<i>Bifidobacterium longum </i>NCIMB 41676

Abstract

Bifidobacterium longum strain NCIMB 41676 is useful for improving or sustaining vitality and/or reducing mental fatigue in stressful situations.

Claims

1. A method of treating a human subject, the method comprising administering to the subject an oral formulation comprising Bifidobacterium longum strain having the accession number NCIMB 41676 and an ingestible carrier, wherein the formulation improves or sustains vitality in the subject as measured by an increase in theta band resting state brain oscillations in the frontal and cingulate cortex and a decrease in beta-3 resting state brain oscillations in the hippocampus, fusiform, and temporal cortex.

2. A method of treating a healthy human subject, the method comprising administering to the subject an oral formulation comprising Bifidobacterium longum strain having the accession number NCIMB 41676 and an ingestible carrier, wherein the formulation improves vitality and reduces mental fatigue in the subject as measured by neural activity, compared to a corresponding healthy human subject orally administered with only the ingestible carrier, and wherein the neural activity includes an increase in theta band resting state brain oscillations of the cingulate cortex, or a decrease in beta-3 resting state brain oscillations in the hippocampus, fusiform, or temporal cortex.

3. The method of claim 1, wherein the formulation is administered daily.

4. The method of claim 1, wherein the strain is in the form of viable cells.

5. The method of claim 1, wherein the strain is in the form of non-viable cells.

6. The method of claim 1, wherein the formulation is in the form of a capsule, a tablet, or a powder.

7. The method of claim 1, wherein the ingestible carrier is a food product chosen from acidified milk, a yoghurt, a frozen food, a gum, a candy, a milk powder, a milk concentrate, a cheese spread, a nutritional composition, a nutritional supplement, a cereal bar, a dressing, or a beverage.

8. The method of claim 1, wherein the formulation is an infant food.

9. The method of claim 1, wherein the formulation further comprises a protein, a peptide, a lipid, a carbohydrate, a vitamin, a mineral, a trace element, or a combination thereof.

10. The method of claim 1, wherein the strain is present in an amount of more than 10.sup.6 cfu.

11. The method of claim 1, wherein the strain is present in the formulation in an amount of from 10.sup.7 cfu to 10.sup.10 cfu.

12. The method of claim 1, wherein the strain is present in the formulation in an amount of from 10.sup.8 cfu to 10.sup.9 cfu.

13. The method of claim 1, wherein the subject is at least 60 years old.

14. The method of claim 1, wherein the formulation improves vitality in the subject as measured by the 36-item short-form health survey (SF36).

15. The method of claim 1, wherein the formulation reduces mental fatigue in the subject as measured by neural activity.

16. The method of claim 2, wherein the strain is in the form of viable cells or non-viable cells.

17. The method of claim 2, wherein the formulation is in the form of a capsule, a tablet, or a powder.

18. The method of claim 2, wherein the ingestible carrier is a food product chosen from acidified milk, a yoghurt, a frozen food, a gum, a candy, a milk powder, a milk concentrate, a cheese spread, a nutritional composition, a nutritional supplement, a cereal bar, a dressing, or a beverage.

19. The method of claim 2, wherein the strain is present in the formulation in an amount of more than 10.sup.6 cfu.

20. The method of claim 2, wherein the formulation further comprises a protein, a peptide, a lipid, a carbohydrate, a vitamin, a mineral, a trace element, or a combination thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention will be more clearly understood from the following description thereof, given by way of example only, in which:—

(2) FIG. 1 is a CONSORT flow diagram of a clinical trial;

(3) FIG. 2 are images that illustrate the difference of neural activity change during resting-state comparing B. longum 1714 vs. placebo. A. After the intervention, an increased theta band (6 Hz) power was obtained in a cluster including regions of bilateral IFC, MFC, and the bilateral ACC and MCC, comparing B. longum 1714 with the placebo group (p<0.05). B. After the intervention, reduced beta-2 band (26 Hz) power was obtained in a cluster, consisting of the bilateral FFG and HIPP, left ITC and STC, bilateral MTC and left CBL, comparing B. longum 1714 with the placebo group (p<0.05);

(4) FIG. 3 is a chart of the correlation between neural activity change and SF36 change. In all groups, a positive correlation was obtained between changes of SF36 item “Energy/Vitality” with changes of theta band power in the cluster (r=0.33, p=0.04). In only B. longum 1714™ group, changes of SF36 item ‘Energy/Vitality’ positively correlated with change of averaged theta band power (r=0.61, p=0.007), and negatively correlated with change of beta-3 band power in the activated clusters during the resting state, respectively (r=−0.50, p=0.04);

(5) FIG. 4 is a schematic outline of a trial in a Cyberball game;

(6) FIG. 5 is a bar chart of the main effects of condition on the Need Threat Scale (NTS) and the ‘Exclusion Perception’ (SEP). Participants in all groups reported increased scores of NTS and SEP in the exclusion condition compared to inclusion condition, after 4 weeks' intervention; inferior, middle and superior frontal cortex (IFC, MFC and SFC), and the bilateral anterior and middle cingulate cortex (ACC and MCC);

(7) FIG. 6 are images that illustrate the difference of neural activity change during the CBG comparing B. longum 1714™ vs. placebo. A. Theta band showed an increased power in a cluster, consisting of the right inferior, middle and superior frontal cortex (IFC, MFC and SFC), the left bilateral anterior and middle cingulate cortex (ACC and MCC) and the right supramarginal gyrus (SMG), comparing B. longum 1714™ group and the placebo group in both conditions (p=0.03). B. Alpha band power also showed an increased power in cluster, including regions of the right IFC the bilateral MFC and SFC, the bilateral ACC and MCC, and the right SMG, comparing B. longum 1714™ group and the placebo group in both conditions (p=0.04). No main effect of condition or interaction of intervention and condition were observed;

(8) FIG. 7 is a chart of the correlation between neural activity change during the CBG and subjective score changes. Only in B. longum 1714™ group and only during the exclusion condition, NTS changes positively correlated with changes of the theta band power (r=0.62, p=0.008) and alpha band power (r=0.54, p=0.03); and

(9) FIG. 8 are graphs illustrating the effect of B. longum 1714™ strain on energy/vitality, concentration and stress in an online study with healthy volunteers.

DETAILED DESCRIPTION

(10) A deposit of B. longum 1714 strain was made under the terms of the Budapest Treaty at the National Collections of Industrial and Marine Bacteria Limited (NCIMB) Ferguson Building, Craibstone Estate, Bucksburg, Aberdeen, AB21 9YA, Scotland, UK on Nov. 5, 2009 and accorded the accession number NCIMB 41676.

(11) WO2011/05853A, the entire contents of which are incorporated herein by reference, describes B. longum 1714™ strain—NCIMB 41676.

(12) In a randomized, double-blinded, placebo-controlled trial, the effects of B. longum 1714™ strain was studied in comparison to placebo on 1) brain activity and quality of life measures in healthy subjects and 2) brain activity and coping skills and mental well-being associated with an induced social stressor. Forty-three healthy volunteers received either B. longum 1714™ or placebo for four weeks. Their health status was assessed using the 36-item short-form health survey (SF36) quality of life tool, and brain activation was studied before and after the application of a social stress test using magnetoencephalography (MEG). The 36-item short-form health survey (SF36) is a highly validated quality of life (QOL) tool to understand vitality, physical, social, emotional and mental functioning. Specifically, the SF36 includes eight subscales: physical functioning, bodily pain, role limitations due to physical health problems, role limitations due to personal or emotional problems, emotional well-being, social functioning, energy/vitality, and general health perceptions. Magnetoencephalography (MEG) is a functional neuroimaging technique for mapping brain activity by recording magnetic fields produced by electrical currents occurring naturally in the brain using very sensitive magnetic techniques. B. longum 1714™ strain was selected as it had previously been demonstrated to modulate stress related behaviours in animals (8, 9). Supporting evidence is also provided by a clinical study that showed reduced stress responses in response to an acute physical test—the cold pressor test and improved cognitive activity following treatment (10). However, the cold pressor test which is a physical induced stressor is different to a social exclusion test which is an emotional challenge. The cold pressor test is a cardiovascular test performed by immersing the hand into an ice water container, usually for one minute, and measuring changes in blood pressure and heart rate as well as cortisol responses. These changes relate to acute vascular and pulse excitability. Other measures can also be obtained from the cold pressor such as pain threshold and pain tolerances. In fact, a social stress induces an emotional response but not a change in blood pressure, heart rate or cortisol response.

Example 1—the Effects of B. longum 1714™ Strain in Comparison to Placebo on Brain Activity and Quality of Life Measures in Healthy Subjects Prior to any Induced Stressor being Applied

(13) Compared to placebo the B. longum 1714™ strain was effective in altering brain activity at particular wavelengths in certain regions of the brain (FIG. 2). Specifically, the B. longum 1714™ affected resting state brain oscillations with an increase in theta band in the frontal and cingulate cortex (p<0.05) and a decrease in beta-3 band in the hippocampus, fusiform, and temporal cortex (p<0.05). These areas represent important regions of the brain which are critical to human emotions, learning and memory functions. Correlation analysis showed that there were no correlations between these changes in brain activity and any SP36 parameter in the placebo fed group whereas surprisingly there were significant correlations between change in brain activity and SF36 sub-scale vitality scores in the B. longum 1714™ fed subjects (FIG. 3 and Table 3). This has never been seen before. Vitality questions included: ‘Did you feel full of pep?’, ‘Did you have a lot of energy?’, ‘Did you feel worn out?’, ‘Did you feel tired?

(14) These results indicate that B. longum 1714™ affects brain activity in healthy subjects in important regions of the brain which are related to increased vitality and reduced mental fatigue.

(15) TABLE-US-00001 TABLE 1 Correlations between brain activity and Energy Vitality Measurements Functional MEG Resting state MEG during Cyberball Subjective Theta band Beta-3 band Theta band Alpha band Groups item power change power change power change power change B. longum SF36- r = 0.61 r = −0.50 — — group Energy/ p = 0.007 p = 0.04 Vitality Abbreviations: SF36, 36-item short-form health survey; NTS, Need Threat Scale; MEG, magnetoencephalography.

Example 2—Effects of B. longum 1714™ on Brain Activity and Coping Skills and Mental Well-being Associated with an Induced Social Stressor

(16) Numerous studies have utilized a standardized paradigm, called the “Cyberball Game”, to study the effects of social stress and ostracism (exclusion), and the corresponding responses in the brain (11). However, this is the first study to test the effect of a probiotic on social stress and ostracism in humans.

(17) The Cyberball Game is “an online ball-tossing game that participants believe they are playing with two or three players” (11). In the Cyberball Game, the participants are asked to play a ball tossing game with two other virtual players programmed by the experimenter using an on-line game. They were made believe that the two players were real and were playing the game.

(18) During the game, the participant is included in tossing the ball to each other and then excluded from the game (see FIG. 4). Participants complete three questionnaires to assess their level of distress, coping and mental well-being during exclusion from the game and inclusion. The questionnaires used included Need Threat Scale, the Mood Questionnaire and the Subjective ‘Exclusion Perception’); all these scales are validated standards for the Cyberball Game (11). The Need Threat Scale was designed to measure the feelings and emotional consequences of social rejection, and higher scores related to higher distress level. Its four items (rated between 1 and 5 for “weak” to “strong”) comprised self-esteem, belonging, meaningful existence and control, and combined ratings have been used as a measure of social distress in previous studies. The Mood Questionnaire was used to assess mood, using 8 questions (are you feeling bad, good, happy, sad, pleasant, angry, friendly and unfriendly), all rated between 1 and 5. The Subjective ‘Exclusion Perception was to record participants’ feeling of being included/ostracized by asking them to rate two statements (‘I was ignored’ and ‘I was excluded’) between 1 and 5. In general, with this game, participants feel more distressed in periods when they are excluded from the game compared to the periods in which they are included in the game Indeed, social stress during the Cyberball Game was consistently associated with altered brain activity in certain brain areas involved in emotions, (12).

(19) In this study, the Cyberball Game resulted in increased distress after 4-weeks intervention in the healthy subjects as measured by the validated Need Threat Scale and Subjective ‘Exclusion Perception’ questionnaires (FIG. 5). This means that the model worked and when subjects were excluded from the game their mood worsened and their stress increased. When included in the game, they felt better.

(20) Interestingly, the group that received B. longum 1714™ showed brain activity changes compared to placebo in response to the Cyberball game. Specifically, only in the B. longum 1714™ group and not in the placebo group, brain activity during the Cyberball game showed increases in theta and alpha bands power in the frontal and temporal cortex and supramarginal gyrus (p=0.03; 0.04) (FIG. 6).

(21) Furthermore, in the B. longum 1714™ fed group that were left out of the game, correlation analysis shows that the more the change in brain activity the better they were able to cope compared to the placebo fed group, as measured by the Need Threat Scale, (FIG. 7). The Need Threat Scale is designed to measure the feelings and emotional consequences of social rejection, and higher scores related to higher distress level. Its four items (rated between 1 and 5 for “weak” to “strong”) comprised self-esteem, belonging, meaningful existence and control, and combined ratings have been used as a measure of social distress in previous studies. Specifically, only with B. longum 1714™ and only during exclusion from the game was there a positive correlation with changes of the theta band power (r=0.62, p=0.008) and alpha band power (r=0.54, p=0.03) with change in mental well-being (FIG. 7, Table 2). This shows that B. longum 1714™ affecting an individuals' neurophysiology in a way that may help in coping better with a social stress challenge. This is a novel finding in humans and was unexpected as emotional regulation is complex. It is surprising that a probiotic could impact such a complicated brain function in such a way.

(22) TABLE-US-00002 TABLE 2 Correlations between brain activity and Social Ostracism/Rejection Measurements Functional MEG Resting state MEG during Cyberball Subjective Theta band Beta-3 band Theta band Alpha band Groups item power change power change power change power change B. longum Need — r = 0.62 − r = 0.54 − group Threat p = 0.008 p = 0.03 Scale Abbreviations: NTS, Need Threat Scale; MEG, magnetoencephalography.

Example 3—Effect of B. longum 1714™ on Energy and Concentration in Healthy Humans

(23) An on-line study was performed with 42 healthy volunteers taking B. longum 1714™ for 5 weeks.

(24) Participants were asked the following questions: 1. Reflecting over the last week how would you rate your Energy levels? (0=very low energy, 9=very high energy) 2. Reflecting over the last week how would you rate your ability to concentrate? (0=completely distracted, 9=excellent concentration)

(25) The results of this study showed that energy levels and ability to concentrate increased progressively over the course of the study (FIG. 8). This supports our work which shows that B. longum 1714™ increases vitality/energy and reduces mental fatigue.

(26) 2) Materials and Methods

(27) The Effects of B. longum 1714™ Strain in Comparison to Placebo on Brain Activity and Quality of Life Measures and Mental Well-being Associated with an Induced Social Stressor in Healthy Subjects.

(28) 2.1 Participants

(29) Based on previously published data (11), we estimated that—with a power of 0.95 for a 2×2 repeated measure ANOVA—a minimum sample size of 34 was required to demonstrate an effect size f=0.2 at α=0.05 in a parallel-group designed study. The study was completed with 40 healthy volunteers, after having recruited initially sixty-one participants. Eighteen participants were excluded because they did not meet the inclusion criteria, and three of them could not be included in the final analysis because of the intake of an antibiotic during the intervention period (see FIG. 1 for detailed trial profile). Criteria for inclusion were: 1) non-smoker for at least 3 months, 2) a body mass index (BMI) of 18-30, 3) no chronic allergies, 4) willing to discontinue their normal consumption of probiotics and prebiotic-containing foods or potentially immune-enhancing dietary supplements, 5) receiving no immune-suppressing intervention and not having any immunosuppressive illness within the last year, 6) receiving no antibiotic therapy within the last 2 months, 7) having no chronic psychiatric or gastrointestinal disorder, 8) and having no non-removable metal parts in the body. Informed consent was obtained from all participants prior to joining the study. The protocol had been approved by the Ethics Board of the University of Tübingen Medical School (No. 503/2015B01, as of Aug. 26, 2015), and was registered at ClinicalTrials.gov (identifier No. NCT02793193).

(30) 2.2 Design

(31) A randomized, double-blinded, and parallel-group design was employed. Participants were screened to exclude for the irritable bowel syndrome and psychiatric disorders using the Rome III criteria (13) and the include healthy subjects using the Patient Health Questionnaire (PHQ) (14). Demographic and baseline psychological information was also recorded. After screening, participants were randomly allocated in different intervention groups and took either probiotic or placebo for 4 weeks (28 days). The probiotic and placebo preparations in identical sachets were provided by Alimentary Health Ltd, Cork, Ireland. The randomization scheme was only unblinded after completion of the experiment and complete data evaluation. At baseline and one day after the intervention period, participants visited our lab for the MEG measurements. In addition, they visited the lab for acquiring structural MR images on a different day, regardless of their intervention schedule.

(32) During the intervention period, participants were instructed to avoid consumption of food containing probiotics/prebiotics, or potentially immune-enhancing dietary supplements. This was supported by providing them with a list of “prohibited” foods.

(33) 2.3 Materials

(34) Each 2 g probiotic sachet contained 1×10.sup.9 colony-forming units B. longum 1714™ strain with maltodextrin and magnesium stearate; each placebo sachet contained only 2 g of maltodextrin and magnesium stearate. Participants were instructed to consume one sachet every morning with food within fifteen minutes, by mixing the content into 50 ml of water.

(35) 2.4 Questionnaires

(36) To record participants health status, the 36-item short-form health survey (SF36) was used (15). The SF36 includes eight subscales: physical functioning, bodily pain, role limitations due to physical health problems, role limitations due to personal or emotional problems, emotional well-being, social functioning, energy/vitality, and general health perceptions. Participants were asked to finish the SF36 at the beginning of each of the two visits.

(37) 2.5 Cyberball Game

(38) In the CBG, the participants were asked to play a ball tossing game with two other virtual players programmed by the experimenter. They were made believe that the two players were real and were playing the game. To minimize gender effects, male participants played with 2 female players, and female participants played with 2 male players. During the game, the other players were depicted as cartoon characters with their photos aside and names below. The volunteering participant was represented by a cartoon in the middle lower part of the screen and could throw the ball to either of the other two players on the left or right, by pressing the left or right button on the response box (FIG. 2).

(39) The CBG consisted of 4 blocks: inclusion-exclusion-inclusion-exclusion conditions; this order was maintained for all volunteers. In each inclusion block, there were 108 trials, during ⅓ (36 trials) of which the participant received the ball from the other players (for another ⅓ the ball was played to one of the other players, and for the other ⅓ between the other two players). The order of the ball throwing to the participant was pseudo-randomized. The ⅓ of trials in the inclusion block when the virtual players threw the ball to each other and not to the participant, were called “not my turn” events.

(40) To equalize the numbers of analysed trials when the virtual players threw the ball to each other and not to the participant, we set 47 total trials in each exclusion block. The participants received the ball 3 times (trials 14, 25, or 26, and 39, 40, or 41 in block 2 and 4, respectively) to maintain their attention. The first 5 exclusion trials, the 3 trials the participant receiving the ball, and the 3 trials the participant was throwing the ball were not analysed but discarded. The remaining 36 so-called “rejection” events were used for comparison with the 36 “not my turn” events in the inclusion block. Visual stimuli of these trials did not differ in two conditions, so any difference of brain activities was supposed to be due to the participants' inner state. The trial began with the ball being presented in the cartoon for 500-2000 ms randomly to imitate a real life situation. Then the ball was moving for 2000 ms before reaching the target player (FIG. 2).

(41) After each block of the CBG, participants needed to complete three questionnaires to assess their acute level of distress. We employed the self-report measures of the Need Threat Scale (NTS), the Mood Questionnaire (MQ) and the Subjective ‘Exclusion Perception’ (SEP); all these scales are validated standards for the CBG (13, 37). The NTS was designed to measure the feelings and emotional consequences of social rejection, and higher scores related to higher distress level. Its four items (rated between 1 and 5 for “weak” to “strong”) comprised self-esteem, belonging, meaningful existence and control, and combined ratings have been used as a measure of social distress in previous studies. The MQ was used to assess mood, using 8 questions (are you feeling bad, good, happy, sad, pleasant, angry, friendly and unfriendly), all rated between 1 and 5. The SEP was to record participants' feeling of being included/ostracized by asking them to rate two statements (‘I was ignored’ and ‘I was excluded’) between 1 and 5.

(42) Assessment of Need Threat Scale, Mood Questionnaire and Exclusion Perception.

(43) All items need to be rated on a scale from 1 (‘not at all’) to 5 (‘very much’). (R)=reversed scored.

(44) Need

(45) Belonging:

(46) 1. I felt disconnected with one or more players.

(47) 2. I felt rejected by other players.

(48) 3. I felt like an outsider.

(49) 4. I felt belonged to the group. (R)

(50) 5. The other players interacted with me a lot. (R)

(51) Self-Esteem:

(52) 6. I felt good about myself. (R)

(53) 7. My self-esteem was high. (R)

(54) 8. I felt I was liked. (R)

(55) 9. I felt insecure.

(56) 10. I felt satisfied. (R)

(57) Meaningful Existence:

(58) 11. I felt invisible.

(59) 12. I felt meaningless.

(60) 13. I felt non-existent.

(61) 14. I felt important. (R)

(62) 15. I felt useful. (R)

(63) Control:

(64) 16. I felt powerful. (R)

(65) 17. I felt I had control over the course of the game. (R)

(66) 18. I felt I had the ability to significantly alter events. (R)

(67) 19. I felt I was unable to influence the actions of others.

(68) 20. I felt the other players decided everything.

(69) Mood

(70) During the game I felt:

(71) 1. Good (R)

(72) 2. Bad

(73) 3. Happy (R)

(74) 4. Sad

(75) 5. Pleasant (R)

(76) 6. Angry

(77) 7. Friendly (R)

(78) 8. Unfriendly

(79) After each of the inclusion and exclusion blocks, participants completed the NTS, the MQ, and the SEP.

(80) 2.6 Magnetoencephalography Recording

(81) Brain magnetic fields were measured with a 275-channel whole-head magnetoencephalograph (CTF Omega, Port Coquitlam, Canada). Participants were studied in supine position. During each recording session, 5 minutes resting state was recorded prior to recording while playing the CBG. During the resting state, participants were instructed to move as little as possible and to be awake, while keeping their eyes closed. During the CBG, task instructions were projected onto a screen in front of the participants via a video projector and a mirror system. Participants were asked to fixate the screen and hold a response box to get ready for the task. Participants were also instructed to move as little as possible. MEG signals were sampled at a rate of 585.94 Hz with an anti-aliasing filter set to 292.97 Hz.

(82) In order to overlay the brain activity derived from MEG on anatomical scans, high-resolution (1 mm, isotropic) T1-weighted structural MR images were acquired using an MPRAGE sequence with a Siemens MAGNETOM Trio 3T scanner (Siemens AG, Erlangen, Germany) (12-channel array head coil) for each participant, but at a separate occasion.

(83) 2.7 Data Analysis

(84) 2.7.1 Data Analysis: Questionnaires

(85) Data analysis was conducted using SPSS 21 (IBM, Armonk, N.Y., USA). To examine whether there was a significant difference in health status between groups at baseline, scores of SF36 during the first visit were entered into a non-parametric two-independent-sample Mann-Whitney U test of Intervention as between factor (B. longum 1714™ vs. Placebo), as parametric assumptions of these data were violated. To test the intervention-related changes in participants' health status scored by SF36, changes from before to after the 4-weeks intervention were computed by subtracting the baseline assessment from the corresponding post-intervention values. Non-parametric two-independent-sample Mann-Whitney U test was used to examine the change of SF36 between Intervention (B. longum 1714™ vs. Placebo).

(86) To examine whether subjective ratings for the CBG were different between groups at baseline, scores of NTS, MQ, and SEP acquired during the first visit were entered into an independent T-test with Intervention as between factor (B. longum 1714™ vs. Placebo). To control the intervention-related changes of the NTS, the MQ and SEP during the CBG, changes after each intervention were computed for each condition and entered into a 2×2 repeated measure ANOVA with Intervention as a between-factor (B. longum 1714™ vs. Placebo)×Condition as a within-factor (exclusion vs. inclusion). Where significant main effects or interaction were observed, pairwise post-hoc comparisons were used with a Bonferroni adjusted threshold (α=0.025). Mean data are reported as M+SD.

(87) 2.7.2 Data Analysis: MEG-Data

(88) Preprocessing

(89) Analysis of the MEG data was carried out using Matlab (Mathworks, Natick, USA) and the open-source toolboxes Fieldtrip (38). The resting state dataset were cut into time windows of 2 s. Data in this time window were filtered using a 4 Hz high pass frequency filter. Non-physiological jumps in the MEG signal and trials with jump and muscle artifacts were excluded by an automatic rejection algorithm that excluded all trial in which the variance exceeded 10.sup.−25 in any channel.

(90) The continuously recorded dataset during the CBG was segmented in epochs of 3 s with 1 s of pre-stimulus interval time-locked to the moment at which the players started to throw the ball). Trials in which one of the virtual players threw the ball towards the other virtual player during the inclusion blocks were defined as ‘inclusion’ condition, and those during exclusion blocks were defined as ‘exclusion’ condition.

(91) Time Frequency Analysis

(92) The time-frequency analysis used the multitaper windowed fast fourier transform ‘MTMFFT’ implemented in Fieldtrip. The ‘multitaper method’ (MTM) is based on Slepian sequences as tapers. The frequency of interest ranged from 4 to 30 Hz with step of 2 Hz. The frequency smoothing window is +/−3 Hz:

(93) Source Analysis

(94) Using the time-frequency determined by the analysis described above, oscillatory sources of theta, alpha, beta-1, beta-2 and beta-3 bands (6, 11, 16, 21, and 26 Hz) were localized using beamformer techniques. We applied the Dynamical Imaging of Coherent Sources (DICS) method. In order to estimate the individual source activity, each participant's brain recorded as T1-MR image was divided in a regular three dimensional grid with a 1 cm resolution. A spatial inverse filter was computed from both conditions and both visits, as common filter. The common filter was applied to each condition and each visit separately in order to obtain the respective source power. The MEG data in each condition were co-registered with the respective individual structural MR images respectively.

(95) Source Statistics

(96) We performed source-level statistics to assess effects of intervention on the data obtained from the resting-state condition and the CBG, respectively. To check if there was any difference between groups at baseline prior to any intervention, resting-state at baseline source power was compared with an independent T-test with Intervention (B. longum 1714™ vs. placebo) as between factor. Then, intervention-induced changes in source power were computed in each frequency band by subtracting the baseline from the post-intervention. The changes of the source power were entered into an independent T-test with Intervention (B. longum 1714™ vs. placebo) as between factor. For the CBG, source power at baseline in each frequency band was also tested with an independent T-test with Interventions (B. longum 1714™ vs. placebo) as between factor to check if brain activations showed differences between groups. Subsequently, changes in the source power after intervention were computed by subtracting the baseline values from the post-intervention for each condition in each frequency band. Changes of source power were entered in a two-way ANOVA of interventions (B. longum 1714™ vs. placebo)×conditions (exclusion vs. inclusion). The statistical analysis was done separately for each frequency band. To localize significant activations, the cluster-based permutation method for multiple comparisons was used with a significance level of alpha of 0.05.

(97) Correlation Between Questionnaire and MEG Data

(98) To investigate the relationship between changes in neural activity and changes in the subjective reports induced by B. longum 1714™ correlations analyses were carried out for both, the resting state recording and the CBG, respectively. For the resting state recording, averaged source power within clusters was calculated for the clusters differing significantly between both visits. The averaged source power was correlated with changes in health status (SF36) for each group separately. For the CBG, for each condition and each intervention, source power within the clusters that differed significantly between both visits was averaged for each condition and each group. The averaged source power was correlated with changes in the scores of NTS, MQ and SEP separately for each condition and each group, using Pearson correlations.

Example 4—Effect of B. longum 1714™ on Energy in Healthy Subjects Using an On-Line Study

(99) 42 subjects were recruited from the general population to take part in an on-line study. Subjects were provided in a capsule format containing 1×10.sup.9 colony-forming units B. longum 1714™. Subjects were asked to take 1 capsule per day for the duration of the study (5 weeks) and complete an on-line questionnaire on their energy, concentration and stress levels.

(100) 3 Result

(101) A total of 40 participants were included in the analysis with half of them receiving B. longum 1714™ intervention. Sex of participants was matched between groups. Age and BMI of participants were not significantly different between groups (See Table 3 for details).

(102) TABLE-US-00003 TABLE 3 Demographic and baseline information B. longum 1714 ™ Placebo P value Sex Male N = 7  N = 7  n/a Female N = 13 N = 13 Birth delivery Caesarean section N = 2  N = 2  n/a Vaginal delivery N = 18 N = 18 Age 31.00 ± 2.28 33.00 ± 2.83 ns. BMI 23.00 ± 0.68 22.00 ± 0.55 ns. SF36 Physical functioning 96.84 ± 1.03 97.63 ± 0.80 Role limitations due 100.00 ± 0.00  100.00 ± 0.00  to physical health Role limitations due 100.00 ± 0.00  95.00 ± 5.00 to emotion problems Energy/Vitality 72.25 ± 2.42 75.25 ± 2.94 ns. Emotional well-being 85.78 ± 1.22 84.42 ± 1.75 Social functioning 100.00 ± 0.00  94.74 ± 2.20 Pain 88.75 ± 2.67 89.75 ± 2.80 General health 82.50 ± 2.31 87.37 ± 2.40 Cyberball game NTS Inclusion −28.30 ± 3.55  −36.90 ± 2.28  Exclusion 14.33 ± 4.55 26.90 ± 3.48 MQ Inclusion 13.80 ± 1.47 18.03 ± 0.99 ns. Exclusion  2.38 ± 2.25 −5.48 ± 2.17 SEP Inclusion −4.68 ± 0.28 −5.30 ± 0.21 Exclusion  0.15 ± 0.92  2.23 ± 0.61 Abbreviations: BMI, body mass index; SF36, 36-item short-form health survey NTS, Need Threat Scale; MQ, mood questionnaire; SEP, subjective exclusion perception; ns. not significant.
3.2 MEG Data

(103) Summaries of frequency bands and neuroanatomical areas found to be related to B. longum 1714™ intervention, and associations of neural activity changes Table 4, FIG. 2 and FIG. 6 and subjective effects are provided in Table 5, FIG. 5 and FIG. 7 respectively.

(104) TABLE-US-00004 TABLE 4 Summarized neuroanatomical areas and frequency bands of changed neural activities influenced by effect of condition, intervention and/or interaction of condition and intervention Frequency Comparison band Brain region Hemisphere P vale Intervention Theta ↑ IFC B <0.05 effect on MFC B resting state: SFC B B. longum ACC B group vs. MCC B Placebe group Beta-3 ↓ FFG B <0.05 HIPP B ITC L MTC B STC L CBL L Intervention Theta ↑ IFC R 0.03 effect on the MFC B Cyberball game SFC B for all ACC L conditions: group MCC B vs. B. longum SMG R Placebo group Alpha ↑ IFC R 0.04 MFC B SFC B ACC B MCC B SMG R Abbreviations: IFC, inferior frontal cortex; MFC, middle frontal cortex; ACC, anterior cingulate cortex; MCC, middle cingulate cortex; FFG, fusiform gyrus; HIPP, hippocampus; ITC, inferior temporal cortex; STC, superior temporal cortex; MTC, middle temporal cortex; CBL, cerebellum; SMG, supramarginal gyrus; B, bilateral; L, left; R, right.

(105) TABLE-US-00005 TABLE 5 Correlations between brain activity and mental well-being Functional MEG Resting state MEG during Cyberball Subjective Theta band Beta-3 band Theta band Alpha band Groups item power change power change power change power change Both SF36- r = 0.33 — — — groups Energy/ p = 0.04 Vitality B. longum SF36- r = 0.61 r = −0.50 — — group Energy/ p = 0.007 p = 0.04 Vitality Need r = 0.62 r = 0.54 Threat p = 0.008 p = 0.03 Scale Abbreviations: SF36, 36-item short-form health survey; NTS, Need Threat Scale; MEG, magnetoencephalography.

Example 5—Effect of B. longum 1714™ on Energy in Healthy Subjects Using an On-Line Study

(106) An on-line study was performed with 42 healthy volunteers taking B. longum 1714™ for 5 weeks.

(107) Participants were asked the following questions: 1. Reflecting over the last week how would you rate your Energy levels? (0=very low energy, 9=very high energy) 2. Reflecting over the last week how would you rate your ability to concentrate? (0=completely distracted, 9=excellent concentration)

(108) The results of this study showed that energy levels and ability to concentrate increased progressively over the course of the study (FIG. 8). This supports our work which shows that B. longum 1714™ increases vitality/energy and reduces mental fatigue.

(109) Recent evidence suggest that probiotics may have a role mental health and well-being (1). However, while there is increasing interest in the role of the gut in communicating with the brain, this field is in its infancy and very little is known about the effect of probiotic on mental well-being in humans. In this work, we showed that surprisingly B. longum 1714™ affects the brain and increased vitality and reduced mental fatigue in healthy subjects as demonstrated in 2 human studies. This is novel and not seen before. Furthermore, the B. longum 1714™ affected resting state brain oscillations with an increase in theta band in the frontal and cingulate cortex (p<0.05) and a decrease in beta-3 band in the hippocampus, fusiform, and temporal cortex (p<0.05). These areas represent in important regions of the brain such which are critical to human emotions, learning and memory functions.

(110) B. longum 1714™ also affecting an individuals' neurophysiology in a way that may help in coping better with a social exclusion challenge. This is a novel finding in humans as no other probiotic has shown an impact on social ostracism. This finding was unexpected as emotional regulation is complex and it is surprising that a probiotic could impact such a complicated brain function in such a way. Previous work shows that B. longum 1714™ was effective in altering response to an acute stressor test (the Cold Pressor Test) when subjects were asked to perform under pressure (10). The Cold Pressor Test involves immersing your non-dominant hand into icewater until it becomes painful (usually between 1 and 2 minutes) and is a pain stimulus. This test is designed to induce a physiological response such as elevated heart beat and cortisol levels—the ‘fight or flight response’. The Cyber Ball Game works with social exclusion (ostracism) among peers, induces feelings of not being liked. It is unexpected that a probiotic would influence emotional and mental wellbeing in a model of social exclusion/ostracism.

(111) The invention is not limited to the embodiments hereinbefore described which may be varied in detail.

REFERENCES

(112) 1. Dinan T G, Stanton C, Cryan J F. Psychobiotics: a novel class of psychotropic. Biol Psychiatry. 2013; 74(10):720-6 2. Bravo J A, Forsythe P, Chew M V, Escaravage E, Savignac H M, Dinan T G, et al. Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve. Proc Natl Acad Sci USA. 2011; 108(38):16050-5. 3. Pinto-Sanchez M I, Hall G B, Ghajar K, Nardelli A, Bolino C, Lau J T, et al. Probiotic Bifidobacterium longum NCC3001 Reduces Depression Scores and Alters Brain Activity: A Pilot Study in Patients With Irritable Bowel Syndrome. Gastroenterology. 2017; 153(2):448-59.e8. 4. Rao A V, Bested A C, Beaulne T M, Katzman M A, Iorio C, Berardi J M, et al. A randomized, double-blind, placebo-controlled pilot study of a probiotic in emotional symptoms of chronic fatigue syndrome. Gut Pathog. 2009; 1(1):6. 5. Steenbergen L, Sellaro R, van Hemert S, Bosch J A, Colzato L S. A randomized controlled trial to test the effect of multispecies probiotics on cognitive reactivity to sad mood. Brain Behav Immun. 2015; 48:258-64. 6. Romijn A R, Rucklidge J J. Systematic review of evidence to support the theory of psychobiotics. Nutr Rev. 2015; 73(10):675-93. 7. Krieger N. Theories for social epidemiology in the 21st century: an ecosocial perspective. Int J Epidemiol. 2001; 30(4):668-77. 8. Savignac H M, Kiely B, Dinan T G, Cryan J F. Bifidobacteria exert strain-specific effects on stress-related behavior and physiology in BALB/c mice. Neurogastroenterology and motility: the official journal of the European Gastrointestinal Motility Society. 2014; 26(11):1615-27. 9. Savignac H M, Tramullas M, Kiely B, Dinan T G, Cryan J F. Bifidobacteria modulate cognitive processes in an anxious mouse strain. Behav Brain Res. 2015; 287:59-72. 10. Allen A P, Hutch W, Bone Y E, Kennedy P J, Temko A, Boylan G, et al. Bifidobacterium longum 1714 as a translational psychobiotic: modulation of stress, electrophysiology and neurocognition in healthy volunteers. Translational psychiatry. 2016; 6(11):e939. 11. Williams K D, Jarvis B. Cyberball: a program for use in research on interpersonal ostracism and acceptance. Behavior research methods. 2006; 38(1):174-80. 12. Cristofori I, Harquel S, Isnard J, Mauguiere F, Sirigu A. Monetary reward suppresses anterior insula activity during social pain. Soc Cogn Affect Neurosci. 2015; 10(12):1668-76. 13. Longstreth G F, Thompson W G, Chey W D, Houghton L A, Mearin F, Spiller R C. Functional bowel disorders. Gastroenterology. 2006; 130(5):1480-91. 14. Kroenke K, Spitzer R L, Williams J B, Lowe B. The Patient Health Questionnaire Somatic, Anxiety, and Depressive Symptom Scales: a systematic review. General hospital psychiatry. 2010; 32(4):345-59. 15. Ware J E, Jr., Sherbourne C D. The MOS 36-item short-form health survey (SF-36). I. Conceptual framework and item selection. Med Care. 1992; 30(6):473-83. 16. Van Schrojenstein L M, Mackus M, Otten L S, De Kruijff dD, Van de Loo A J, Kraneveld A D, Garssen J, Verster jIC; Mental resilience, perceived immune functioning, and health. J Multidiscip Healthcare. 2017 Mar. 21; 10:107-112.