The timing and method of reintroduction of cow's milk is based on the type of allergy (IgE or non-IgE), clinical presentation, history of ingestion or accidental ingestion, and laboratory findings, when relevant [7][8][9]. The process of gradual reintroduction of food allergens into an individual's diet, in order to facilitate the development of natural tolerance, is referred to as a food allergen "ladder" and was historically used in mild to moderate non-IgE mediated food allergies only [8,10], but more recent data support its safe use in IgE mediated food allergies as well [11].
Food allergen ladders are being used increasingly in clinical practice, when it is assumed that the allergy is resolving, but the levels of allergens in many cases are uncharacterized, and probably depend on the degree of heating and/or processing of the milk [12]. Caretakers should consult their physician and/or dietitian for reintroduction guidance, including recipes, steps of reintroduction, and identification and treatment of allergic symptoms, as well as for advice on the importance of carrying rescue medication in the case of IgE mediated food allergies [13][14][15]. In addition to the currently available ladders, there is a need for standardized food ladders that include culturally appropriate, healthy food items for successful and safe introduction of food allergens, depending on the region [16,17].
For reintroduction of milk, specifically, although it is generally considered safe to carry out this process outside of the office or hospital setting, and, in some countries [18][19][20], this demands a careful selection of patients based on their type of allergy, specific IgE levels, and prognosis of resolution [21]. The original Milk Allergy in Primary Care (MAP) Guidelines were transformed into the International Milk Allergy in Primary Care (IMAP) Guidelines in 2017, which is a shortened, improved, and international version of the guidelines, with specific recipes, indicating the exact milk protein content, and the recommended duration and temperature of heating for each step [8].
The aim of the current study was to develop a Mediterranean milk ladder based on the principles of the Mediterranean eating pattern as a healthy version of the IMAP for milk reintroduction in the Mediterranean region.

Recipes Development
Based on a bilingual Mediterranean recipe book dedicated to people with food allergy [22], we aimed to develop recipes with local ingredients that are representative of the Mediterranean diet, including whole grain flour, brown rice, olive oil, fresh and dried fruit, legumes, and vegetables.
The initial design and selection of the recipes for the ladder was based on their total milk protein content, as well as the temperature and duration of heating. At least two different recipes were prepared for each step in an attempt to increase the variety and the acceptability and for users. The protein content delivered in a portion of the food products finally selected for each step of the ladder corresponded to that provided in the IMAP ladder.

Milk Protein Content Analysis
Two samples from each recipe were analyzed for their total milk protein content with the Veratox Total Milk Allergen Enzyme-linked immunosorbent assay (ELISA) kit for casein with Veratox Casein Allergens ELISA Kit and for beta-lactoglobulin by SENSISpec ELISA Beta-Lactoglobulin Eurofins, provided by the Food Allergens Laboratory (www. foodallergenslab.com, accessed on 10 September 2022). The aim was to establish both the total milk protein content and the levels of the components that are denatured by heating, i.e., beta-lactoglobulin and the stable components, i.e., casein in our recipes [8,9].

Results
The recipes, as shown in Table 1, were created, based on the principal steps outlined above, to reflect culturally appropriate foods, and they contain local ingredients highlighted in the traditional Mediterranean diet. These ingredients include: vegetables, such as carrots, tomatoes and onions; fresh fruit, such as banana and dried fruit, such as raisins; whole grains, such as whole meal flour, oats and brown rice; and non-animal protein sources, such as lentils. Preheat the oven to 180 • C, in the air setting 6. Cover a baking tray with non-stick paper. 7. Form 10 small balls with your hands and press them in the middle. 8. You will make 10 cookies of 55 g each (~40 g raw). 9. Bake for about 12-15 min, until they turn light brown at the edges. 10. Take them out of the oven and let them cool down completely.

Sweet whole wheat muffins with berries or raisins.
Ingredients 250 g whole grain flour 1 tsp baking soda 6. Add the grated cheese and mix. 7. Cover the bowl and chill in the fridge for an hour. 8. Shape into small balls and place them in a baking tray. 9. Bake in a preheated oven at 180 • C for 50 min.

g
Served portion

Preparation
1. Heat the milk on medium heat, stirring continuously so that it does not stick, at 90 • C if it is not pasteurized, or at 52 • C if it is pasteurized. You will need a kitchen thermometer, but if you do not have one, you can turn off the heat just before the unpasteurized milk starts to boil, that is, as soon as we see it that it starts to swell or for the pasteurized milk when it starts to steam (~52 • C). 2. Dissolve the traditional sheep yogurt without the skin in a bowl in 10 tablespoons of warn milk from the pot, so that it becomes smooth, without pieces, then pour it into the rest of the milk and stir the milk slightly.
3. Place the pot immediately after in a warm place, cover it with a clean towel and then a lid, and leave it still for three to five hours. Alternatively, you can put it uncovered in the oven, preheated to 45-50 • C. Caution: do not shake or move the utensil during coagulation. Leave it for at least two to three hours without moving it. When it is ready, place the yogurt in the refrigerator, and consume it within 10 days. Saturated fats (butter) and trans-fats (margarines) were avoided. Avocado and olive oil, key elements of the Mediterranean diet, rich in monounsaturated fatty acids, flavonoids, and polyphenols, were used in the recipes. Sugar and refined carbohydrates were used sparingly. A seven-step milk ladder was created, including the last step with the pasteurized milk. The recipes included thirteen foods for increased dietary variety during milk reintroduction, with alternatives to increase the acceptability and palatability for the users.
Quantification of the total milk protein, casein content, and beta-lactoglobulin with ELISA was able to detect the gradual increase in the concentrations, but the accuracy of the method was affected by the presence of the other ingredients in the mixtures, such as wheat, grains, and fat (Table 2). 3.2 n/a n/a n/a n/a n/a * estimated portion sizes for preschoolers [23]; ** reduction in casein and beta-lactoglobulin levels may not indicate that the allergens are destroyed but may indicate reduced bioaccessibility. n/a: non-applicable. *** ELISA: Enzyme-linked immunosorbent assay.

Discussion
We developed a Mediterranean milk ladder that includes foods based on the principles of the Mediterranean eating pattern [24][25][26]. By developing a milk ladder rooted in the traditions of the Mediterranean eating pattern, not only are we providing a format for the reintroduction of milk at home, but we are doing so in a way that includes local foods that have healthy attributes associated with prevention of chronic non-communicable diseases [24][25][26][27].
A standardized approach was used, including three key components: the food allergen, nutritional factors, and medical aspects [11].
The milk ladder was designed by following the principal step outlined in the IMAP ladder. We considered the dose of milk protein required and the amount of milk needed to reach the appropriate dose for each step, followed by the presence of a wheat matrix for the initial steps, as well as the duration and temperature of heating of the milk in the preparation of the recipe. The recipes also needed to be simple in preparation and contain ingredients that would be culturally accepted and familiar to the users.
The original MAP ladder, when evaluated by users and their carers, which produced criticism of the high amounts of sugars and saturated fat used in the recipes [28]. These were in contrast to healthy eating guidelines, which promote high amounts of fruit, vegetables, and whole grains, as well as monounsaturated and polyunsaturated fats rather that saturated fats. When developing the Mediterranean milk ladder, a key consideration was reduction of the amount of sugar, which was achieved by using limited amounts of brown sugar and substituting sugar with fresh fruit juice or honey for children older than one year of age [29]. As the ladder is being used in a younger population, we also wanted to ensure that the taste and texture of the foods included would be palatable to the target population while continuing to meet the key components of Mediterranean eating pattern.
The Mediterranean milk ladder is intended to be used for children with milk allergy under the supervision of a healthcare professional. The concept of milk-reintroduction through the ladder is similar to that used in the original IMAP [8]. Briefly, the healthcare professional decides when it is appropriate for the child for milk re-introduction based on his/her clinical history and symptoms. In collaboration with the parent/carer, the healthcare professional must identify the appropriate step at which the individual child should start, as some children may already include one or more forms of treated milk in their diet, for instance small portions of baked milk in cake or cookies. Before starting the ladder, the child must feel well and be free of abdominal symptoms, bowel symptoms, and eczema.
Although the Mediterranean milk ladder recommended here consists of seven steps, the healthcare professional may adjust the number of steps or the time spent on each step. The estimated portion size in each step is provided as a guide for preschoolers [11], but this may need personalized modification based on the age and development of the individual child. Finally, when a child tolerates a food at any particular step, he/she should continue to consume this food in addition to foods from the previous steps. Conversely, when a food is not tolerated, the child should go back to the previous tolerated step, and the healthcare professional should advise on the time that this step can be tried again.

Conclusions
CMA is among the most common of food allergies worldwide. We propose here a Mediterranean milk ladder, which includes principles of (a) healthy eating based on the Mediterranean diet, as well as (b) acceptability of foods across different ages based on regional eating habits. We support the concept that this model of milk introduction provides the dietary variety needed for continuous consumption of milk during each step of the ladder, along with a healthy eating pattern that has immunomodulatory effects, which might play a role in the mitigation of the allergy outcomes [30,31]. Investigation of future application in clinical practice is needed to provide supportive evidence.