Nutrients 2023 , 15 , 3466
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as having poor sleep quality if the total score was >5 and good sleep quality with a valueof ≤ 5, thus lower scores indicate a better sleep quality. The Competency Assessment (COMPASS) battery of different cognitive assessment tools was used to investigate cognitive performance before and after the intervention. COMPASS was developed by the Brain, Performance, and Nutrition Research Centre, Northumbria University, and is commercially available (https://www.cognitivetesting.co. uk, accessed on 2 August 2023). It has been used extensively to assess the cognitive benefits of dietary supplements, nutritional compounds, and probiotics. Five tests from COMPASS were used in the study. Attention and vigilance were tested using the four choice reaction time test (FCRT) and rapid visual information processing test (RVIP). In both, an increased percentage of correct responses and decreased reaction time indicate an improvement. The FCRT shows arrows on a screen pointing at different locations, and the subjects are requested to indicate the direction of the arrows as fast as possible. The RVIP shows a series of numbers one by one in quick succession and the subjects are requested to indicate whenever they see three odd or even numbers displayed in a row. Working and spatial memory were tested using the numeric working memory test (NWM) and the Computerized Corsi block test. In the NWM test, subjects are shown one number at a time and instructed to memorize the numbers. Once the series is complete the procedure is repeated, including other numbers, subjects indicate whether the number displayed was in the previous list or not. Improvements are measured by increased accuracy and decreased reaction time. The Corsi block test shows subjects nine blue squares on a black background. The squares change to red, black, and blue again in different sequences, and the subjects recall the response by using the cursor to click the squares. This is repeated in up to fifteen levels as long as the participant selects enough correct responses. An improvement is measured as an increase in span score (average of the last 3 correct responses in a row). Short-term memory was assessed with a word recall test. Subjects are shown one word per second and once 15 words are displayed, they are given 60 s to write down as many words as they remember (immediate recall). Improvement is measured as an increase in accuracy. The Paired-Associate Learning test (PAL) from the Cambridge Neuropsychological Test Automated Battery (CANTAB ® ; Cambridge Cognition, https://cambridgecognition.com, accessed on 2 August 2023) was also included in the study. PAL assesses learning and memory [15]. White boxes are presented on the screen and open in a randomized order. Some of the boxes display a certain pattern, and subjects are requested to learn and remember a specific pattern and where it is located. The number of patterns increases with each level. The test terminates when the subject does not identify the correct patterns within 10 trials. A decrease in errors indicates improvement. 2.5. Awakening Cortisol and Analysis of Biomarkers in Blood The Cortisol Awakening Response (CAR) is the change in cortisol concentration that occurs in the first hour after waking from a night’s sleep [16]. It is different from the rest of the cortisol output during the day and has been related to stress and other psychological symptoms [17]. Cortisol levels were measured in saliva samples collected by passive drooling at T0, T30, T45, and T60 min after waking on the days of the visits at the clinic. The subjects were not allowed to brush or floss their teeth and eat or drink anything until after all samples had been collected. The samples were brought to the clinic and thereafter stored at − 80 ◦ C until analysis. Analysis of cortisol was performed with ELISA (High sensitivity salivary cortisol, Salimetrics, State College, PA, USA), according to the manufacturer’s instructions. Blood samples for assessing biomarkers were collected at baseline and after 12 weeks. Serum and plasma were separated by centrifugation for 10 min at 3000 rpm at 4 ◦ C. The samples were thereafter stored at − 80 ◦ C until analysis. The biomarkers assessed were Transforming Growth Factor β 1 (TGF- β 1), galectin- 3, fractalkine/CX3CL/CX3CL1, brain-derived neurotrophic factor (BDNF), tryptophan, L-kynurenine, and high-sensitivity C-reactive protein (hs-CRP).
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